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Sample records for subcellular localization activation

  1. Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

    Science.gov (United States)

    Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

    2013-01-01

    Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

  2. Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

    Science.gov (United States)

    Stubbs, M; Aiston, S; Agius, L

    2000-12-01

    We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

  3. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  4. Heat shock modulates the subcellular localization, stability, and activity of HIPK2

    International Nuclear Information System (INIS)

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam

    2016-01-01

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

  5. Heat shock modulates the subcellular localization, stability, and activity of HIPK2

    Energy Technology Data Exchange (ETDEWEB)

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam, E-mail: sganesh@iitk.ac.in

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

  6. Subcellular localization of cadmium in hyperaccumulator Populus ...

    African Journals Online (AJOL)

    In this study, subcellular localization of cadmium in hyperaccumulator grey poplar (Populus × canescens) was investigated by the transmission electron microscopy (TEM) method. Young Populus × canescens were grown and hydroponic experiments were conducted under four Cd2+ concentrations (10, 30, 50, and 70 μM) ...

  7. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence tolerant rice (Oryza sativa L.) cultivar FR13A. In the public databases, this protein was designated as putative Os02g0465900 protein. The cDNA containing the full-length sequence of OsARP gene was ...

  8. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

    Science.gov (United States)

    Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

    2013-11-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  9. The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

    2012-10-01

    Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

  10. Subcellular Iron Localization Mechanisms in Plants

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    Emre Aksoy

    2017-12-01

    Full Text Available The basic micro-nutrient element iron (Fe is present as a cofactor in the active sites of many metalloproteins with important roles in the plant. On the other hand, since it is excessively reactive, excess accumulation in the cell triggers the production of reactive oxygen species, leading to cell death. Therefore, iron homeostasis in the cell is very important for plant growth. Once uptake into the roots, iron is distributed to the subcellular compartments. Subcellular iron transport and hence cellular iron homeostasis is carried out through synchronous control of different membrane protein families. It has been discovered that expression levels of these membrane proteins increase under iron deficiency. Examination of the tasks and regulations of these carriers is very important in terms of understanding the iron intake and distribution mechanisms in plants. Therefore, in this review, the transporters responsible for the uptake of iron into the cell and its subcellular distribution between organelles will be discussed with an emphasis on the current developments about these transporters.

  11. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  12. Tau regulates the subcellular localization of calmodulin

    International Nuclear Information System (INIS)

    Barreda, Elena Gomez de; Avila, Jesus

    2011-01-01

    Highlights: → In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  13. Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity.

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    Jamin, Augusta; Wicklund, April; Wiebe, Matthew S

    2014-05-01

    Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways

  14. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

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    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  15. Subcellular localization and photodynamic activity of Photodithazine (glucosamine salt of chlorin e6) in murine melanoma B16-F10: an in vitro and in vivo study

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    Ono, Bruno Andrade; Pires, Layla; Nogueira, Marcelo Saito; Kurachi, Cristina; Pratavieira, Sebastião.

    2018-02-01

    Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.

  16. Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus

    International Nuclear Information System (INIS)

    Claudiani, Pamela; Riano, Elena; Errico, Alessia; Andolfi, Gennaro; Rugarli, Elena I.

    2005-01-01

    Most cases of autosomal-dominant hereditary spastic paraplegia are linked to mutations in SPG4 encoding spastin, a protein involved in microtubule dynamics and membrane trafficking. In pyramidal neurons of the motor cortex and in immortalized motor neurons, spastin is localized to the synaptic terminals and growth cones. However, in other neurons and in proliferating cells spastin is prevalently nuclear. The mechanisms that determine targeting of spastin to the nucleus or the cytoplasm are unknown. We show here that the SPG4 mRNA is able to direct synthesis of two spastin isoforms, 68 and 60 kDa, respectively, through usage of two different translational start sites. Both isoforms are imported into the nucleus, but the 68-kDa isoform contains two nuclear export signals that efficiently drive export to the cytoplasm. Nuclear export is leptomycin-B sensitive. The cytoplasmic 68-kDa spastin isoform is more abundant in the brain and the spinal cord than in other tissues. Our data indicate that spastin function is modulated through usage of alternative translational start sites and active nuclear import and export, and open new perspectives for the pathogenesis of hereditary spastic paraplegia

  17. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

    Science.gov (United States)

    Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

  18. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  19. Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization

    NARCIS (Netherlands)

    Dong, L.; Miettinen, K.; Verstappen, F.W.A.; Voster, A.; Jongsma, M.A.; Memelink, J.; Krol, van der S.; Bouwmeester, H.J.

    2013-01-01

    Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and

  20. Subcellular localization of the antidepressant-sensitive norepinephrine transporter

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    Winder Danny G

    2009-06-01

    Full Text Available Abstract Background Reuptake of synaptic norepinephrine (NE via the antidepressant-sensitive NE transporter (NET supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons. Results We elucidate NET localization in brain and superior cervical ganglion (SCG neurons, aided by a new NET monoclonal antibody, subcellular immunoisolation techniques and quantitative immunofluorescence approaches. We present evidence that axonal NET extensively colocalizes with syntaxin 1A, and to a limited degree with SCAMP2 and synaptophysin. Intracellular NET in SCG axons and boutons also quantitatively segregates from the vesicular monoamine transporter 2 (VMAT2, findings corroborated by organelle isolation studies. At the surface of SCG boutons, NET resides in both lipid raft and non-lipid raft subdomains and colocalizes with syntaxin 1A. Conclusion Our findings support the hypothesis that SCG NET is segregated prior to transport from the cell body from proteins comprising large dense core vesicles. Once localized to presynaptic boutons, NET does not recycle via VMAT2-positive, small dense core vesicles. Finally, once NET reaches presynaptic plasma membranes, the transporter localizes to syntaxin 1A-rich plasma membrane domains, with a portion found in cholera toxin-demarcated lipid rafts. Our findings indicate that activity-dependent insertion of NET into the SCG plasma membrane derives from vesicles distinct from those that deliver NE. Moreover, NET is localized in presynaptic membranes in a manner that can take advantage of regulatory processes targeting lipid raft subdomains.

  1. Subcellular localization of hepatitis E virus (HEV) replicase

    International Nuclear Information System (INIS)

    Rehman, Shagufta; Kapur, Neeraj; Durgapal, Hemlata; Panda, Subrat Kumar

    2008-01-01

    Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of ∼ 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication

  2. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

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    Tam Michael WC

    2010-03-01

    Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

  3. Protein subcellular localization prediction using artificial intelligence technology.

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2008-01-01

    Proteins perform many important tasks in living organisms, such as catalysis of biochemical reactions, transport of nutrients, and recognition and transmission of signals. The plethora of aspects of the role of any particular protein is referred to as its "function." One aspect of protein function that has been the target of intensive research by computational biologists is its subcellular localization. Proteins must be localized in the same subcellular compartment to cooperate toward a common physiological function. Aberrant subcellular localization of proteins can result in several diseases, including kidney stones, cancer, and Alzheimer's disease. To date, sequence homology remains the most widely used method for inferring the function of a protein. However, the application of advanced artificial intelligence (AI)-based techniques in recent years has resulted in significant improvements in our ability to predict the subcellular localization of a protein. The prediction accuracy has risen steadily over the years, in large part due to the application of AI-based methods such as hidden Markov models (HMMs), neural networks (NNs), and support vector machines (SVMs), although the availability of larger experimental datasets has also played a role. Automatic methods that mine textual information from the biological literature and molecular biology databases have considerably sped up the process of annotation for proteins for which some information regarding function is available in the literature. State-of-the-art methods based on NNs and HMMs can predict the presence of N-terminal sorting signals extremely accurately. Ab initio methods that predict subcellular localization for any protein sequence using only the native amino acid sequence and features predicted from the native sequence have shown the most remarkable improvements. The prediction accuracy of these methods has increased by over 30% in the past decade. The accuracy of these methods is now on par with

  4. Evaluation and comparison of mammalian subcellular localization prediction methods

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    Fink J Lynn

    2006-12-01

    Full Text Available Abstract Background Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER, peroxisome, and lysosome. The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE

  5. Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

    Science.gov (United States)

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

    2017-08-30

    Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (Prock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these

  6. Subcellular localization of ammonium transporters in Dictyostelium discoideum

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    Davis Carter T

    2008-12-01

    Full Text Available Abstract Background With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. Results Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. Conclusion Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not

  7. Gene ontology based transfer learning for protein subcellular localization

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    Zhou Shuigeng

    2011-02-01

    Full Text Available Abstract Background Prediction of protein subcellular localization generally involves many complex factors, and using only one or two aspects of data information may not tell the true story. For this reason, some recent predictive models are deliberately designed to integrate multiple heterogeneous data sources for exploiting multi-aspect protein feature information. Gene ontology, hereinafter referred to as GO, uses a controlled vocabulary to depict biological molecules or gene products in terms of biological process, molecular function and cellular component. With the rapid expansion of annotated protein sequences, gene ontology has become a general protein feature that can be used to construct predictive models in computational biology. Existing models generally either concatenated the GO terms into a flat binary vector or applied majority-vote based ensemble learning for protein subcellular localization, both of which can not estimate the individual discriminative abilities of the three aspects of gene ontology. Results In this paper, we propose a Gene Ontology Based Transfer Learning Model (GO-TLM for large-scale protein subcellular localization. The model transfers the signature-based homologous GO terms to the target proteins, and further constructs a reliable learning system to reduce the adverse affect of the potential false GO terms that are resulted from evolutionary divergence. We derive three GO kernels from the three aspects of gene ontology to measure the GO similarity of two proteins, and derive two other spectrum kernels to measure the similarity of two protein sequences. We use simple non-parametric cross validation to explicitly weigh the discriminative abilities of the five kernels, such that the time & space computational complexities are greatly reduced when compared to the complicated semi-definite programming and semi-indefinite linear programming. The five kernels are then linearly merged into one single kernel for

  8. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

    Science.gov (United States)

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

  9. Identifying essential proteins based on sub-network partition and prioritization by integrating subcellular localization information.

    Science.gov (United States)

    Li, Min; Li, Wenkai; Wu, Fang-Xiang; Pan, Yi; Wang, Jianxin

    2018-06-14

    Essential proteins are important participants in various life activities and play a vital role in the survival and reproduction of living organisms. Identification of essential proteins from protein-protein interaction (PPI) networks has great significance to facilitate the study of human complex diseases, the design of drugs and the development of bioinformatics and computational science. Studies have shown that highly connected proteins in a PPI network tend to be essential. A series of computational methods have been proposed to identify essential proteins by analyzing topological structures of PPI networks. However, the high noise in the PPI data can degrade the accuracy of essential protein prediction. Moreover, proteins must be located in the appropriate subcellular localization to perform their functions, and only when the proteins are located in the same subcellular localization, it is possible that they can interact with each other. In this paper, we propose a new network-based essential protein discovery method based on sub-network partition and prioritization by integrating subcellular localization information, named SPP. The proposed method SPP was tested on two different yeast PPI networks obtained from DIP database and BioGRID database. The experimental results show that SPP can effectively reduce the effect of false positives in PPI networks and predict essential proteins more accurately compared with other existing computational methods DC, BC, CC, SC, EC, IC, NC. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Expression and subcellular localization of ORC1 in Leishmania major

    International Nuclear Information System (INIS)

    Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

    2008-01-01

    The mechanism of DNA replication is highly conserved in eukaryotes, with the process being preceded by the ordered assembly of pre-replication complexes (pre-RCs). Pre-RC formation is triggered by the association of the origin replication complex (ORC) with chromatin. Leishmania major appears to have only one ORC ortholog, ORC1. ORC1 in other eukaryotes is the largest of the ORC subunits and is believed to play a significant role in modulating replication initiation. Here we report for the first time, the cloning of ORC1 from L. major, and the analysis of its expression in L. major promastigotes. In human cells ORC1 levels have been found to be upregulated in G1 and subsequently degraded, thus playing a role in controlling replication initiation. We examine the subcellular localization of L. major ORC1 in relation to the different stages of the cell cycle. Our results show that, unlike what is widely believed to be the case with ORC1 in human cells, ORC1 in L. major is nuclear at all stages of the cell cycle

  11. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

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    Xia eGuo

    2016-01-01

    Full Text Available Histone deacetylases (HDACs are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2 and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12 remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  12. Predicting the subcellular localization of viral proteins within a mammalian host cell

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    Thomas DY

    2006-04-01

    Full Text Available Abstract Background The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. Results Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. Conclusion Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.

  13. ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

    DEFF Research Database (Denmark)

    la Cour, Jonas Marstrand; Mollerup, Jens; Berchtold, Martin Werner

    2007-01-01

    discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate...

  14. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2015-01-01

    was used. Various statistical and machine learning algorithms are used with all three approaches, and various measures and standards are employed when reporting the performances of the developed methods. This chapter presents a number of available methods for prediction of sorting signals and subcellular...

  15. Subcellular localization for Gram positive and Gram negative bacterial proteins using linear interpolation smoothing model.

    Science.gov (United States)

    Saini, Harsh; Raicar, Gaurav; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

    2015-12-07

    Protein subcellular localization is an important topic in proteomics since it is related to a protein׳s overall function, helps in the understanding of metabolic pathways, and in drug design and discovery. In this paper, a basic approximation technique from natural language processing called the linear interpolation smoothing model is applied for predicting protein subcellular localizations. The proposed approach extracts features from syntactical information in protein sequences to build probabilistic profiles using dependency models, which are used in linear interpolation to determine how likely is a sequence to belong to a particular subcellular location. This technique builds a statistical model based on maximum likelihood. It is able to deal effectively with high dimensionality that hinders other traditional classifiers such as Support Vector Machines or k-Nearest Neighbours without sacrificing performance. This approach has been evaluated by predicting subcellular localizations of Gram positive and Gram negative bacterial proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. SMYD3 interacts with HTLV-1 Tax and regulates subcellular localization of Tax.

    Science.gov (United States)

    Yamamoto, Keiyu; Ishida, Takaomi; Nakano, Kazumi; Yamagishi, Makoto; Yamochi, Tadanori; Tanaka, Yuetsu; Furukawa, Yoichi; Nakamura, Yusuke; Watanabe, Toshiki

    2011-01-01

    HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus. © 2010 Japanese Cancer Association.

  17. Prediction of protein subcellular localization using support vector machine with the choice of proper kernel

    Directory of Open Access Journals (Sweden)

    Al Mehedi Hasan

    2017-07-01

    Full Text Available The prediction of subcellular locations of proteins can provide useful hints for revealing their functions as well as for understanding the mechanisms of some diseases and, finally, for developing novel drugs. As the number of newly discovered proteins has been growing exponentially, laboratory-based experiments to determine the location of an uncharacterized protein in a living cell have become both expensive and time-consuming. Consequently, to tackle these challenges, computational methods are being developed as an alternative to help biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging problem, particularly when query proteins may have multi-label characteristics, i.e. their simultaneous existence in more than one subcellular location, or if they move between two or more different subcellular locations as well. At this point, to get rid of this problem, several types of subcellular localization prediction methods with different levels of accuracy have been proposed. The support vector machine (SVM has been employed to provide potential solutions for problems connected with the prediction of protein subcellular localization. However, the practicability of SVM is affected by difficulties in selecting its appropriate kernel as well as in selecting the parameters of that selected kernel. The literature survey has shown that most researchers apply the radial basis function (RBF kernel to build a SVM based subcellular localization prediction system. Surprisingly, there are still many other kernel functions which have not yet been applied in the prediction of protein subcellular localization. However, the nature of this classification problem requires the application of different kernels for SVM to ensure an optimal result. From this viewpoint, this paper presents the work to apply different kernels for SVM in protein

  18. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction

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    Kohlbacher Oliver

    2009-09-01

    Full Text Available Abstract Background Knowledge of subcellular localization of proteins is crucial to proteomics, drug target discovery and systems biology since localization and biological function are highly correlated. In recent years, numerous computational prediction methods have been developed. Nevertheless, there is still a need for prediction methods that show more robustness and higher accuracy. Results We extended our previous MultiLoc predictor by incorporating phylogenetic profiles and Gene Ontology terms. Two different datasets were used for training the system, resulting in two versions of this high-accuracy prediction method. One version is specialized for globular proteins and predicts up to five localizations, whereas a second version covers all eleven main eukaryotic subcellular localizations. In a benchmark study with five localizations, MultiLoc2 performs considerably better than other methods for animal and plant proteins and comparably for fungal proteins. Furthermore, MultiLoc2 performs clearly better when using a second dataset that extends the benchmark study to all eleven main eukaryotic subcellular localizations. Conclusion MultiLoc2 is an extensive high-performance subcellular protein localization prediction system. By incorporating phylogenetic profiles and Gene Ontology terms MultiLoc2 yields higher accuracies compared to its previous version. Moreover, it outperforms other prediction systems in two benchmarks studies. MultiLoc2 is available as user-friendly and free web-service, available at: http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc2.

  19. Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

    Science.gov (United States)

    Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

    2011-01-01

    Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

  20. Detrended cross-correlation coefficient: Application to predict apoptosis protein subcellular localization.

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2016-12-01

    Apoptosis, or programed cell death, plays a central role in the development and homeostasis of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful for understanding the apoptosis mechanism. The prediction of subcellular localization of an apoptosis protein is still a challenging task, and existing methods mainly based on protein primary sequences. In this paper, we introduce a new position-specific scoring matrix (PSSM)-based method by using detrended cross-correlation (DCCA) coefficient of non-overlapping windows. Then a 190-dimensional (190D) feature vector is constructed on two widely used datasets: CL317 and ZD98, and support vector machine is adopted as classifier. To evaluate the proposed method, objective and rigorous jackknife cross-validation tests are performed on the two datasets. The results show that our approach offers a novel and reliable PSSM-based tool for prediction of apoptosis protein subcellular localization. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging

    International Nuclear Information System (INIS)

    Wolff, Horst; Hadian, Kamyar; Ziegler, Manja; Weierich, Claudia; Kramer-Hammerle, Susanne; Kleinschmidt, Andrea; Erfle, Volker; Brack-Werner, Ruth

    2006-01-01

    The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors

  2. The cellular and subcellular localization of zinc transporter 7 in the mouse spinal cord

    Science.gov (United States)

    The present work addresses the cellular and subcellular localization of the zinc transporter 7 (ZNT7, SLC30a7) protein and the distribution of zinc ions (Zn2+) in the mouse spinal cord. Our results indicated that the ZNT7 immunoreactive neurons were widely distributed in the Rexed’s laminae of the g...

  3. A novel representation for apoptosis protein subcellular localization prediction using support vector machine.

    Science.gov (United States)

    Zhang, Li; Liao, Bo; Li, Dachao; Zhu, Wen

    2009-07-21

    Apoptosis, or programmed cell death, plays an important role in development of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful to understand the apoptosis mechanism. In this paper, based on the concept that the position distribution information of amino acids is closely related with the structure and function of proteins, we introduce the concept of distance frequency [Matsuda, S., Vert, J.P., Ueda, N., Toh, H., Akutsu, T., 2005. A novel representation of protein sequences for prediction of subcellular location using support vector machines. Protein Sci. 14, 2804-2813] and propose a novel way to calculate distance frequencies. In order to calculate the local features, each protein sequence is separated into p parts with the same length in our paper. Then we use the novel representation of protein sequences and adopt support vector machine to predict subcellular location. The overall prediction accuracy is significantly improved by jackknife test.

  4. Requirements of cyclin a for mitosis are independent of its subcellular localization.

    Science.gov (United States)

    Dienemann, Axel; Sprenger, Frank

    2004-06-22

    Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.

  5. Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

    International Nuclear Information System (INIS)

    Dolci, Susanna; Belmonte, Alessia; Santone, Rocco; Giorgi, Mauro; Pellegrini, Manuela; Carosa, Eleonora; Piccione, Emilio; Lenzi, Andrea; Jannini, Emmanuele A.

    2006-01-01

    We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for First time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype

  6. Thyroid states regulate subcellular glucose phosphorylation activity in male mice

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    Flavia Letícia Martins Peçanha

    2017-07-01

    Full Text Available The thyroid hormones (THs, triiodothyronine (T3 and thyroxine (T4, are very important in organism metabolism and regulate glucose utilization. Hexokinase (HK is responsible for the first step of glycolysis, catalyzing the conversion of glucose to glucose 6-phosphate. HK has been found in different cellular compartments, and new functions have been attributed to this enzyme. The effects of hyperthyroidism on subcellular glucose phosphorylation in mouse tissues were examined. Tissues were removed, subcellular fractions were isolated from eu- and hyperthyroid (T3, 0.25 μg/g, i.p. during 21 days mice and HK activity was assayed. Glucose phosphorylation was increased in the particulate fraction in soleus (312.4% ± 67.1, n = 10, gastrocnemius (369.2% ± 112.4, n = 10 and heart (142.2% ± 13.6, n = 10 muscle in the hyperthyroid group compared to the control group. Hexokinase activity was not affected in brain or liver. No relevant changes were observed in HK activity in the soluble fraction for all tissues investigated. Acute T3 administration (single dose of T3, 1.25 μg/g, i.p. did not modulate HK activity. Interestingly, HK mRNA levels remained unchanged and HK bound to mitochondria was increased by T3 treatment, suggesting a posttranscriptional mechanism. Analysis of the AKT pathway showed a 2.5-fold increase in AKT and GSK3B phosphorylation in the gastrocnemius muscle in the hyperthyroid group compared to the euthyroid group. Taken together, we show for the first time that THs modulate HK activity specifically in particulate fractions and that this action seems to be under the control of the AKT and GSK3B pathways.

  7. Fast subcellular localization by cascaded fusion of signal-based and homology-based methods

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    Wang Wei

    2011-10-01

    Full Text Available Abstract Background The functions of proteins are closely related to their subcellular locations. In the post-genomics era, the amount of gene and protein data grows exponentially, which necessitates the prediction of subcellular localization by computational means. Results This paper proposes mitigating the computation burden of alignment-based approaches to subcellular localization prediction by a cascaded fusion of cleavage site prediction and profile alignment. Specifically, the informative segments of protein sequences are identified by a cleavage site predictor using the information in their N-terminal shorting signals. Then, the sequences are truncated at the cleavage site positions, and the shortened sequences are passed to PSI-BLAST for computing their profiles. Subcellular localization are subsequently predicted by a profile-to-profile alignment support-vector-machine (SVM classifier. To further reduce the training and recognition time of the classifier, the SVM classifier is replaced by a new kernel method based on the perturbational discriminant analysis (PDA. Conclusions Experimental results on a new dataset based on Swiss-Prot Release 57.5 show that the method can make use of the best property of signal- and homology-based approaches and can attain an accuracy comparable to that achieved by using full-length sequences. Analysis of profile-alignment score matrices suggest that both profile creation time and profile alignment time can be reduced without significant reduction in subcellular localization accuracy. It was found that PDA enjoys a short training time as compared to the conventional SVM. We advocate that the method will be important for biologists to conduct large-scale protein annotation or for bioinformaticians to perform preliminary investigations on new algorithms that involve pairwise alignments.

  8. Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

    Science.gov (United States)

    Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

    2015-02-01

    Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

  9. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    DEFF Research Database (Denmark)

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga

    2006-01-01

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD...

  10. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  11. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    International Nuclear Information System (INIS)

    Tachibana, Keisuke; Gotoh, Eiko; Kawamata, Natsuko; Ishimoto, Kenji; Uchihara, Yoshie; Iwanari, Hiroko; Sugiyama, Akira; Kawamura, Takeshi; Mochizuki, Yasuhiro; Tanaka, Toshiya; Sakai, Juro; Hamakubo, Takao; Kodama, Tatsuhiko

    2015-01-01

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  12. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    International Nuclear Information System (INIS)

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-01-01

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm

  13. Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

    International Nuclear Information System (INIS)

    Clarke, J.T.; Cook, H.W.; Spence, M.W.

    1985-01-01

    To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-[1- 3 H]galactose or [ 3 H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [ 3 H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid

  14. Iron in seeds – loading pathways and subcellular localization

    Directory of Open Access Journals (Sweden)

    Louis eGrillet

    2014-01-01

    Full Text Available Iron (Fe is one of the most abundant elements on earth, but its limited bioavailability poses a major constraint for agriculture and constitutes a serious problem in human health. Due to an improved understanding of the mechanisms that control Fe homeostasis in plants, major advances towards engineering biofortified crops have been made during the past decade. Examples of successful biofortification strategies are, however, still scarce and the process of Fe loading into seeds is far from being well understood in most crop species. In particular in grains where the embryo represents the main storage compartment such as legumes, increasing the seed Fe content remains a challenging task. This review aims at placing the recently identified actors in Fe transport into the unsolved puzzle of grain filling, taking the differences of Fe distribution between various species into consideration. We summarize the current knowledge on Fe transport between symplasmic and apoplasmic compartments, and provide models for Fe trafficking and localization in different seed types that may help to develop high seed Fe germplasms.

  15. DeepLoc: prediction of protein subcellular localization using deep learning

    DEFF Research Database (Denmark)

    Almagro Armenteros, Jose Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae

    2017-01-01

    The prediction of eukaryotic protein subcellular localization is a well-studied topic in bioinformatics due to its relevance in proteomics research. Many machine learning methods have been successfully applied in this task, but in most of them, predictions rely on annotation of homologues from...... knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict...... current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc . Example code is available at https://github.com/JJAlmagro/subcellular_localization . The dataset is available at http...

  16. ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes

    Directory of Open Access Journals (Sweden)

    King Brian R

    2012-07-01

    Full Text Available Abstract Background Understanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of organelles in the cell. Additionally, the majority of methods predict only a single location for a sequence, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function. Findings We present a software package and a web server for predicting the subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively. Conclusions ngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.

  17. Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

    Science.gov (United States)

    Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

    2002-04-12

    The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

  18. Rechecking the Centrality-Lethality Rule in the Scope of Protein Subcellular Localization Interaction Networks.

    Directory of Open Access Journals (Sweden)

    Xiaoqing Peng

    Full Text Available Essential proteins are indispensable for living organisms to maintain life activities and play important roles in the studies of pathology, synthetic biology, and drug design. Therefore, besides experiment methods, many computational methods are proposed to identify essential proteins. Based on the centrality-lethality rule, various centrality methods are employed to predict essential proteins in a Protein-protein Interaction Network (PIN. However, neglecting the temporal and spatial features of protein-protein interactions, the centrality scores calculated by centrality methods are not effective enough for measuring the essentiality of proteins in a PIN. Moreover, many methods, which overfit with the features of essential proteins for one species, may perform poor for other species. In this paper, we demonstrate that the centrality-lethality rule also exists in Protein Subcellular Localization Interaction Networks (PSLINs. To do this, a method based on Localization Specificity for Essential protein Detection (LSED, was proposed, which can be combined with any centrality method for calculating the improved centrality scores by taking into consideration PSLINs in which proteins play their roles. In this study, LSED was combined with eight centrality methods separately to calculate Localization-specific Centrality Scores (LCSs for proteins based on the PSLINs of four species (Saccharomyces cerevisiae, Homo sapiens, Mus musculus and Drosophila melanogaster. Compared to the proteins with high centrality scores measured from the global PINs, more proteins with high LCSs measured from PSLINs are essential. It indicates that proteins with high LCSs measured from PSLINs are more likely to be essential and the performance of centrality methods can be improved by LSED. Furthermore, LSED provides a wide applicable prediction model to identify essential proteins for different species.

  19. Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

    Science.gov (United States)

    Scherer, G F

    1984-03-01

    A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

  20. ClubSub-P: Cluster-based subcellular localization prediction for Gram-negative bacteria and Archaea.

    Directory of Open Access Journals (Sweden)

    Nagarajan eParamasivam

    2011-11-01

    Full Text Available The subcellular localization of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned subcellular localizations. This and other problems in subcellular localization prediction, such as the relatively high false positive and false negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing subcellular localization prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false positive and false negative predictions. ClubSub-P can assign the subcellular localization of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the subcellular localization prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/

  1. The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer

    International Nuclear Information System (INIS)

    Akkiprik, Mustafa; Hu, Limei; Sahin, Aysegul; Hao, Xishan; Zhang, Wei

    2009-01-01

    Insulin-like growth factor binding protein 5 (IGFBP5) has been shown to be associated with breast cancer metastasis in clinical marker studies. However, a major difficulty in understanding how IGFBP5 functions in this capacity is the paradoxical observation that ectopic overexpression of IGFBP5 in breast cancer cell lines results in suppressed cellular proliferation. In cancer tissues, IGFBP5 resides mainly in the cytoplasm; however, in transfected cells, IGFBP5 is mainly located in the nucleus. We hypothesized that subcellular localization of IGFBP5 affects its functions in host cells. To test this hypothesis, we generated wild-type and mutant IGFBP5 expression constructs. The mutation occurs within the nuclear localization sequence (NLS) of the protein and is generated by site-directed mutagenesis using the wild-type IGFBP5 expression construct as a template. Next, we transfected each expression construct into MDA-MB-435 breast cancer cells to establish stable clones overexpressing either wild-type or mutant IGFBP5. Functional analysis revealed that cells overexpressing wild-type IGFBP5 had significantly lower cell growth rate and motility than the vector-transfected cells, whereas cells overexpressing mutant IGFBP5 demonstrated a significantly higher ability to proliferate and migrate. To illustrate the subcellular localization of the proteins, we generated wild-type and mutant IGFBP5-pDsRed fluorescence fusion constructs. Fluorescence microscopy imaging revealed that mutation of the NLS in IGFBP5 switched the accumulation of IGFBP5 from the nucleus to the cytoplasm of the protein. Together, these findings imply that the mutant form of IGFBP5 increases proliferation and motility of breast cancer cells and that mutation of the NLS in IGFBP5 results in localization of IGFBP5 in the cytoplasm, suggesting that subcellular localization of IGFBP5 affects its cell growth and migration functions in the breast cancer cells

  2. Differential subcellular localization of insulin receptor substrates depends on C-terminal regions and importin β

    International Nuclear Information System (INIS)

    Kabuta, Tomohiro; Take, Kazumi; Kabuta, Chihana; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2008-01-01

    Insulin receptor substrates (IRSs) play essential roles in signal transduction of insulin and insulin-like growth factors. Previously, we showed that IRS-3 is localized to the nucleus as well as the cytosol, while IRS-1 and 2 are mainly localized to the cytoplasm. In the present study, we found that importin β directly interacts with IRS-3 and is able to mediate nuclear transport of IRS-3. Importin β interacted with the pleckstrin homology domain, the phosphotyrosine binding domain and the C-terminal region of IRS-3; indeed all of these fragments exhibited predominant nuclear localization. By contrast, almost no interaction of importin β with IRS-1 and -2 was observed, and their C-terminal regions displayed discrete spotty images in the cytosol. In addition, using chimeric proteins between IRS-1 and IRS-3, we revealed that the C-terminal regions are the main determinants of the differing subcellular localizations of IRS-1 and IRS-3.

  3. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...... regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P...

  4. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.

    2008-01-01

    . YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

  5. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  6. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  7. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning.

    Science.gov (United States)

    Pärnamaa, Tanel; Parts, Leopold

    2017-05-05

    High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy. Copyright © 2017 Parnamaa and Parts.

  8. Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

    Directory of Open Access Journals (Sweden)

    Amanda Getty

    Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

  9. Protein Subcellular Localization with Gaussian Kernel Discriminant Analysis and Its Kernel Parameter Selection.

    Science.gov (United States)

    Wang, Shunfang; Nie, Bing; Yue, Kun; Fei, Yu; Li, Wenjia; Xu, Dongshu

    2017-12-15

    Kernel discriminant analysis (KDA) is a dimension reduction and classification algorithm based on nonlinear kernel trick, which can be novelly used to treat high-dimensional and complex biological data before undergoing classification processes such as protein subcellular localization. Kernel parameters make a great impact on the performance of the KDA model. Specifically, for KDA with the popular Gaussian kernel, to select the scale parameter is still a challenging problem. Thus, this paper introduces the KDA method and proposes a new method for Gaussian kernel parameter selection depending on the fact that the differences between reconstruction errors of edge normal samples and those of interior normal samples should be maximized for certain suitable kernel parameters. Experiments with various standard data sets of protein subcellular localization show that the overall accuracy of protein classification prediction with KDA is much higher than that without KDA. Meanwhile, the kernel parameter of KDA has a great impact on the efficiency, and the proposed method can produce an optimum parameter, which makes the new algorithm not only perform as effectively as the traditional ones, but also reduce the computational time and thus improve efficiency.

  10. Subcellular localization-dependent decrements in skeletal muscle glycogen and mitochondria content following short-term disuse in young and old men

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Suetta, Charlotte; Hvid, Lars G

    2010-01-01

    of disuse and aging on human skeletal muscle glycogen and mitochondria content in subsarcolemmal (SS), intermyofibrillar (IMF), and intramyofibrillar (intra) localizations. Five young (∼23 yr) and five old (∼66 yr) recreationally active men had their quadriceps muscle immobilized for 2 wk by whole leg...... unchanged. A localization-dependent decrease (P = 0.03) in mitochondria content following immobilization was found in both age groups, where SS mitochondria decreased by 33% (P = 0.02), superficial IMF mitochondria decreased by 20% (P = 0.05), and central IMF mitochondria remained unchanged. In conclusion......Previous studies have shown that skeletal muscle glycogen and mitochondria are distributed in distinct subcellular localizations, but the role and regulation of these subcellular localizations are unclear. In the present study, we used transmission electron microscopy to investigate the effect...

  11. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    Science.gov (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  12. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC imaging method for cadmium ions (Cd2+ was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  13. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

    Science.gov (United States)

    Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X; Wang, Baomin

    2015-01-01

    The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  14. Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments

    Science.gov (United States)

    van Diggelen, Lisa; Khin, Hnin; Conner, Kip; Shao, Jenny; Sweezy, Margaretta; Jung, Anna H.; Isaac, Meden; Simonis, Ursula

    2009-06-01

    Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out. Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di

  15. Physiological aspects of the subcellular localization of glycogen in skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Ørtenblad, Niels

    2013-01-01

    Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electron microscopy images as roughly spherical particles (known as β-particles of 10-45 nm in diameter), which are distributed in distinct localizations within the myofibers and are physically associated...... investigated the role and regulation of these distinct deposits of glycogen. In this report, we review the available literature regarding the subcellular localization of glycogen in skeletal muscle as investigated by electron microscopy studies and put this into perspective in terms of the architectural......, topological, and dynamic organization of skeletal muscle fibers. In summary, the distribution of glycogen within skeletal muscle fibers has been shown to depend on the fiber phenotype, individual training status, short-term immobilization, and exercise and to influence both muscle contractility...

  16. CellMap visualizes protein-protein interactions and subcellular localization

    Science.gov (United States)

    Dallago, Christian; Goldberg, Tatyana; Andrade-Navarro, Miguel Angel; Alanis-Lobato, Gregorio; Rost, Burkhard

    2018-01-01

    Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers. PMID:29497493

  17. ESLpred2: improved method for predicting subcellular localization of eukaryotic proteins

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2008-11-01

    Full Text Available Abstract Background The expansion of raw protein sequence databases in the post genomic era and availability of fresh annotated sequences for major localizations particularly motivated us to introduce a new improved version of our previously forged eukaryotic subcellular localizations prediction method namely "ESLpred". Since, subcellular localization of a protein offers essential clues about its functioning, hence, availability of localization predictor would definitely aid and expedite the protein deciphering studies. However, robustness of a predictor is highly dependent on the superiority of dataset and extracted protein attributes; hence, it becomes imperative to improve the performance of presently available method using latest dataset and crucial input features. Results Here, we describe augmentation in the prediction performance obtained for our most popular ESLpred method using new crucial features as an input to Support Vector Machine (SVM. In addition, recently available, highly non-redundant dataset encompassing three kingdoms specific protein sequence sets; 1198 fungi sequences, 2597 from animal and 491 plant sequences were also included in the present study. First, using the evolutionary information in the form of profile composition along with whole and N-terminal sequence composition as an input feature vector of 440 dimensions, overall accuracies of 72.7, 75.8 and 74.5% were achieved respectively after five-fold cross-validation. Further, enhancement in performance was observed when similarity search based results were coupled with whole and N-terminal sequence composition along with profile composition by yielding overall accuracies of 75.9, 80.8, 76.6% respectively; best accuracies reported till date on the same datasets. Conclusion These results provide confidence about the reliability and accurate prediction of SVM modules generated in the present study using sequence and profile compositions along with similarity search

  18. Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

    Science.gov (United States)

    Nielsen, Joachim; Holmberg, Hans-Christer; Schrøder, Henrik D; Saltin, Bengt; Ørtenblad, Niels

    2011-01-01

    Abstract Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, = 68 ± 5 ml kg−1 min−1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. PMID:21486810

  19. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    Science.gov (United States)

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  20. Prediction of essential proteins based on subcellular localization and gene expression correlation.

    Science.gov (United States)

    Fan, Yetian; Tang, Xiwei; Hu, Xiaohua; Wu, Wei; Ping, Qing

    2017-12-01

    Essential proteins are indispensable to the survival and development process of living organisms. To understand the functional mechanisms of essential proteins, which can be applied to the analysis of disease and design of drugs, it is important to identify essential proteins from a set of proteins first. As traditional experimental methods designed to test out essential proteins are usually expensive and laborious, computational methods, which utilize biological and topological features of proteins, have attracted more attention in recent years. Protein-protein interaction networks, together with other biological data, have been explored to improve the performance of essential protein prediction. The proposed method SCP is evaluated on Saccharomyces cerevisiae datasets and compared with five other methods. The results show that our method SCP outperforms the other five methods in terms of accuracy of essential protein prediction. In this paper, we propose a novel algorithm named SCP, which combines the ranking by a modified PageRank algorithm based on subcellular compartments information, with the ranking by Pearson correlation coefficient (PCC) calculated from gene expression data. Experiments show that subcellular localization information is promising in boosting essential protein prediction.

  1. Tissue and subcellular localizations of 3H-cyclosporine A in mice

    International Nuclear Information System (INIS)

    Baeckman, L.; Brandt, I.; Appelkvist, E.-L.; Dallner, G.

    1988-01-01

    The tissue and subcellular localizations of 3 H-cyclosporine A after administration to mice were determined with whole-body autoradiography and scintillation counting of lipid extracts of tissues and subcellular fractions. The radioactivity was widely distributed in the body and the pattern of distribution after oral or parenteral administration was the same, except that tissue levels were generatlly lower after oral administration. Pretreatment of the animals with a diet containing cyclosporine A for 30 days before the injection of radioactive cyclosporine A did not change the pattern of distribution substantially. No significant radioactivity was found in the central nervous system, except for the choroidal plexus and the area postrema region of the brain. In pregnant mice no passage of radioactivity from the placentas to fetuses was observed after a single injection. 3 H-cyclosporine A and/or its metabolites showed a high affinity for the lympho-myeloid tissues, with a marked long-term retention in bone marrow and lymph nodes. There was massive excretion in the intestinal tract after parenteral administration, and the liver, bile, pancreas and salivary glands contained high levels of radioactivity. In the kidney radioactivity was confined to the outer zone of the outer kidney medulla. In liver homogenates no quantitatively significant binding of 3 H-cyclosporine A and/or its metabolites to cellular molecules such as proteins, DNA, phospho- or neutral lipids was found. After lipid extraction with organic solvents, almost all radioactivity was recovered in the organic phase. (author)

  2. Comparison of expressed human and mouse sodium/iodide sym-porters reveals differences in transport properties and subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Dayem, M.; Basquin, C.; Navarro, V.; Carrier, P.; Marsault, R.; Lindenthal, S.; Pourcher, T. [Univ Nice Sophia Antipolis, Sch Med, CEA, DSV, iBEB, SBTN, TIRO, F-06107 Nice (France); Chang, P. [CNRS, UPMC Biol Dev, UMR 7009, F-06230 Villefranche Sur Mer (France); Huc, S.; Darrouzet, E. [CEA Valrho, DSV, iBEB, SBTN, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    The active transport of iodide from the blood stream into thyroid follicular cells is mediated by the Na{sup +}/I{sup -} sym-porter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference,we compared {sup 125}I, uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2-5-fold lower for hNIS than mNIS. We also performed immuno-cyto-localization studies and observed that the subcellular distribution of the two ortho-logs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortho-log was intracellular in {approx} 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2-5-fold higher than that of the human protein, and therefore cannot alone account for,x values. We reasoned that the difference in the obtained Vmax the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein. (authors)

  3. Comparison of expressed human and mouse sodium/iodide sym-porters reveals differences in transport properties and subcellular localization

    International Nuclear Information System (INIS)

    Dayem, M.; Basquin, C.; Navarro, V.; Carrier, P.; Marsault, R.; Lindenthal, S.; Pourcher, T.; Chang, P.; Huc, S.; Darrouzet, E.

    2008-01-01

    The active transport of iodide from the blood stream into thyroid follicular cells is mediated by the Na + /I - sym-porter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference,we compared 125 I, uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2-5-fold lower for hNIS than mNIS. We also performed immuno-cyto-localization studies and observed that the subcellular distribution of the two ortho-logs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortho-log was intracellular in ∼ 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2-5-fold higher than that of the human protein, and therefore cannot alone account for,x values. We reasoned that the difference in the obtained Vmax the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein. (authors)

  4. The subcellular localization of natural 210Po in the hepatopancreas of the rock lobster (Jasus lalandii)

    International Nuclear Information System (INIS)

    Heyraud, M.; Dowdle, E.B.; Cherry, R.D.

    1987-01-01

    The subcellular localization of the naturally occurring nuclide 210 Po in the hepatopancreas of the South African rock lobster, Jasus lalandii, has been studied using centrifugation, ultrafiltration and chromatography. Just over half of the 210 Po was found to be associated with a component in the microsomal pellet. Most of the 210 Po was tightly bound to a component of high molecular mass. Dissociation of the 210 Po from this component required incubation with sulphydryl-reducing reagents, after which the 210 Po appeared to associate with a fraction having a molecular mass of 1500 daltons or less. A search for negatively-charged, hydrophobic, sulphur-containing membrane proteins which bind 210 Po is suggested. (author)

  5. Subcellular localization of natural /sup 210/Po in the hepatopancreas of the rock lobster (Jasus lalandii)

    Energy Technology Data Exchange (ETDEWEB)

    Heyraud, M; Dowdle, E B; Cherry, R D

    1987-01-01

    The subcellular localization of the naturally occurring nuclide /sup 210/Po in the hepatopancreas of the South African rock lobster, Jasus lalandii, has been studied using centrifugation, ultrafiltration and chromatography. Just over half of the /sup 210/Po was found to be associated with a component in the microsomal pellet. Most of the /sup 210/Po was tightly bound to a component of high molecular mass. Dissociation of the /sup 210/Po from this component required incubation with sulphydryl-reducing reagents, after which the /sup 210/Po appeared to associate with a fraction having a molecular mass of 1500 daltons or less. A search for negatively-charged, hydrophobic, sulphur-containing membrane proteins which bind /sup 210/Po is suggested.

  6. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

    Directory of Open Access Journals (Sweden)

    Miguel A. Ibeas

    2017-12-01

    Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  7. CoBaltDB: Complete bacterial and archaeal orfeomes subcellular localization database and associated resources

    Directory of Open Access Journals (Sweden)

    Lucchetti-Miganeh Céline

    2010-03-01

    Full Text Available Abstract Background The functions of proteins are strongly related to their localization in cell compartments (for example the cytoplasm or membranes but the experimental determination of the sub-cellular localization of proteomes is laborious and expensive. A fast and low-cost alternative approach is in silico prediction, based on features of the protein primary sequences. However, biologists are confronted with a very large number of computational tools that use different methods that address various localization features with diverse specificities and sensitivities. As a result, exploiting these computer resources to predict protein localization accurately involves querying all tools and comparing every prediction output; this is a painstaking task. Therefore, we developed a comprehensive database, called CoBaltDB, that gathers all prediction outputs concerning complete prokaryotic proteomes. Description The current version of CoBaltDB integrates the results of 43 localization predictors for 784 complete bacterial and archaeal proteomes (2.548.292 proteins in total. CoBaltDB supplies a simple user-friendly interface for retrieving and exploring relevant information about predicted features (such as signal peptide cleavage sites and transmembrane segments. Data are organized into three work-sets ("specialized tools", "meta-tools" and "additional tools". The database can be queried using the organism name, a locus tag or a list of locus tags and may be browsed using numerous graphical and text displays. Conclusions With its new functionalities, CoBaltDB is a novel powerful platform that provides easy access to the results of multiple localization tools and support for predicting prokaryotic protein localizations with higher confidence than previously possible. CoBaltDB is available at http://www.umr6026.univ-rennes1.fr/english/home/research/basic/software/cobalten.

  8. Subcellular fractionation and localization studies reveal a direct interaction of the Fragile X Mental Retardation Protein (FMRP) with nucleolin

    NARCIS (Netherlands)

    Taha, M.S.; Nouri, K.; Milroy, L.G.; Moll, J.M.; Herrmann, C.; Brunsveld, L.; Piekorz, R.P.; Ahmadian, M.R.

    2014-01-01

    Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its

  9. Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart

    DEFF Research Database (Denmark)

    Rasmussen, Hanne Borger; Møller, Morten; Knaus, Hans-Günther

    2003-01-01

    In the heart, several K(+) channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K(+) currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K(+) channels, KCNQ1 and ether...

  10. Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

    Science.gov (United States)

    Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

    2016-11-04

    In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Concentration of 17 Elements in Subcellular Fractions of Beef Heart Tissue Determined by Neutron Activation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Wester, P O

    1964-12-15

    Subcellular fractions of beef heart tissue are investigated, by means of neutron activation analysis, with respect to their concentration of 17 different elements. A recently developed ion-exchange technique combined with gamma spectrometry is used. The homogeneity of the subcellular fractions is examined electron microscopically. The following elements are determined: As, Ba, Br, Cas Co, Cs, Cu, Fe, Hg, La, Mo, P, Rb, Se, Sm, W and Zn. The determination of Ag, Au, Cd, Ce, Cr, Sb and Sc is omitted, in view of contamination. Reproducible and characteristic patterns of distribution are obtained for all elements studied.

  12. Concentration of 17 Elements in Subcellular Fractions of Beef Heart Tissue Determined by Neutron Activation Analysis

    International Nuclear Information System (INIS)

    Wester, P.O.

    1964-12-01

    Subcellular fractions of beef heart tissue are investigated, by means of neutron activation analysis, with respect to their concentration of 17 different elements. A recently developed ion-exchange technique combined with gamma spectrometry is used. The homogeneity of the subcellular fractions is examined electron microscopically. The following elements are determined: As, Ba, Br, Cas Co, Cs, Cu, Fe, Hg, La, Mo, P, Rb, Se, Sm, W and Zn. The determination of Ag, Au, Cd, Ce, Cr, Sb and Sc is omitted, in view of contamination. Reproducible and characteristic patterns of distribution are obtained for all elements studied

  13. Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli.

    Science.gov (United States)

    Rondón-Mercado, Rocío; Acosta, Héctor; Cáceres, Ana J; Quiñones, Wilfredo; Concepción, Juan Luis

    2017-09-01

    Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The

  14. [Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

    Science.gov (United States)

    Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

    2017-03-01

    Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

  15. Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

    Science.gov (United States)

    Sato, Shinya; Fukagawa, Takashi; Tachibanaki, Shuji; Yamano, Yumiko; Wada, Akimori; Kawamura, Satoru

    2013-12-20

    Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

  16. Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yan [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Lv, Liyang [Department of Health, Jinan Military Area Command, Jinan 250022 (China); Du, Juan; Yue, Longtao [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Cao, Lili, E-mail: cllly22@163.com [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China)

    2013-09-20

    Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizations may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.

  17. Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

    International Nuclear Information System (INIS)

    Song, Yan; Lv, Liyang; Du, Juan; Yue, Longtao; Cao, Lili

    2013-01-01

    Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizations may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis

  18. Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity.

    Science.gov (United States)

    Studzian, Kazimierz; Kik, Krzysztof; Lukawska, Malgorzata; Oszczapowicz, Irena; Strek, Malgorzata; Szmigiero, Leszek

    2015-10-01

    In this study, we compared the cellular uptake, intracellular localization and cytotoxicity of two groups of anthracycline derivatives in cultured H9c2(2-1) rat cardiomyoblasts. The first group consisted of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N = CH-N<) at the C-3' position with a morpholine (DOXM) or a hexamethyleneimine (DOXH) ring. The second group consisted of daunorubicin (DRB) and its derivatives containing a morpholine (DRBM) or a hexamethyleneimine (DRBH) ring. DOXH and DRBH were taken up by cardiomyoblasts more efficiently than estimated for other tested anthracyclines. The cellular uptakes of DOXM and DRBM were reduced compared to those of the parent compounds. Applied structural modifications of DOX and DRB influenced the subcellular localization of the tested derivatives. DOX and DOXH were localized primarily in nuclei, whereas the other anthracyclines were found in the nuclei and cytoplasm. The percentages of the compounds that accumulated in the nuclei were 80.2 and 54.2 % for DOX and DOXH, respectively. The lowest nuclear accumulation values were observed for DRBM (19.9 %), DRBH (21.9 %) and DOXM (23.7 %). The ability of anthracyclines to accumulate in the nuclei correlated with their DNA binding constants (r = 0.858, P = 0.029). A correlation was found between the accumulation of the tested anthracyclines in the nuclei of cardiomyoblasts and their cardiotoxicity in vivo, which was observed in our previous study. We suggest that cytotoxicity and the anthracycline accumulation level in the nuclei of cultured cardiomyoblasts could be used for early prediction of their cardiotoxicity.

  19. Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

    Science.gov (United States)

    Zheng, Wu; Blake, Catherine

    2015-10-01

    Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

    Science.gov (United States)

    Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

    2013-01-01

    Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

  1. Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos.

    Science.gov (United States)

    Carjuzaa, P; Castellión, M; Distéfano, A J; del Vas, M; Maldonado, S

    2008-01-01

    The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences

  2. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes.

    Science.gov (United States)

    Yu, Nancy Y; Wagner, James R; Laird, Matthew R; Melli, Gabor; Rey, Sébastien; Lo, Raymond; Dao, Phuong; Sahinalp, S Cenk; Ester, Martin; Foster, Leonard J; Brinkman, Fiona S L

    2010-07-01

    PSORTb has remained the most precise bacterial protein subcellular localization (SCL) predictor since it was first made available in 2003. However, the recall needs to be improved and no accurate SCL predictors yet make predictions for archaea, nor differentiate important localization subcategories, such as proteins targeted to a host cell or bacterial hyperstructures/organelles. Such improvements should preferably be encompassed in a freely available web-based predictor that can also be used as a standalone program. We developed PSORTb version 3.0 with improved recall, higher proteome-scale prediction coverage, and new refined localization subcategories. It is the first SCL predictor specifically geared for all prokaryotes, including archaea and bacteria with atypical membrane/cell wall topologies. It features an improved standalone program, with a new batch results delivery system complementing its web interface. We evaluated the most accurate SCL predictors using 5-fold cross validation plus we performed an independent proteomics analysis, showing that PSORTb 3.0 is the most accurate but can benefit from being complemented by Proteome Analyst predictions. http://www.psort.org/psortb (download open source software or use the web interface). psort-mail@sfu.ca Supplementary data are available at Bioinformatics online.

  3. Role of the EHD2 unstructured loop in dimerization, protein binding and subcellular localization.

    Directory of Open Access Journals (Sweden)

    Kriti Bahl

    Full Text Available The C-terminal Eps 15 Homology Domain proteins (EHD1-4 play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2 might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting

  4. Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes.

    Science.gov (United States)

    Duong, Michelle; Yu, Xuejiao; Teng, Beina; Schroder, Patricia; Haller, Hermann; Eschenburg, Susanne; Schiffer, Mario

    2017-07-21

    Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of β-catenin, which promotes its degradation. β-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with β-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower β-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, β-catenin showed a reversed subcellular localization pattern: Although β-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates β-catenin expression and stabilization in a glycogen synthase kinase 3β-independent manner. Further, β-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the β-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in β-catenin that are required for full β-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and β-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of β-catenin levels and define PKCϵ as an important β-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

    Science.gov (United States)

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  6. Expression, purification, characterization and subcellular localization of the goose parvovirus rep1 protein.

    Science.gov (United States)

    Chen, Zongyan; Li, Chuanfeng; Peng, Gaojing; Liu, Guangqing

    2013-07-01

    The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl β-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.

  7. Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

    Science.gov (United States)

    Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

  8. Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

    Science.gov (United States)

    Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

    2010-05-15

    During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

  9. Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages

    International Nuclear Information System (INIS)

    Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.; Zenebergh, A.; Tulkens, P.M.

    1987-01-01

    beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14 C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14 C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al., in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections

  10. Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells.

    Science.gov (United States)

    DeRose, Robert; Pohlmeyer, Christopher; Umeda, Nobuhiro; Ueno, Tasuku; Nagano, Tetsuo; Kuo, Scot; Inoue, Takanari

    2012-03-09

    Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).

  11. Subcellular Localization of Cadmium in Chlorella vulgaris Beijerinck Strain Bt-09

    Directory of Open Access Journals (Sweden)

    P.B. Lintongan

    2004-06-01

    Full Text Available Growth response curves of Chlorella vulgaris Beijerinck strain Bt-09 to sublethal concentrations of cadmium were evaluated. The growth responses of this microalgal isolate was determined through analysis of chlorophyll a levels. Cadmium was effectively taken up by the cells as determined by Flame Atomic Absorption Spectrophotometry (F-AAS. Subcellular fractionation was undertaken to locate sites that accumulate cadmium.

  12. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca(2+)/PKA crosstalk.

    Science.gov (United States)

    Schott, Micah B; Gonowolo, Faith; Maliske, Benjamin; Grove, Bryon

    2016-04-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca(2+)]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium "switch" that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca(2+) and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca(2+)/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca(2+) and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  13. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

    Science.gov (United States)

    Schott, Micah; Gonowolo, Faith; Maliske, Ben; Grove, Bryon

    2016-01-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca2+]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium “switch” that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca2+ and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca2+/PKA crosstalk was not observed in cells expressing a gravin mutant that resists calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  14. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available One of the fundamental goals in proteomics and cell biology is to identify the functions of proteins in various cellular organelles and pathways. Information of subcellular locations of proteins can provide useful insights for revealing their functions and understanding how they interact with each other in cellular network systems. Most of the existing methods in predicting plant protein subcellular localization can only cover three or four location sites, and none of them can be used to deal with multiplex plant proteins that can simultaneously exist at two, or move between, two or more different location sits. Actually, such multiplex proteins might have special biological functions worthy of particular notice. The present study was devoted to improve the existing plant protein subcellular location predictors from the aforementioned two aspects. A new predictor called "Plant-mPLoc" is developed by integrating the gene ontology information, functional domain information, and sequential evolutionary information through three different modes of pseudo amino acid composition. It can be used to identify plant proteins among the following 12 location sites: (1 cell membrane, (2 cell wall, (3 chloroplast, (4 cytoplasm, (5 endoplasmic reticulum, (6 extracellular, (7 Golgi apparatus, (8 mitochondrion, (9 nucleus, (10 peroxisome, (11 plastid, and (12 vacuole. Compared with the existing methods for predicting plant protein subcellular localization, the new predictor is much more powerful and flexible. Particularly, it also has the capacity to deal with multiple-location proteins, which is beyond the reach of any existing predictors specialized for identifying plant protein subcellular localization. As a user-friendly web-server, Plant-mPLoc is freely accessible at http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to

  15. MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

    International Nuclear Information System (INIS)

    Ziaei, Samira; Shimada, Naoko; Kucharavy, Herman; Hubbard, Karen

    2012-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence. -- Highlights: ► MNK1 and not MAPKAPK2 phosphorylates hnRNP A1. ► MNK1 has elevated levels in senescent cells, this has not been reported previously. ► MNK1 activity induces cytoplasmic accumulation of hnRNP A1 in senescent cells. ► Altered cytoplasmic localization of hnRNP A1 may alter gene expression patterns. ► Our studies may increase our understanding of RNA metabolism during cellular aging.

  16. In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

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    Yunxiao Liu

    2018-04-01

    Full Text Available Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83 could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.

  17. Optogenetic Tools for Subcellular Applications in Neuroscience.

    Science.gov (United States)

    Rost, Benjamin R; Schneider-Warme, Franziska; Schmitz, Dietmar; Hegemann, Peter

    2017-11-01

    The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

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    Xiuzhi Jia

    Full Text Available Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652 between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

  19. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

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    Soledad Natalia Gonzalez

    Full Text Available The enzyme of the pentose phosphate pathway (PPP ribulose-5-phosphate-epimerase (RPE is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å. Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological

  20. Dynamic changes to survivin subcellular localization are initiated by DNA damage

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    Maritess Gay Asumen

    2010-07-01

    Full Text Available Maritess Gay Asumen1, Tochukwu V Ifeacho2, Luke Cockerham3, Christina Pfandl4, Nathan R Wall31Touro University’s College of Osteopathic Medicine, Vallejo, CA, USA; 2University of Southern California, Los Angeles, CA, USA; 3Center for Health Disparities Research and Molecular Medicine, Loma Linda University, CA, USA; 4Green Mountain Antibodies, Burlington, VT, USAAbstract: Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light- initiated DNA damage and that its distribution may be responsible for its multifunctionality.Keywords: survivin, PIK kinases, ATM, ATR, DNA-PK

  1. The in vitro sub-cellular localization and in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.

    Science.gov (United States)

    Trickler, W J; Nagvekar, A A; Dash, A K

    2009-08-01

    To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol). The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors. The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously. Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664.

  2. HybridGO-Loc: mining hybrid features on gene ontology for predicting subcellular localization of multi-location proteins.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2014-01-01

    Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO) have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS) between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM) classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/.

  3. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

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    Bo Fu

    2015-12-01

    Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

  4. Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization

    International Nuclear Information System (INIS)

    Heibein, Allan D; Guo, Baoqing; Sprowl, Jason A; MacLean, David A; Parissenti, Amadeo M

    2012-01-01

    Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the “hit list” was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify

  5. The SubCons webserver: A user friendly web interface for state-of-the-art subcellular localization prediction.

    Science.gov (United States)

    Salvatore, M; Shu, N; Elofsson, A

    2018-01-01

    SubCons is a recently developed method that predicts the subcellular localization of a protein. It combines predictions from four predictors using a Random Forest classifier. Here, we present the user-friendly web-interface implementation of SubCons. Starting from a protein sequence, the server rapidly predicts the subcellular localizations of an individual protein. In addition, the server accepts the submission of sets of proteins either by uploading the files or programmatically by using command line WSDL API scripts. This makes SubCons ideal for proteome wide analyses allowing the user to scan a whole proteome in few days. From the web page, it is also possible to download precalculated predictions for several eukaryotic organisms. To evaluate the performance of SubCons we present a benchmark of LocTree3 and SubCons using two recent mass-spectrometry based datasets of mouse and drosophila proteins. The server is available at http://subcons.bioinfo.se/. © 2017 The Protein Society.

  6. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    Science.gov (United States)

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

  7. Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

    Science.gov (United States)

    Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

    2014-10-01

    Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft

  8. CerebralWeb: a Cytoscape.js plug-in to visualize networks stratified by subcellular localization.

    Science.gov (United States)

    Frias, Silvia; Bryan, Kenneth; Brinkman, Fiona S L; Lynn, David J

    2015-01-01

    CerebralWeb is a light-weight JavaScript plug-in that extends Cytoscape.js to enable fast and interactive visualization of molecular interaction networks stratified based on subcellular localization or other user-supplied annotation. The application is designed to be easily integrated into any website and is configurable to support customized network visualization. CerebralWeb also supports the automatic retrieval of Cerebral-compatible localizations for human, mouse and bovine genes via a web service and enables the automated parsing of Cytoscape compatible XGMML network files. CerebralWeb currently supports embedded network visualization on the InnateDB (www.innatedb.com) and Allergy and Asthma Portal (allergen.innatedb.com) database and analysis resources. Database tool URL: http://www.innatedb.com/CerebralWeb © The Author(s) 2015. Published by Oxford University Press.

  9. [L-arginine metabolism enzyme activities in rat liver subcellular fractions under condition of protein deprivation].

    Science.gov (United States)

    Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

    The features of arginase and NO-synthase pathways of arginine's metabolism have been studied in rat liver subcellular fractions under condition of protein deprivation. During the experimental period (28 days) albino male rats were kept on semi synthetic casein diet AIN-93. The protein deprivation conditions were designed as total absence of protein in the diet and consumption of the diet partially deprived with 1/2 of the casein amount compared to in the regular diet. Daily diet consumption was regulated according to the pair feeding approach. It has been shown that the changes of enzyme activities, involved in L-arginine metabolism, were characterized by 1.4-1.7 fold decrease in arginase activity, accompanied with unchanged NO-synthase activity in cytosol. In mitochondrial fraction the unchanged arginase activity was accompanied by 3-5 fold increase of NO-synthase activity. At the terminal stages of the experiment the monodirectional dynamics in the studied activities have been observed in the mitochondrial and cytosolfractions in both experimental groups. In the studied subcellular fractions arginase activity decreased (2.4-2.7 fold with no protein in the diet and 1.5 fold with partly supplied protein) and was accompanied by NO-synthase activity increase by 3.8 fold in cytosole fraction, by 7.2 fold in mitochondrial fraction in the group with no protein in the diet and by 2.2 and 3.5 fold in the group partialy supplied with protein respectively. The observed tendency is presumably caused by the switch of L-arginine metabolism from arginase into oxidizing NO-synthase parthway.

  10. A comparative antibody analysis of Pannexin1 expression in four rat brain regions reveals varying subcellular localizations

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    Angela C Cone

    2013-02-01

    Full Text Available Pannexin1 (Panx1 channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide-field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on

  11. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    International Nuclear Information System (INIS)

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table

  12. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    Science.gov (United States)

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity. PMID:19726875

  13. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.

    Science.gov (United States)

    Benoit, Stephen C; Kemp, Christopher J; Elias, Carol F; Abplanalp, William; Herman, James P; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G; Holland, William L; Clegg, Deborah J

    2009-09-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.

  14. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Olivier Poupel

    Full Text Available The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their

  15. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  16. Tissue distribution and subcellular localizations determine in vivo functional relationship among prostasin, matriptase, HAI-1, and HAI-2 in human skin.

    Science.gov (United States)

    Lee, Shiao-Pieng; Kao, Chen-Yu; Chang, Shun-Cheng; Chiu, Yi-Lin; Chen, Yen-Ju; Chen, Ming-Hsing G; Chang, Chun-Chia; Lin, Yu-Wen; Chiang, Chien-Ping; Wang, Jehng-Kang; Lin, Chen-Yong; Johnson, Michael D

    2018-01-01

    The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors.

  17. Subcellular localization of class II HDAs in Arabidopsis thaliana: nucleocytoplasmic shuttling of HDA15 is driven by light.

    Directory of Open Access Journals (Sweden)

    Malona V Alinsug

    Full Text Available Class II histone deacetylases in humans and other model organisms undergo nucleocytoplasmic shuttling. This unique functional regulatory mechanism has been well elucidated in eukaryotic organisms except in plant systems. In this study, we have paved the baseline evidence for the cytoplasmic and nuclear localization of Class II HDAs as well as their mRNA expression patterns. RT-PCR analysis on the different vegetative parts and developmental stages reveal that Class II HDAs are ubiquitously expressed in all tissues with minimal developmental specificity. Moreover, stable and transient expression assays using HDA-YFP/GFP fusion constructs indicate cytoplasmic localization of HDA5, HDA8, and HDA14 further suggesting their potential for nuclear transport and deacetylating organellar and cytoplasmic proteins. Organelle markers and stains confirm HDA14 to abound in the mitochondria and chloroplasts while HDA5 localizes in the ER. HDA15, on the other hand, shuttles in and out of the nucleus upon light exposure. In the absence of light, it is exported out of the nucleus where further re-exposition to light treatments signals its nuclear import. Unlike HDA5 which binds with 14-3-3 proteins, HDA15 fails to interact with these chaperones. Instead, HDA15 relies on its own nuclear localization and export signals to navigate its subcellular compartmentalization classifying it as a Class IIb HDA. Our study indicates that nucleocytoplasmic shuttling is indeed a hallmark for all eukaryotic Class II histone deacetylases.

  18. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    Science.gov (United States)

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  19. pLoc-mAnimal: predict subcellular localization of animal proteins with both single and multiple sites.

    Science.gov (United States)

    Cheng, Xiang; Zhao, Shu-Guang; Lin, Wei-Zhong; Xiao, Xuan; Chou, Kuo-Chen

    2017-11-15

    Cells are deemed the basic unit of life. However, many important functions of cells as well as their growth and reproduction are performed via the protein molecules located at their different organelles or locations. Facing explosive growth of protein sequences, we are challenged to develop fast and effective method to annotate their subcellular localization. However, this is by no means an easy task. Particularly, mounting evidences have indicated proteins have multi-label feature meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Unfortunately, most of the existing computational methods can only be used to deal with the single-label proteins. Although the 'iLoc-Animal' predictor developed recently is quite powerful that can be used to deal with the animal proteins with multiple locations as well, its prediction quality needs to be improved, particularly in enhancing the absolute true rate and reducing the absolute false rate. Here we propose a new predictor called 'pLoc-mAnimal', which is superior to iLoc-Animal as shown by the compelling facts. When tested by the most rigorous cross-validation on the same high-quality benchmark dataset, the absolute true success rate achieved by the new predictor is 37% higher and the absolute false rate is four times lower in comparison with the state-of-the-art predictor. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mAnimal/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. xxiao@gordonlifescience.org or kcchou@gordonlifescience.org. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  20. iLoc-Animal: a multi-label learning classifier for predicting subcellular localization of animal proteins.

    Science.gov (United States)

    Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

    2013-04-05

    Predicting protein subcellular localization is a challenging problem, particularly when query proteins have multi-label features meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multi-label proteins should not be ignored because they usually bear some special function worthy of in-depth studies. By introducing the "multi-label learning" approach, a new predictor, called iLoc-Animal, has been developed that can be used to deal with the systems containing both single- and multi-label animal (metazoan except human) proteins. Meanwhile, to measure the prediction quality of a multi-label system in a rigorous way, five indices were introduced; they are "Absolute-True", "Absolute-False" (or Hamming-Loss"), "Accuracy", "Precision", and "Recall". As a demonstration, the jackknife cross-validation was performed with iLoc-Animal on a benchmark dataset of animal proteins classified into the following 20 location sites: (1) acrosome, (2) cell membrane, (3) centriole, (4) centrosome, (5) cell cortex, (6) cytoplasm, (7) cytoskeleton, (8) endoplasmic reticulum, (9) endosome, (10) extracellular, (11) Golgi apparatus, (12) lysosome, (13) mitochondrion, (14) melanosome, (15) microsome, (16) nucleus, (17) peroxisome, (18) plasma membrane, (19) spindle, and (20) synapse, where many proteins belong to two or more locations. For such a complicated system, the outcomes achieved by iLoc-Animal for all the aforementioned five indices were quite encouraging, indicating that the predictor may become a useful tool in this area. It has not escaped our notice that the multi-label approach and the rigorous measurement metrics can also be used to investigate many other multi-label problems in molecular biology. As a user-friendly web-server, iLoc-Animal is freely accessible to the public at the web-site .

  1. Expression and subcellular localization of p70S6 kinase under heart failure

    Directory of Open Access Journals (Sweden)

    Usenko V. S.

    2010-11-01

    Full Text Available The PI3K/PDK/Akt/mTOR/p70S6K signaling pathway is primary associated with the activation of insulin receptors and is important for cardiomyocytes survival. p70S6K is a key regulator of the speed and efficiency of protein biosynthesis within the cell. Recently the pro-apoptotic protein BAD has been identified as a new target for р70S6K1. BAD is inactivated in normal cardiomyocytes by р70S6K1 phosphorylation which prevents the cardiomyocytes apoptosis. Aim. To study possible changes in р70S6K1 expression and/or cellular localization at heart failure progression – in DCM- affected human myocardia and murine hearts with experimental DCM-like pathology. Methods. Western-blot analysis and immunohystochemistry. Results. The substantial decrease in р70S6K1 level was observed at the final stage of pathology progression and in the dynamics of DCM pathogenesis as well. For the first time relocalization of the protein to the connective tissue was shown according to the Western-blot results. Conclusions. The data obtained allow us to understand a possible role of р70S6K1 in the regulation of stress-induced apoptotic signaling in cardiomyocytes.

  2. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

    Science.gov (United States)

    The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING...

  3. Skeletal muscle glycogen content and particle size of distinct subcellular localizations in the recovery period after a high-level soccer match

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Krustrup, Peter; Nybo, Lars

    2012-01-01

    Whole muscle glycogen levels remain low for a prolonged period following a soccer match. The present study was conducted to investigate how this relates to glycogen content and particle size in distinct subcellular localizations. Seven high-level male soccer players had a vastus lateralis muscle...... biopsy collected immediately after and 24, 48, 72 and 120 h after a competitive soccer match. Transmission electron microscopy was used to estimate the subcellular distribution of glycogen and individual particle size. During the first day of recovery, glycogen content increased by ~60% in all...

  4. Subcellular distribution of histone-degrading enzyme activities from rat liver

    International Nuclear Information System (INIS)

    Heinrich, P.C.; Raydt, G.; Puschendorf, B.; Jusic, M.

    1976-01-01

    Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. (orig.) [de

  5. Modeling curvature-dependent subcellular localization of a small sporulation protein in Bacillus subtilis

    Science.gov (United States)

    Wasnik, Vaibhav; Wingreen, Ned; Mukhopadhyay, Ranjan

    2012-02-01

    Recent experiments suggest that in the bacterium, B. subtilis, the cue for the localization of small sporulation protein, SpoVM, that plays a central role in spore coat formation, is curvature of the bacterial plasma membrane. This curvature-dependent localization is puzzling given the orders of magnitude difference in lengthscale of an individual protein and radius of curvature of the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane absorption of SpoVM and clustering of membrane-associated SpoVM and compare our results with experiments.

  6. Active Neural Localization

    OpenAIRE

    Chaplot, Devendra Singh; Parisotto, Emilio; Salakhutdinov, Ruslan

    2018-01-01

    Localization is the problem of estimating the location of an autonomous agent from an observation and a map of the environment. Traditional methods of localization, which filter the belief based on the observations, are sub-optimal in the number of steps required, as they do not decide the actions taken by the agent. We propose "Active Neural Localizer", a fully differentiable neural network that learns to localize accurately and efficiently. The proposed model incorporates ideas of tradition...

  7. Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

    Science.gov (United States)

    Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

    1998-01-01

    Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

  8. Subcellular localization of Bombyx mori ribosomal protein S3a and ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... In the present study, using a BV/PH-Bms3a-EGFP, we found that Bombyx mori ribosomal protein S3a. (BmS3a) with EGFP fused to its C-terminal, was predominantly localized in the cytoplasm of B. mori cells. Subsequently, to investigate the effect of BmS3a over-expression on BmNPV infection both at the.

  9. Subcellular Localization of Patched and Smoothened, the Receptors for Sonic Hedgehog Signaling, in the Hippocampal Neuron

    OpenAIRE

    Petralia, Ronald S.; Schwartz, Catherine M.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

    2011-01-01

    Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level i...

  10. The subcellular localization of yeast glycogen synthase is dependent upon glycogen content

    OpenAIRE

    Wilson, Wayne A.; Boyer, Michael P.; Davis, Keri D.; Burke, Michael; Roach, Peter J.

    2010-01-01

    The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilize Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase is located within cells. We demonstrate that the localization pattern of Gsy2-GFP depends upon the glycogen content of the cell. When glyco...

  11. Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

    International Nuclear Information System (INIS)

    Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan; Bi, Jing; Wang, Dang; Xiao, Shaobo

    2016-01-01

    Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

  12. Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Bi, Jing [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Department of Immunology and Aetology, College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065 (China); Wang, Dang [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Xiao, Shaobo, E-mail: vet@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China)

    2016-12-15

    Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

  13. Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

    Science.gov (United States)

    Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

    2013-09-01

    Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

    Science.gov (United States)

    Schotte, A; Rostène, W; Laduron, P M

    1988-04-01

    The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

  15. Tissue distribution, subcellular localization and endocrine disruption patterns induced by Cr and Mn in the crab Ucides cordatus

    International Nuclear Information System (INIS)

    Correa, Jose Dias; Ramos da Silva, Miguel; Bastos da Silva, Antonio Carlos; Araujo de Lima, Silene Maria; Malm, Olaf; Allodi, Silvana

    2005-01-01

    The essential trace elements Cr and Mn are toxic at high concentrations and information about low concentration is insufficient in the literature. In polluted mangroves, the crab Ucides cordatus can represent a useful tool to assess information on the potential impact of trace elements like Cr and Mn on the environment, since this species is comestible and thus, commercially negotiated. Therefore, U. cordatus crabs were exposed in vivo to different concentrations of Cr and Mn solved in seawater and had their tissue distribution and subcellular deposits evaluated. The gill, hepatopancreas and muscle concentrations were determined by atomic absorption spectroscopy and the results showed that Cr and Mn presented the highest values in the gills rather than in the hepatopancreas and muscular tissue. Electron microscopy and analytical X-ray microanalysis revealed Cr precipitates on the gill surface, co-localized with epiphyte bacteria. In addition, since Cr and Mn did not equally accumulate in most of the tissues studied, glycemic rate of animals, which received injections of extracts of eyestalks of the contaminated crabs, were measured in order to evaluate whether the studied concentrations of Cr and Mn could produce any metabolic alteration. The results indicated that extracts of the eyestalks of crabs submitted to Cr and Mn salts and injected into normal crabs markedly influenced crustacean hyperglycemic hormone synthesis and/or release. The results are discussed with respect to sensitivity of the employed methods and the possible significance of the concentrations of Cr and Mn in the organisms

  16. ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

    Science.gov (United States)

    Paramasivam, Nagarajan; Linke, Dirk

    2011-01-01

    The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

  17. Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

    International Nuclear Information System (INIS)

    Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H.

    1990-01-01

    Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell

  18. Endogenous spar tin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons

    International Nuclear Information System (INIS)

    Robay, Dimitri; Patel, Heema; Simpson, Michael A.; Brown, Nigel A.; Crosby, Andrew H.

    2006-01-01

    Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker α-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction

  19. Subcellular localization of human neutral ceramidase expressed in HEK293 cells

    International Nuclear Information System (INIS)

    Hwang, Young-ha; Tani, Motohiro; Nakagawa, Tetsuto; Okino, Nozomu; Ito, Makoto

    2005-01-01

    We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence

  20. The Subcellular Localization and Functional Analysis of Fibrillarin2, a Nucleolar Protein in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Luping Zheng

    2016-01-01

    Full Text Available Nucleolar proteins play important roles in plant cytology, growth, and development. Fibrillarin2 is a nucleolar protein of Nicotiana benthamiana (N. benthamiana. Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled with yellow fluorescent protein (YFP. The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells of N. benthamiana. The N. benthamiana fibrillarin2 (NbFib2 protein has three functional domains (i.e., glycine and arginine rich domain, RNA-binding domain, and α-helical domain and a nuclear localization signal (NLS in C-terminal. The protein 3D structure analysis predicted that NbFib2 is an α/β protein. In addition, the virus induced gene silencing (VIGS approach was used to determine the function of NbFib2. Our results showed that symptoms including growth retardation, organ deformation, chlorosis, and necrosis appeared in NbFib2-silenced N. benthamiana.

  1. Anks3 alters the sub-cellular localization of the Nek7 kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandran, Haribaskar; Engel, Christina; Müller, Barbara [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Dengjel, Jörn [Department of Dermatology, University Freiburg Medical Center and Center of Biological Systems Analysis, Habsburgerstr. 49, 79104 Freiburg (Germany); Walz, Gerd [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Center for Biological Signaling Studies (BIOSS), Albertstr. 19, 79104 Freiburg (Germany); Yakulov, Toma A., E-mail: toma.antonov.yakulov@uniklinik-freiburg.de [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany)

    2015-08-28

    Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7. - Highlights: • Anks3 interacted with Nek7 kinase, and was heavily modified in the presence of Nek7. • Anks3 N-terminal ankyrin repeats, but not SAM domain required for Nek7 interaction. • Nek7 increased Ser/Thr phosphorylation of Anks3 primarily within ankyrin domain. • Interaction with Anks3 led to cytoplasmic retention and nuclear exclusion of Nek7.

  2. Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

    Science.gov (United States)

    Tarquini, Giulia; Ermacora, Paolo; Bianchi, Gian Luca; De Amicis, Francesca; Pagliari, Laura; Martini, Marta; Loschi, Alberto; Saldarelli, Pasquale; Loi, Nazia; Musetti, Rita

    2018-05-01

    Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.

  3. Nucleolin modulates the subcellular localization of GDNF-inducible zinc finger protein 1 and its roles in transcription and cell proliferation

    International Nuclear Information System (INIS)

    Dambara, Atsushi; Morinaga, Takatoshi; Fukuda, Naoyuki; Yamakawa, Yoshinori; Kato, Takuya; Enomoto, Atsushi; Asai, Naoya; Murakumo, Yoshiki; Matsuo, Seiichi; Takahashi, Masahide

    2007-01-01

    GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin

  4. CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

    International Nuclear Information System (INIS)

    Neyton, Sophie; Lespinasse, Francoise; Lahaye, Francois; Staccini, Pascal; Paquis-Flucklinger, Veronique; Santucci-Darmanin, Sabine

    2007-01-01

    MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions

  5. Local Worlds of Activation

    DEFF Research Database (Denmark)

    Jacobsson, Kerstin; Hollertz, Katarina; Garsten, Christina

    2017-01-01

    arrangements and the role of private services and actors in service delivery differed significantly too, ranging from strictly market-based forms of governance to classical public administration. The article moreover shows how the different activation approaches were reflected in the radically different usages...... from local politics, established local traditions, patterns of networking and modes of collaborating, as the notion of ‘local words of activation’ intends to capture....

  6. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    International Nuclear Information System (INIS)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

    2013-01-01

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  7. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  8. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    OpenAIRE

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of ...

  9. Subcellular localization of Patched and Smoothened, the receptors for Sonic hedgehog signaling, in the hippocampal neuron.

    Science.gov (United States)

    Petralia, Ronald S; Schwartz, Catherine M; Wang, Ya-Xian; Mattson, Mark P; Yao, Pamela J

    2011-12-15

    Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level in young postnatal and adult brains. By using immunofluorescence light microscopy and immunoelectron microscopy, we examine the spatial distribution of Ptch and Smo within the hippocampal neurons. In young developing neurons, Ptch and Smo are present in the processes and are clustered at their growth cones. In mature neurons, Ptch and Smo are concentrated in dendrites, spines, and postsynaptic sites. Synaptic Ptch and Smo often co-exist with unusual structures-synaptic spinules and autophagosomes. Our results reveal the anatomical organization of the Shh receptors within both the young and the mature hippocampal neurons. Copyright © 2011 Wiley-Liss, Inc.

  10. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

    Science.gov (United States)

    Carmo, Lívia A S; Dias, Felipe F; Malta, Kássia K; Amaral, Kátia B; Shamri, Revital; Weller, Peter F; Melo, Rossana C N

    2015-10-01

    SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

    Energy Technology Data Exchange (ETDEWEB)

    Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B. [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Shamri, Revital; Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States)

    2015-10-01

    Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High

  12. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

    International Nuclear Information System (INIS)

    Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B.; Shamri, Revital; Weller, Peter F.; Melo, Rossana C.N.

    2015-01-01

    Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High

  13. 2-Bromo-5-hydroxyphenylporphyrins for photodynamic therapy: photosensitization efficiency, subcellular localization and in vivo studies.

    Science.gov (United States)

    Laranjo, Mafalda; Serra, Arménio C; Abrantes, Margarida; Piñeiro, Marta; Gonçalves, Ana C; Casalta-Lopes, João; Carvalho, Lina; Sarmento-Ribeiro, Ana B; Rocha-Gonsalves, António; Botelho, Filomena

    2013-02-01

    Photodynamic therapy (PDT) is a therapeutic modality capable of inducing cell death by oxidative stress through activation of a sensitizer by light. Aryl-porphyrin with hydroxyl groups are good photosensitizers and presence of bromine atoms can enhance the photodynamic activity through heavy atom effect. These facts and our previous work made pertinent to compare the photodynamic capacity of tetraaryl brominated porphyrin (TBr4) with the corresponding diaryl (BBr2) derivative. Cell cultures were incubated with the sensitizers, ranging from 50nM to 10μM and irradiated until 10J. Cell proliferation was analysed by MTT assay. Flow cytometry studies evaluated cell death pathways, mitochondrial membrane potential and ROS. For in vivo studies Balb/c nu/nu mice were injected with 4×10(6)cells. After PDT, monitoring was carried out for 12 days to establish Kaplan-Meier survival curves. Tumours were excised and histological analysis was performed. Both sensitizers seem to accumulate in the mitochondria. The molecules have no intrinsic cytotoxicity or in non-tumour cells at therapeutic concentrations. Both sensitizers induced a significant decrease of cell proliferation and growth of xenografts of melanoma and colorectal adenocarcinoma. Diaryl BBr2 is more efficient than tetraaryl TBr4, concerning intracellular ROS production, mitochondrial disruption and induction of cell death. The main cell death pathway is necrosis. TBr2 and BBr4 are promising sensitizers with good photodynamic properties and have the ability to induce cell death in human melanoma and colorectal adenocarcinoma in vitro and in vivo. We consider that BBr2 is a molecule that should be the subject of extensive studies towards clinical use. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Subcellular Targeting of Methylmercury Lyase Enhances Its Specific Activity for Organic Mercury Detoxification in Plants1

    Science.gov (United States)

    Bizily, Scott P.; Kim, Tehryung; Kandasamy, Muthugapatti K.; Meagher, Richard B.

    2003-01-01

    Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments. PMID:12586871

  15. Subcellular Localization of Large Yellow Croaker ( Larimichthys crocea) TLR21 and Expression Profiling of Its Gene in Immune Response

    Science.gov (United States)

    Sun, Qingxue; Fan, Zejun; Yao, Cuiluan

    2018-04-01

    Toll-like receptor 21 (TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from large yellow croaker, Larimichthys crocea and was termed as LcTLR21. It consists of 3365 bp, including a 5'-terminal untranslated region (UTR) of 97 bp, a 3'-terminal UTR of 331 bp, and an open reading frame (ORF) of 2937 bp encoding a polypeptide of 978 amino acid residues. The deduced LcTLR21 contains a signal peptide domain at N-terminal, 12 leucine-rich repeats (LRRs) at the extracellular region, a transmembrane domain and a cytoplasmic toll-interleukin-1 receptor (TIR) domain at the C-terminal. Subcellular localization analysis revealed that the LcTLR21-GFP was constitutively expressed in cytoplasm. Tissue expression analysis indicated that LcTLR21 gene broadly expressed in most of the examined tissues, with the most predominant abundance in spleen, followed by head-kidney and liver, while the weakest expression was detected in brain. The expression level of LcTLR21 after LPS, poly I:C and Vibrio parahaemolyticus challenges was investigated in spleen, head-kidney and liver. LcTLR21 gene transcripts increased significantly in all examined tissues after the challenges, and the highest expression level was detected in liver at 24 h after poly I:C stimulation ( P < 0.05), suggesting that LcTLR21 might play a crucial role in fish resistance to viral and bacterial infections.

  16. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  17. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    International Nuclear Information System (INIS)

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-01-01

    Highlights: ► The study presents cloning and characterization of TCP1γ gene from L. donovani. ► TCP1γ is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. ► LdTCPγ exhibited differential expression in different stages of promastigotes. ► LdTCPγ co-localized with actin, a cytoskeleton protein. ► The data suggests that this gene may have a role in differentiation/biogenesis. ► First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

  18. pLoc-mHum: predict subcellular localization of multi-location human proteins via general PseAAC to winnow out the crucial GO information.

    Science.gov (United States)

    Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

    2018-05-01

    For in-depth understanding the functions of proteins in a cell, the knowledge of their subcellular localization is indispensable. The current study is focused on human protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions that are particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called 'pLoc-mHum' by extracting the crucial GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validations on a same stringent benchmark dataset have indicated that the proposed pLoc-mHum predictor is remarkably superior to iLoc-Hum, the state-of-the-art method in predicting the human protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mHum/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. xcheng@gordonlifescience.org. Supplementary data are available at Bioinformatics online.

  19. Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking.

    Science.gov (United States)

    Zirngibl, R; Schulze, D; Mirski, S E; Cole, S P; Greer, P A

    2001-05-15

    The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking. Copyright 2001 Academic Press.

  20. Activation analysis study on subcellular distribution of trace elements in human brain tumor

    International Nuclear Information System (INIS)

    Zheng Jian; Zhuan Guisun; Wang Yongji; Dong Mo; Zhang Fulin

    1992-01-01

    The concentrations of up to 11 elements in subcellular fractions of human brain (normal and malignant tumor) have been determined by a combination of gradient centrifugation and INAA methods. Samples of human brain were homogenized in a glass homogenizer tube, the homogenate was separated into nuclei, mitochondrial, myelin, synaptosome fractions, and these fractions were then analyzed using the INAA method. The discussions of elemental subcelleular distributions in human brain malignant tumor are presented in this paper. (author) 11 refs.; 2 figs.; 4 tabs

  1. Immunohistochemical analysis based Ep-ICD subcellular localization index (ESLI) is a novel marker for metastatic papillary thyroid microcarcinoma

    International Nuclear Information System (INIS)

    Kunavisarut, Tada; Kak, Ipshita; MacMillan, Christina; Ralhan, Ranju; Walfish, Paul G

    2012-01-01

    Thyroid cancer is among the fastest growing malignancies; almost fifty-percent of these rapidly increasing incidence tumors are less than or equal to 1cm in size, termed papillary thyroid microcarcinoma (PTMC). The management of PTMC remains a controversy due to differing natural history of these patients. Epithelial cell adhesion molecule (EpCAM) is comprised of an extracellular domain (EpEx), a single transmembrane domain and an intracellular domain (Ep-ICD). Our group reported nuclear Ep-ICD correlated with poor prognosis in thyroid cancer (Ralhan et al., BMC Cancer 2010,10:331). Here in, we hypothesized nuclear and cytoplasmic accumulation of Ep-ICD and loss of membranous EpEx may aid in distinguishing metastatic from non-metastatic PTMC, which is an important current clinical challenge. To test our hypothesis, Ep-ICD and EpEx expression levels were analyzed in PTMC and the staining was correlated with metastatic potential of these carcinomas. Thirty-six PTMC patients (tumor size 0.5 - 1cm; metastatic 8 cases and non-metastatic 28 cases) who underwent total thyroidectomy were selected. The metastatic group consisted of patients who developed lymph node or distant metastasis at diagnosis or during follow up. The patients’ tissues were stained for Ep-ICD and EpEx using domain specific antibodies by immunohistochemistry and evaluated. PTMC patients with metastasis had higher scores for nuclear and cytoplasmic Ep-ICD immunostaining than the patients without metastasis (1.96 ± 0.86 vs. 1.22 ± 0.45; p = 0.007 and 5.37 ± 0.33 vs. 4.72 ± 1.07; p = 0.016, respectively). Concomitantly, the former had lower scores for membrane EpEx than the non-metastatic group (4.64 ± 1.08 vs. 5.64 ± 1.51; p = 0.026). An index of aggressiveness, Ep-ICD subcellular localization index (ESLI), was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep − ICD nuc + Ep − ICD cyt + loss of membranous EpEx]. Notably

  2. pLoc-mVirus: Predict subcellular localization of multi-location virus proteins via incorporating the optimal GO information into general PseAAC.

    Science.gov (United States)

    Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

    2017-09-10

    Knowledge of subcellular locations of proteins is crucially important for in-depth understanding their functions in a cell. With the explosive growth of protein sequences generated in the postgenomic age, it is highly demanded to develop computational tools for timely annotating their subcellular locations based on the sequence information alone. The current study is focused on virus proteins. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex proteins is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mVirus" by extracting the optimal GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on a same stringent benchmark dataset indicated that the proposed pLoc-mVirus predictor is remarkably superior to iLoc-Virus, the state-of-the-art method in predicting virus protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mVirus/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. pLoc-mPlant: predict subcellular localization of multi-location plant proteins by incorporating the optimal GO information into general PseAAC.

    Science.gov (United States)

    Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

    2017-08-22

    One of the fundamental goals in cellular biochemistry is to identify the functions of proteins in the context of compartments that organize them in the cellular environment. To realize this, it is indispensable to develop an automated method for fast and accurate identification of the subcellular locations of uncharacterized proteins. The current study is focused on plant protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most of the existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex protein is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mPlant" by extracting the optimal GO (Gene Ontology) information into the Chou's general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on the same stringent benchmark dataset indicated that the proposed pLoc-mPlant predictor is remarkably superior to iLoc-Plant, the state-of-the-art method for predicting plant protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at , by which users can easily get their desired results without the need to go through the complicated mathematics involved.

  4. Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization.

    Science.gov (United States)

    Veeranki, Sudhakar; Choubey, Divaker

    2012-01-01

    The interferon (IFN)-inducible p200-protein family includes structurally related murine (for example, p202a, p202b, p204, and Aim2) and human (for example, AIM2 and IFI16) proteins. All proteins in the family share a partially conserved repeat of 200-amino acid residues (also called HIN-200 domain) in the C-terminus. Additionally, most proteins (except the p202a and p202b proteins) also share a protein-protein interaction pyrin domain (PYD) in the N-terminus. The HIN-200 domain contains two consecutive oligosaccharide/oligonucleotide binding folds (OB-folds) to bind double stranded DNA (dsDNA). The PYD domain in proteins allows interactions with the family members and an adaptor protein ASC. Upon sensing cytosolic dsDNA, Aim2, p204, and AIM2 proteins recruit ASC protein to form an inflammasome, resulting in increased production of proinflammatory cytokines. However, IFI16 protein can sense cytosolic as well as nuclear dsDNA. Interestingly, the IFI16 protein contains a nuclear localization signal (NLS). Accordingly, the initial studies had indicated that the endogenous IFI16 protein is detected in the nucleus and within the nucleus in the nucleolus. However, several recent reports suggest that subcellular localization of IFI16 protein in nuclear versus cytoplasmic (or both) compartment depends on cell type. Given that the IFI16 protein can sense cytosolic as well as nuclear dsDNA and can initiate different innate immune responses (production of IFN-β versus proinflammatory cytokines), here we evaluate the experimental evidence for the regulation of subcellular localization of IFI16 protein in various cell types. We conclude that further studies are needed to understand the molecular mechanisms that regulate the subcellular localization of IFI16 protein. Published by Elsevier Ltd.

  5. Role of NH2-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    International Nuclear Information System (INIS)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke; Kostsin, Dzmitry G.; Kashiwayama, Yoshinori; Takanashi, Kojiro; Yazaki, Kazufumi; Imanaka, Tsuneo; Morita, Masashi

    2014-01-01

    Highlights: • ABCD proteins classifies based on with or without NH 2 -terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH 2 -terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH 2 -terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH 2 -terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH 2 -terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH 2 -terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH 2 -terminal H0 motif in organelle targeting is widely conserved in living organisms

  6. Hum-mPLoc 3.0: prediction enhancement of human protein subcellular localization through modeling the hidden correlations of gene ontology and functional domain features.

    Science.gov (United States)

    Zhou, Hang; Yang, Yang; Shen, Hong-Bin

    2017-03-15

    Protein subcellular localization prediction has been an important research topic in computational biology over the last decade. Various automatic methods have been proposed to predict locations for large scale protein datasets, where statistical machine learning algorithms are widely used for model construction. A key step in these predictors is encoding the amino acid sequences into feature vectors. Many studies have shown that features extracted from biological domains, such as gene ontology and functional domains, can be very useful for improving the prediction accuracy. However, domain knowledge usually results in redundant features and high-dimensional feature spaces, which may degenerate the performance of machine learning models. In this paper, we propose a new amino acid sequence-based human protein subcellular location prediction approach Hum-mPLoc 3.0, which covers 12 human subcellular localizations. The sequences are represented by multi-view complementary features, i.e. context vocabulary annotation-based gene ontology (GO) terms, peptide-based functional domains, and residue-based statistical features. To systematically reflect the structural hierarchy of the domain knowledge bases, we propose a novel feature representation protocol denoted as HCM (Hidden Correlation Modeling), which will create more compact and discriminative feature vectors by modeling the hidden correlations between annotation terms. Experimental results on four benchmark datasets show that HCM improves prediction accuracy by 5-11% and F 1 by 8-19% compared with conventional GO-based methods. A large-scale application of Hum-mPLoc 3.0 on the whole human proteome reveals proteins co-localization preferences in the cell. www.csbio.sjtu.edu.cn/bioinf/Hum-mPLoc3/. hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  7. Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

    Science.gov (United States)

    Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

    2015-01-01

    It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible

  8. mPLR-Loc: an adaptive decision multi-label classifier based on penalized logistic regression for protein subcellular localization prediction.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2015-03-15

    Proteins located in appropriate cellular compartments are of paramount importance to exert their biological functions. Prediction of protein subcellular localization by computational methods is required in the post-genomic era. Recent studies have been focusing on predicting not only single-location proteins but also multi-location proteins. However, most of the existing predictors are far from effective for tackling the challenges of multi-label proteins. This article proposes an efficient multi-label predictor, namely mPLR-Loc, based on penalized logistic regression and adaptive decisions for predicting both single- and multi-location proteins. Specifically, for each query protein, mPLR-Loc exploits the information from the Gene Ontology (GO) database by using its accession number (AC) or the ACs of its homologs obtained via BLAST. The frequencies of GO occurrences are used to construct feature vectors, which are then classified by an adaptive decision-based multi-label penalized logistic regression classifier. Experimental results based on two recent stringent benchmark datasets (virus and plant) show that mPLR-Loc remarkably outperforms existing state-of-the-art multi-label predictors. In addition to being able to rapidly and accurately predict subcellular localization of single- and multi-label proteins, mPLR-Loc can also provide probabilistic confidence scores for the prediction decisions. For readers' convenience, the mPLR-Loc server is available online (http://bioinfo.eie.polyu.edu.hk/mPLRLocServer). Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Filippidis, G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kouloumentas, C [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kapsokalyvas, D [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Voglis, G [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Tavernarakis, N [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Papazoglou, T G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece)

    2005-08-07

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT) (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  10. Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae.

    Science.gov (United States)

    Roosendaal, E; Jacobs, A A; Rathman, P; Sondermeyer, C; Stegehuis, F; Oudega, B; de Graaf, F K

    1987-09-01

    Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

  11. Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

    International Nuclear Information System (INIS)

    Hu, Xiaolan; Yu, Yingnian; Li, Qian; Wu, Danxiao; Tan, Zhengning; Wang, Cheng; Wang, Jvping; Wu, Meiping

    2011-01-01

    Highlights: → MNNG-induced appearance of DUT-N in the extracellular fluid has cellular specificity. → MNNG alters the subcellular distribution of DUT-N in human cells in different ways. → DUT-N may be a potential biomarker to assess the risk of alkylating agents exposure. -- Abstract: Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

  12. Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

    Science.gov (United States)

    Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

    2015-09-11

    Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

  13. Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

    Directory of Open Access Journals (Sweden)

    Jianjun He

    Full Text Available It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

  14. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  15. Establishing the subcellular localization of photodynamically-induced ROS using 3,3′-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration

    DEFF Research Database (Denmark)

    Stockert, Juan Carlos; Blazquez-Castro, Alfonso

    2016-01-01

    The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions...... is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation....... between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose...

  16. Effect of cadmium on the physiological parameters and the subcellular cadmium localization in the potato (Solanum tuberosum L.).

    Science.gov (United States)

    Xu, Dongyu; Chen, Zhifan; Sun, Ke; Yan, Dong; Kang, Mingjie; Zhao, Ye

    2013-11-01

    The pollution of agricultural soils with cadmium (Cd) has become a serious problem worldwide. The potato (Solanum tuberosum L.) was used to investigate how different concentrations of Cd (1, 5, and 25mgkg(-1)) affected the physiological parameters and the subcellular distribution of Cd in the potato. The analyses were conducted using scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX). The results suggest that the leaf is the organ with the highest accumulation of Cd. The malondialdehyde (MDA) content increased and the chlorophyll content decreased in response to high level of Cd. The SEM-EDX microanalysis revealed that Cd was primarily deposited in the spongy and palisade tissues of the leaf. Furthermore, Cd was also detected in the cortex and the adjacent phloem and was observed inside the intercellular space, the interior surface of the plasma membrane, and on the surface of the elliptical starch granules in the tubers of the potato. Although low concentrations of Cd migrated from the root to the tuber, the accumulation of Cd in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be seriously evaluated because Cd-containing potatoes might present high health risk to humans. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Cui, Huaqing; Wu, Feng; Sun, Yanling; Fan, Guocai; Wang, Qingming

    2010-01-01

    Hepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown. Recombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry. We found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased. This work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed

  18. Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT through the erythrocytic cycle of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mitchell Sarah L

    2010-12-01

    the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release. Conclusions Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

  19. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

    Energy Technology Data Exchange (ETDEWEB)

    Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

    2010-06-01

    chromosome is located. Two other proteins - Thiosulfate reductase and ATP binding protein were found to be cytoplasmically distributed, whereas a molybdenum transporter was found to locate to the cell periphery. We judge labeling outcome by (1) SDS gel electrophoresis, followed by direct fluorescence imaging of the gel to address specificity of labeling/confirm expected molecular weight, and subsequent Coomassie analysis to ensure comparable protein levels (2) fluorescence intensity of culture by plate reader for statistical sampling (after adjustment for respective cell numbers) and (3) fluorescence microscopy for addressing cell-to-cell signal variation and potential localization patterns. All three assays were usually found to be consistent with one another. While we have been able to improve the efficacy of photoconversion by drastically reducing (eliminating) non-specific binding with our altered labeling protocol, we are currently working on reducing non-specific photoconversion reaction arising occasionally in non-labeled cells. In addition, we have confirmed the presence of SNAP tagged constructs in three recently cloned E.coli strains under promotor control, and are in the process of utilizing them for evaluating the sensitivity of the photoconversion protocol. Fluorescent Activated Cell Sorting was successfully applied to labeled E.coli cells containing SNAP tagged AtpA protein. Different batches of sorted cells, representing low and high labeling intensity, were re-grown and re-labeled and displayed a labeling efficiency similar to the starter culture, supporting the notion that cell-to-cell differences in labeling reflect difference in protein expression, rather then genetic differences.

  20. Glycolytic pathway (GP), kreb's cycle (KC), and hexose monophosphate shunt (HMS) activity in myocardial subcellular fractions exposed to cannabinoids

    International Nuclear Information System (INIS)

    Watson, A.T.; Manno, B.R.; King, J.W.; Fowler, M.R.; Dempsey, C.A.; Manno, J.E.

    1986-01-01

    Delta-9-tetrahydrocannabinol (Δ 9 -THC), the primary psychoactive component of marihuana, and its active metabolite 11-hydroxy-Δ 9 -tetrahydrocannabinol (11-OH-Δ 9 -THC) have been reported to produce a direct cardiac depressant effect. Studies in isolated perfused rat hearts have indicated a decreased force of contraction (inotropic response) when Δ 9 -THC or 11-OH-Δ 9 -THC was administered in microgram amounts. The mechanism and site of action have not been explained or correlated with associated metabolic pathways. The purpose of this study was to investigate the effects of cannabinoids on major myocardial energy producing pathways, GP and KC, and a non-energy producing pathway, HMS. Cardiac ventricular tissue from male Sprague-Dawley rats (250-300 g) was excised and homogenized for subcellular fractionation. KC, GP and HMS activity was assayed in the appropriate fractions by measuring 14 CO 2 generation from 14 C-2-pyruvate, 14 C-6-glucose and 14 C-1-glucose respectively. Duplicate assays (n=8) were performed on tissue exposed to saline (control), empty liposomes (vehicle) and four doses each of Δ 9 -THC and 11-OH-Δ 9 -THC. Changes in metabolic activity and decreases in cardiac contractile performance may be associated

  1. Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.

    Science.gov (United States)

    Domínguez-Escobar, Julia; Wolf, Diana; Fritz, Georg; Höfler, Carolin; Wedlich-Söldner, Roland; Mascher, Thorsten

    2014-05-01

    The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage. © 2014 John Wiley & Sons Ltd.

  2. Effect of gamma irradiation on the activity of alanine and aspartate transaminases in subcellular fractions of the brain and heart in white rats

    Energy Technology Data Exchange (ETDEWEB)

    Plenin, A E

    1973-01-01

    In experiments on rats, the activity of alanine (I) and aspartate transaminases (II) was studied in homogenates and subcellular fractions of the brain and myocardium under normal conditions and for 30 days after ..gamma.. irradiation at 40 rads. The activity of II in brain homogenates increased 1 hour after irradiation but decreased by 20 percent on day 3; it decreased again on days 7 and 15. The activity of brain I increased after 1 hour and 3 days but then returned to normal. The activity of I in heart homogenates increased in all the periods after irradiation. The subcellular fractions exhibited phase changes in the activity of the enzymes. These changes were different in nature from those observed after X and ..gamma.. irradiation at the same dose.

  3. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    development of subcellularly targeted DDSs that will deliver specific drugs to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs.

  4. Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Hashiguchi, Kohtaro; Ozaki, Masumi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Kuraoka, Isao [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, Kumamoto (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

  5. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    Energy Technology Data Exchange (ETDEWEB)

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina); Vas, Mariana del, E-mail: mdelvas@cnia.inta.gov.ar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina)

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  6. Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1

    International Nuclear Information System (INIS)

    Finsterbusch, T.; Steinfeldt, T.; Caliskan, R.; Mankertz, A.

    2005-01-01

    Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs

  7. Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Costello, F.; Huhtanen, M.

    1996-01-01

    Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP...

  8. hnRNPs H, H' and F behave differently with respect to posttranslational cleavage and subcellular localization

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H; Baandrup, U

    1999-01-01

    cytoplasmic localization in other cells. The different fates may reflect differences in functional roles that so far only have included nuclear functions. The presence of significant quantities of hnRNP F in the cytoplasm of many cells indicates that it also may have a functional role here. Udgivelsesdato...

  9. Loss of Subcellular Lipid Transport Due to ARV1 Deficiency Disrupts Organelle Homeostasis and Activates the Unfolded Protein Response*

    Science.gov (United States)

    Shechtman, Caryn F.; Henneberry, Annette L.; Seimon, Tracie A.; Tinkelenberg, Arthur H.; Wilcox, Lisa J.; Lee, Eunjee; Fazlollahi, Mina; Munkacsi, Andrew B.; Bussemaker, Harmen J.; Tabas, Ira; Sturley, Stephen L.

    2011-01-01

    The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1Δ strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR. PMID:21266578

  10. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    Directory of Open Access Journals (Sweden)

    Irene L Ibañez

    Full Text Available The Cyclin-dependent kinase inhibitor 1B (p27Kip1 is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2O(2 in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p at serine 10 (S10 and at threonine 198 (T198 because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2O(2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2O(2 (0.1 µM to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2O(2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization

  11. Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina.

    Science.gov (United States)

    López-Begines, Santiago; Plana-Bonamaisó, Anna; Méndez, Ana

    2018-02-13

    Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

  12. Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.

    Science.gov (United States)

    Ben-Shimon, Lilach; Paul, Viktoria D; David-Kadoch, Galit; Volpe, Marina; Stümpfig, Martin; Bill, Eckhard; Mühlenhoff, Ulrich; Lill, Roland; Ben-Aroya, Shay

    2018-05-30

    Fe-S clusters act as co-factors of proteins with diverse functions, e.g. in DNA repair. Down-regulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability by the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, co-localize with components of the mitotic machinery. Down-regulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon down-regulation of the CIA targeting complex contributes to the mitotic defects. © 2018. Published by The Company of Biologists Ltd.

  13. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against α-transforming growth factor

    International Nuclear Information System (INIS)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-01-01

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human α-transforming growth factor (α-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting α-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native α-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of α-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of α-TGF has a cellular role beyond that of an autocrine growth factor

  14. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against. cap alpha. -transforming growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-04-07

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human ..cap alpha..-transforming growth factor (..cap alpha..-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting ..cap alpha..-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native ..cap alpha..-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of ..cap alpha..-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of ..cap alpha..-TGF has a cellular role beyond that of an autocrine growth factor.

  15. Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

    DEFF Research Database (Denmark)

    Chen, Z. W.; Jiang, C. Y.; She, Qunxin

    2005-01-01

    ). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant......Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

  16. FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

    NARCIS (Netherlands)

    Zelazny, E.; Borst, J.W.; Muylaert, M.; Batoko, H.; Hemminga, M.A.; Chaumont, F.

    2007-01-01

    Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient,

  17. Genome-wide identification, subcellular localization and gene expression analysis of the members of CESA gene family in common tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Xu, Zong-Chang; Kong, Yingzhen

    2017-06-20

    Cellulose-synthase proteins (CESAs) are membrane localized proteins and they form protein complexes to produce cellulose in the plasma membrane. CESA proteins play very important roles in cell wall construction during plant growth and development. In this study, a total of 21 NtCESA gene sequences were identified by using PF03552 conserved protein sequence and 10 AtCESA protein sequences of Arabidopsis thaliana to blast against the common tobacco (Nicotiana tabacum L.) genome database with TBLASTN protocol. We analyzed the physical and chemical properties of protein sequences based on some software or on-line analysis tools. The results showed that there were no significant variances in terms of the physical and chemical properties of the 21 NtCESA proteins. First, phylogenetic tree analysis showed that 21 NtCESA genes and 10 AtCESA genes were clustered into five groups, and the gene structures were similar among the genes that are clustered into the same group. Second, in all of the 21 NtCESA proteins the conserved zinc finger domain was identified in the N-terminus, transmembrane domains were identified in the C-terminus and the DDD-QXXRW conserved domains were also identified. Third, gene expression analysis results indicated that most NtCESA genes were expressed in roots and leaves of seedling or mature tissues of tobacco, seeds and callus tissues. The genes that clustered into the same group share similar expression patterns. Importantly, NtCESA proteins that are involved in secondary cell wall cellulose synthesis have two extra transmembrane domains compared with that involved in primary cell wall cellulose biosynthesis. In addition, subcellular localization results showed that NtCESA9 and NtCESA14 were two plasma membrane anchored proteins. This study will lay a foundation for further functional characterization of these NtCESA genes.

  18. Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

    Science.gov (United States)

    Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

    2005-08-01

    Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

  19. Localization of Glucose Oxidase and Catalase Activities in Aspergillus niger

    NARCIS (Netherlands)

    Witteveen, Cor F.B.; Veenhuis, Marten; Visser, Jaap

    The subcellular localization of glucose oxidase (EC 1.1.3.4) in Aspergillus niger N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were

  20. Sub-cellular Electrical Heterogeneity Revealed by Loose Patch Recording Reflects Differential Localization of Sarcolemmal Ion Channels in Intact Rat Hearts

    Directory of Open Access Journals (Sweden)

    Igor V. Kubasov

    2018-02-01

    Full Text Available The cardiac action potential (AP is commonly recoded as an integral signal from isolated myocytes or ensembles of myocytes (with intracellular microelectrodes and extracellular macroelectrodes, respectively. These signals, however, do not provide a direct measure of activity of ion channels and transporters located in two major compartments of a cardiac myocyte: surface sarcolemma and the T-tubule system, which differentially contribute to impulse propagation and excitation-contraction (EC coupling. In the present study we investigated electrical properties of myocytes within perfused intact rat heart employing loose patch recording with narrow-tip (2 μm diameter extracellular electrodes. Using this approach, we demonstrated two distinct types of electric signals with distinct waveforms (single peak and multi-peak AP; AP1 and AP2, respectively during intrinsic pacemaker activity. These two types of waveforms depend on the position of the electrode tip on the myocyte surface. Such heterogeneity of electrical signals was lost when electrodes of larger pipette diameter were used (5 or 10 μm, which indicates that the electric signal was assessed from a region of <5 μm. Importantly, both pharmacological and mathematical simulation based on transverse (T-tubular distribution suggested that while the AP1 and the initial peak of AP2 are predominantly attributable to the fast, inward Na+ current in myocyte's surface sarcolemma, the late components of AP2 are likely representative of currents associated with L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX currents which are predominantly located in T-tubules. Thus, loose patch recording with narrow-tip pipette provides a valuable tool for studying cardiac electric activity on the subcellular level in the intact heart.

  1. A20 Functional Domains Regulate Subcellular Localization and NF-Kappa B Activation

    Science.gov (United States)

    2013-08-15

    endothelial cells or if the effects of A20 are limited strictly to the process of apoptosis. The Ferran group began by taking bovine aortic endothelial...protein (64, 67, 81, 118, 122). Interestingly, A20 is also involved in the regulation of intracellular parasite infection (109, 123). Given the...was supplemented with a combination of heat inactivated 10% fetal bovine serum and 1% penicillin G/streptomycin sulfate/gentamycin sulfate

  2. Two-Photon Irradiation of an Intracellular Singlet Oxygen Photosensitizer: Achieving Localized Sub-Cellular Excitation in Spatially-Resolved Experiments

    DEFF Research Database (Denmark)

    Pedersen, Brian Wett; Breitenbach, Thomas; Redmond, Robert W.

    2010-01-01

    The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defi ned by the dimensions of the laser...

  3. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    Directory of Open Access Journals (Sweden)

    Karunesh Kumar

    Full Text Available 14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3, sorghum (Sb14-3-3 and maize (Zm14-3-3, respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize

  4. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    Science.gov (United States)

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  5. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    Science.gov (United States)

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

  6. Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

    Science.gov (United States)

    Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

    2011-01-01

    NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

  7. Effect of Content of Sulfate Groups in Seaweed Polysaccharides on Antioxidant Activity and Repair Effect of Subcellular Organelles in Injured HK-2 Cells

    Directory of Open Access Journals (Sweden)

    Xiao-Tao Ma

    2017-01-01

    Full Text Available This study aims to investigate the repair effect of subcellular structure injuries of the HK-2 cells of four degraded seaweed polysaccharides (DSPs, namely, the degraded Porphyra yezoensis, Gracilaria lemaneiformis, Sargassum fusiform, and Undaria pinnatifida polysaccharides. The four DSPs have similar molecular weight, but with different content of sulfate groups (i.e., 17.9%, 13.3%, 8.2%, and 5.5%, resp.. The damaged model was established using 2.8 mmol/L oxalate to injure HK-2 cells, and 60 μg/mL of various DSPs was used to repair the damaged cells. With the increase of sulfate group content in DSPs, the scavenging activity of radicals and their reducing power were all improved. Four kinds of DSPs have repair effect on the subcellular organelles of damaged HK-2 cells. After being repaired by DSPs, the release amount of lactate dehydrogenase was decreased, the integrity of cell membrane and lysosome increased, the Δψm increased, the cell of G1 phase arrest was inhibited, the proportion of S phase increased, and cell apoptotic and necrosis rates were significantly reduced. The greater the content of sulfate group is, the stronger is the repair ability of the polysaccharide. These DSPs, particularly the polysaccharide with higher sulfate group content, may be a potential drug for the prevention and cure of kidney stones.

  8. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    Science.gov (United States)

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  9. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

    Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  10. Biosynthesis of platelet activating factor (PAF) via alternate pathways: subcellular distribution of products in HL-60 cells

    International Nuclear Information System (INIS)

    Record, M.; Snyder, F.

    1986-01-01

    Final steps in the biosynthesis of PAF can be catalyzed by two different routes: CDP-choline:1-alkyl-2-acetyl-Gro cholinephosphotransferase [dithiothrietol (DTT)-insensitive] or acetyl-CoA:1-alkyl-2-lyso-GroPCho acetyltransferase. The authors have investigated the conversion of tritium-labeled 1-alkyl-2-acetyl-Gro and 1-alkyl-2-lyso-GroPCho (lyso-PAF) to PAF and other lipid products in HL-60 cells and in subcellular organelles isolated by centrifugation in a Percoll gradient. When cells are incubated with the labeled precursors (2 μM) the total amount of labeled PAF and 1-alkyl-2-acyl-GroPCho formed was similar from both precursors (60 pmol from 1-alkyl-2-acetyl-Gro and 50 pmol from lyso-PAF). However, PAF formed from 1-alkyl-2-acetyl-Gro represented 70% of the total products, whereas with lyso-PAF the major labeled product was 1-alkyl-2-acyl-GroPCho. Formation of PAF from 1-[ 3 H]alkyl-2-acetyl-Gro was linear to at least 30 min at 20 0 C. After a 15-min incubation of this neutral lipid with HL-60 cells, the labeled PAF produced was located exclusively in the plasma membrane fraction as opposed to the label in the 1-alkyl-2-acyl-GroPCho, which was found only in the endoplasmic reticulum; none of the labeled PAF product was released to the media. The authors results suggest PAF might be synthesized by the DTT-insensitive cholinephosphotransferase at the site of the plasma membrane in HL-60 cells

  11. Japan's nuclear PA activity in local governments

    International Nuclear Information System (INIS)

    Fujii, Nobuyuki

    1995-01-01

    This presentation emphasises some points of PA activities, based on the experience of 'cooperation projects for local governments'. Local governments distribute the public information directly to the residents. This is very important because officers of the local government can be the opinion leaders of the region. Local government exist very close to the residents, while the central government is a distant and faceless existence for the local people. It is believed that the local governments play an imperative role in PA activities. In other words, we must further utilize the organizations and functions of the local governments to implement PA activities. In conclusion, three recommendations are offered. Firstly, enough budget and authority should be given to the local governments as far as PA activities in their areas are concerned, and most of such activities should be entrusted to the local governments. Local governments should place more public relations officers, and continue the manpower development. Second, with regard to highly technical or specialized issues which a local governments cannot treat alone, related organizations like JAERO should support their PA activities. Third, such related organizations should also cooperate with local government including assistance in providing know-how, when their public information activities focus on educators, journalists, or the women. These three points should be given due consideration in our cooperation projects for the local governments, and JAERO is doing its best every day

  12. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    Science.gov (United States)

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  13. Glycolytic pathway (GP), kreb's cycle (KC), and hexose monophosphate shunt (HMS) activity in myocardial subcellular fractions exposed to cannabinoids

    Energy Technology Data Exchange (ETDEWEB)

    Watson, A.T.; Manno, B.R.; King, J.W.; Fowler, M.R.; Dempsey, C.A.; Manno, J.E.

    1986-03-05

    Delta-9-tetrahydrocannabinol (..delta../sup 9/-THC), the primary psychoactive component of marihuana, and its active metabolite 11-hydroxy-..delta../sup 9/-tetrahydrocannabinol (11-OH-..delta../sup 9/-THC) have been reported to produce a direct cardiac depressant effect. Studies in isolated perfused rat hearts have indicated a decreased force of contraction (inotropic response) when ..delta../sup 9/-THC or 11-OH-..delta../sup 9/-THC was administered in microgram amounts. The mechanism and site of action have not been explained or correlated with associated metabolic pathways. The purpose of this study was to investigate the effects of cannabinoids on major myocardial energy producing pathways, GP and KC, and a non-energy producing pathway, HMS. Cardiac ventricular tissue from male Sprague-Dawley rats (250-300 g) was excised and homogenized for subcellular fractionation. KC, GP and HMS activity was assayed in the appropriate fractions by measuring /sup 14/CO/sub 2/ generation from /sup 14/C-2-pyruvate, /sup 14/C-6-glucose and /sup 14/C-1-glucose respectively. Duplicate assays (n=8) were performed on tissue exposed to saline (control), empty liposomes (vehicle) and four doses each of ..delta../sup 9/-THC and 11-OH-..delta../sup 9/-THC. Changes in metabolic activity and decreases in cardiac contractile performance may be associated.

  14. Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction.

    Science.gov (United States)

    Marziali, Federico; Bugnon Valdano, Marina; Brunet Avalos, Clarisse; Moriena, Lucía; Cavatorta, Ana Laura; Gardiol, Daniela

    2017-11-23

    Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain-containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions.

  15. Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction

    Directory of Open Access Journals (Sweden)

    Federico Marziali

    2017-11-01

    Full Text Available Human T cell leukemia virus (HTLV-1 Tax (Tax protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1 domain–containing proteins such as Discs large protein 1 (DLG1 which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET, we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC. This study contributes to understand the biological significance of Tax-PDZ interactions.

  16. Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle.

    Science.gov (United States)

    MacPherson, Rebecca E K; Herbst, Eric A F; Reynolds, Erica J; Vandenboom, Rene; Roy, Brian D; Peters, Sandra J

    2012-01-01

    Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation (n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles (P muscles (P = 0.001, r(2) = 0.99) and linearly in stimulated muscles (slope = -0.0023 ± 0.0006, P muscles (P contraction in isolated skeletal muscle.

  17. Localization and regulation of mouse pantothenate kinase 2 [The PanK2 Genes of Mouse and Human Specify Proteins with Distinct Subcellular Locations

    Energy Technology Data Exchange (ETDEWEB)

    Leonardi, Roberta [St. Jude Children' s Research Hospital, Memphis, TN (United States); Zhang, Yong-Mei [St. Jude Children' s Research Hospital, Memphis, TN (United States); Lykidis, Athanasios [DOE Joint Genome Inst., Walnut Creek, CA (United States); Rock, Charles O. [St. Jude Children' s Research Hospital, Memphis, TN (United States); Jackowski, Suzanne [St. Jude Children' s Research Hospital, Memphis, TN (United States)

    2007-09-07

    Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency.

  18. Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA

    Directory of Open Access Journals (Sweden)

    Lacalandra Giovanni M

    2010-06-01

    Full Text Available Abstract Background Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye. Results We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations. Conclusions The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for

  19. Source localization of rhythmic ictal EEG activity

    DEFF Research Database (Denmark)

    Beniczky, Sándor; Lantz, Göran; Rosenzweig, Ivana

    2013-01-01

    Although precise identification of the seizure-onset zone is an essential element of presurgical evaluation, source localization of ictal electroencephalography (EEG) signals has received little attention. The aim of our study was to estimate the accuracy of source localization of rhythmic ictal...... EEG activity using a distributed source model....

  20. Temporal study of acetaminophen (APAP) and S-adenosyl-L-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity

    International Nuclear Information System (INIS)

    Brown, J. Michael; Ball, John G.; Hogsett, Amy; Williams, Tierra; Valentovic, Monica

    2010-01-01

    Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 g) were treated with vehicle (Veh; water 15 ml/kg ip injections), 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered 1 h after APAP injection (SAMe and SAMe + APAP). Hepatic tissue was collected 2, 4, and 6 h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4 h by APAP overdose, but not at 2 or 6 h. APAP depressed mitochondrial SAMe levels at 4 and 6 h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4 h following APAP administration. SAMe administration following APAP (SAMe + APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity.

  1. Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

    Science.gov (United States)

    Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

    2018-01-25

    The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

  2. Rab21, a Novel PS1 Interactor, Regulates γ-Secretase Activity via PS1 Subcellular Distribution.

    Science.gov (United States)

    Sun, Zhenzhen; Xie, Yujie; Chen, Yintong; Yang, Qinghu; Quan, Zhenzhen; Dai, Rongji; Qing, Hong

    2018-05-01

    γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer's disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.

  3. Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

    Science.gov (United States)

    Wang, Xin; Komatsu, Setsuko

    2016-06-30

    Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of

  4. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

    DEFF Research Database (Denmark)

    Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

    1996-01-01

    substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types...... of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV...

  5. A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Roslyn N.; Sanford, James A.; Park, Jea H.; Deatherage, Brooke L.; Champion, Boyd L.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

    2012-06-01

    Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.

  6. Yersinia pestis insecticidal-like toxin complex (Tc family proteins: characterization of expression, subcellular localization, and potential role in infection of the flea vector

    Directory of Open Access Journals (Sweden)

    Spinner Justin L

    2012-12-01

    Full Text Available Abstract Background Toxin complex (Tc family proteins were first identified as insecticidal toxins in Photorhabdus luminescens and have since been found in a wide range of bacteria. The genome of Yersinia pestis, the causative agent of bubonic plague, contains a locus that encodes the Tc protein homologues YitA, YitB, YitC, and YipA and YipB. Previous microarray data indicate that the Tc genes are highly upregulated by Y. pestis while in the flea vector; however, their role in the infection of fleas and pathogenesis in the mammalian host is unclear. Results We show that the Tc proteins YitA and YipA are highly produced by Y. pestis while in the flea but not during growth in brain heart infusion (BHI broth at the same temperature. Over-production of the LysR-type regulator YitR from an exogenous plasmid increased YitA and YipA synthesis in broth culture. The increase in production of YitA and YipA correlated with the yitR copy number and was temperature-dependent. Although highly synthesized in fleas, deletion of the Tc proteins did not alter survival of Y. pestis in the flea or prevent blockage of the proventriculus. Furthermore, YipA was found to undergo post-translational processing and YipA and YitA are localized to the outer membrane of Y. pestis. YitA was also detected by immunofluorescence microscopy on the surface of Y. pestis. Both YitA and YipA are produced maximally at low temperature but persist for several hours after transfer to 37°C. Conclusions Y. pestis Tc proteins are highly expressed in the flea but are not essential for Y. pestis to stably infect or produce a transmissible infection in the flea. However, YitA and YipA localize to the outer membrane and YitA is exposed on the surface, indicating that at least YitA is present on the surface when Y. pestis is transmitted into the mammalian host from the flea.

  7. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    Science.gov (United States)

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1. © 2011 Blackwell Publishing Ltd.

  8. Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

    Science.gov (United States)

    Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

    2006-01-01

    We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

  9. Go local: morality and international activism

    Directory of Open Access Journals (Sweden)

    Aleksandar Jokic

    2013-03-01

    Full Text Available A step towards constructing an ethics of international activism is proposed by formulating a series of constraints on what would constitute morally permissible agency in the context that involves delivering services abroad, directly or indirectly. Perhaps surprisingly, in this effort the author makes use of the concept of ‘force multiplier’. This idea and its official applications have explanatory importance in considering the correlation between the post-Cold War phenomenal growth in the number of international non-governmental organizations and the emergence of the US as the sole, unchallenged superpower. Four moral constraints useful for morally assessing international activism are formulated and defended. The final outcome is an argument in favor of an overarching duty for any activist-minded Westerner to go local, while developing nations are urged to closely regulate, even criminalize, activities by international activists and ‘human rights organizations’ on their territory when not in solidarity or in support of local movements. The position defended, urging the normative primacy of local over international activism, also finds support in Immanuel Kant's Third Definitive Article for A Perpetual Peace.

  10. EXPENSES FOR ECONOMIC ACTIVITIES FROM LOCAL BUDGETS

    Directory of Open Access Journals (Sweden)

    CRISTINEL ICHIM

    2015-04-01

    Full Text Available In the present article we propose to analyze and deepen significant categories of costs funded from the local budgets, namely the expenditure for economic activities. Our scientific approach begins with determining the place occupied by such expenses in local public expenditure by specifying their content and role. The center of gravity of the study is to treat and deepen the three subgroups of expenses that we consider representative: "The expenses for production, transportation, distribution and supply of heat in a centralized system", "Transport Costs" and Expenditure for agriculture and forestry ". The reaserch is based on the quantitative analysis of the expenses for economic actions, in local budgets, based on the existing data from the Statistical Yearbook of Romania, and highlights the structure of this type of expenses as well as the place they hold in the expediture of local budgets.The study includes an analysis of the dynamics of the share held by economic costs within total expenses from local budgets. From the reaserch carried out, it is shown that the evolution and structure of the expenditures for economic actions from local budgets is determined by the action of certain economical and social factors that vary from one administrative teritorial unit to another: the ray of economical develpoment of the administrative ter itorial unit, urbanization, the number and social structure of the population. The reaserch shows that in the field of expenses for economic actions, the largest share is held by expenditures for transportation (almost 80%, far away from the expenses for fuel and energy (13,66%. During the 1999-2013 the dynamic of expenses for economical actions in the total of expenditures of local budgets, is sinusoidal due to the intervention of certain legislative changes.

  11. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  12. Protein kinase A activation enhances β-catenin transcriptional activity through nuclear localization to PML bodies.

    Directory of Open Access Journals (Sweden)

    Mei Zhang

    Full Text Available The Protein Kinase A (PKA and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.

  13. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Md. Imtiaz Uddin

    2012-02-14

    Feb 14, 2012 ... important factors, the limitation of gas diffusion under water and reduced irradiance, which impair photo- synthesis ... functions as cell expansion, universal stress protein, and putative ..... They catalyze the exchange of Na+.

  14. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G

    1990-01-01

    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  15. Active surveillance for localized prostate cancer

    DEFF Research Database (Denmark)

    Thomsen, Frederik B; Berg, Kasper D; Røder, M Andreas

    2015-01-01

    and costs of AS in patients with localized PCa. MATERIALS AND METHODS: In total, 317 PCa patients were followed in a prospective, single-arm AS cohort. The primary outcomes were number of patient contacts, prostate-specific antigen (PSA) tests, biopsies, hospital admissions due to biopsy complications......OBJECTIVE: Evidence supports active surveillance (AS) as a means to reduce overtreatment of low-risk prostate cancer (PCa). The consequences of close and long-standing follow-up with regard to outpatient visits, tests and repeated biopsies are widely unknown. This study investigated the trajectory...

  16. Active surveillance for localized prostate cancer

    DEFF Research Database (Denmark)

    Thostrup, Mathias; Thomsen, Frederik B; Iversen, Peter

    2018-01-01

    risk of biochemical recurrence were investigated and compared in men with very low-risk, low-risk and intermediate-risk PCa in the cohort. MATERIALS AND METHODS: In total, 451 men were followed on AS and monitored with prostate-specific antigen (PSA) tests, digital rectal examinations and rebiopsies......OBJECTIVE: The purpose of active surveillance (AS) is to reduce overtreatment of men with localized prostate cancer (PCa) without compromising survival. The objective of this study was to update a large Scandinavian single-center AS cohort. Furthermore, the use of curative treatment and subsequent...

  17. Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

    Science.gov (United States)

    Taupin, P; Ben-Ari, Y; Roisin, M P

    1994-05-02

    Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

  18. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Directory of Open Access Journals (Sweden)

    Qian-Quan Sun

    Full Text Available To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2 is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites. We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  19. Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

    Science.gov (United States)

    Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

    2013-06-15

    Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. The subcellular compartmentalization of arginine metabolizing enzymes and their role in endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Feng eChen

    2013-07-01

    Full Text Available The endothelial production of nitric oxide (NO mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle proliferation and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS through the conversion of its substrate, L-arginine to L-citrulline. Reduced access to L-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2 metabolize L-arginine to generate L-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of L-arginine. Indeed, supplemental L-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis suggesting additional mechanisms. The compartmentalization of intracellular L-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of L-arginine. Indeed the subcellular location of L-arginine metabolizing enzymes plays important functional roles. In endothelial cells, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localtion. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, ASL, co-localize with eNOS and facilitate NO release. This review highlights the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease.

  1. Plant sterol metabolism. Δ7-Sterol-C5-Desaturase (STE1/DWARF7), Δ5,7-Sterol-Δ7-Reductase (DWARF5) and Δ24-Sterol-Δ24-Reductase (DIMINUTO/DWARF1) show multiple subcellular localizations in Arabidopsis thaliana (Heynh) L

    DEFF Research Database (Denmark)

    Silvestro, Daniele; Andersen, Tonni Grube; Schaller, Hubert

    2013-01-01

    in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both ¿(5,7)-sterol-¿(7)-reductase and ¿(24)-sterol-¿(24)-reductase are in addition localized to the plasma membrane, whereas ¿(7)-sterol-C(5)-desaturase......Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown...... to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map...

  2. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  3. Localization of cortical areas activated by thinking.

    Science.gov (United States)

    Roland, P E; Friberg, L

    1985-05-01

    These experiments were undertaken to demonstrate that pure mental activity, thinking, increases the cerebral blood flow and that different types of thinking increase the regional cerebral blood flow (rCBF) in different cortical areas. As a first approach, thinking was defined as brain work in the form of operations on internal information, done by an awake subject. The rCBF was measured in 254 cortical regions in 11 subjects with the intracarotid 133Xe injection technique. In normal man, changes in the regional cortical metabolic rate of O2 leads to proportional changes in rCBF. One control study was taken with the subjects at rest. Then the rCBF was measured during three different simple algorithm tasks, each consisting of retrieval of a specific memory followed by a simple operation on the retrieved information. Once started, the information processing went on in the brain without any communication with the outside world. In 50-3 thinking, the subjects started with 50 and then, in their minds only, continuously subtracted 3 from the result. In jingle thinking the subjects internally jumped every second word in a nine-word circular jingle. In route-finding thinking the subjects imagined that they started at their front door and then walked alternatively to the left or the right each time they reached a corner. The rCBF increased only in homotypical cortical areas during thinking. The areas in the superior prefrontal cortex increased their rCBF equivalently during the three types of thinking. In the remaining parts of the prefrontal cortex there were multifocal increases of rCBF. The localizations and intensities of these rCBF increases depended on the type of internal operation occurring. The rCBF increased bilaterally in the angular cortex during 50-3 thinking. The rCBF increased in the right midtemporal cortex exclusively during jingle thinking. The intermediate and remote visual association areas, the superior occipital, posterior inferior temporal, and

  4. Plasma effects on subcellular structures

    International Nuclear Information System (INIS)

    Gweon, Bomi; Kim, Dan Bee; Jung, Heesoo; Choe, Wonho; Kim, Daeyeon; Shin, Jennifer H.

    2010-01-01

    Atmospheric pressure helium plasma treated human hepatocytes exhibit distinctive zones of necrotic and live cells separated by a void. We propose that plasma induced necrosis is attributed to plasma species such as oxygen radicals, charged particles, metastables and/or severe disruption of charged cytoskeletal proteins. Interestingly, uncharged cytoskeletal intermediate filaments are only minimally disturbed by plasma, elucidating the possibility of plasma induced electrostatic effects selectively destroying charged proteins. These bona fide plasma effects, which inflict alterations in specific subcellular structures leading to necrosis and cellular detachment, were not observed by application of helium flow or electric field alone.

  5. Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites.

    Science.gov (United States)

    Dodge, Matthew A; Waller, Ross F; Chow, Larry M C; Zaman, Muhammad M; Cotton, Leanne M; McConville, Malcolm J; Wirth, Dyann F

    2004-03-01

    Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

  6. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    International Nuclear Information System (INIS)

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-01-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification

  7. In vitro antitumour activity, safety testing and subcellular distribution of two poly[oxyethylene(aminophosphonate-co-H-phosphonate]s in Ehrlich ascites carcinoma and BALB/c 3T3 cell culture systems

    Directory of Open Access Journals (Sweden)

    Ani Georgieva

    2016-01-01

    Full Text Available Two polyphosphoesters containing anthracene-derived aminophosphonate and hydrophilic H-phosphonate repeating units, poly[oxyethylene(aminophosphonate-co-H-phosphonate]s (1 and 2, were tested for the in vitro antitumour activity on cell cultures derived from ascitic form of Ehrlich mammary adenocarcinoma by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT-dye reduction assay. The in vitro safety testing of the copolymers was performed by BALB/c 3T3 neutral red uptake assay. A study on their uptake and subcellular distribution in non-tumourigenic and tumour cells was performed by means of fluorescence microscopy. Both copolymers showed significant antitumour activity towards Ehrlich ascites carcinoma (EAC cells. However, the in vitro safety testing revealed significant toxicity of polymer 2 to BALB/c 3T3 mouse embryo cells. In contrast, polymer 1 showed complete absence of cytotoxicity to BALB/c 3T3 cells. The fluorescent studies showed that the substances were diffusely distributed in the cytoplasm in both cell culture systems. As opposed to BALB/c 3T3 cells, in EAC cells, intense fluorescent signal was observed in the nuclei and in the perinuclear region. The tested polyphosphoesters are expected to act under physiological conditions as prodrugs of aminophosphonates.

  8. Evaluation of local anesthetic and antipyretic activities of Cinchona ...

    African Journals Online (AJOL)

    Purpose: To evaluate the local anesthetic and antipyretic activities of an aqueous extract of Cinchona officinalis (C. officinalis) in experimental animal models. Methods: Various doses of the aqueous extract was tested for its local anesthetic activity in guinea pigs and frogs using intracutaneous and plexus anesthesia, ...

  9. Multi-Label Learning via Random Label Selection for Protein Subcellular Multi-Locations Prediction.

    Science.gov (United States)

    Wang, Xiao; Li, Guo-Zheng

    2013-03-12

    Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multi-location proteins to multiple proteins with single location, which doesn't take correlations among different subcellular locations into account. In this paper, a novel method named RALS (multi-label learning via RAndom Label Selection), is proposed to learn from multi-location proteins in an effective and efficient way. Through five-fold cross validation test on a benchmark dataset, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark datasets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multi-locations of proteins. The prediction web server is available at http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

  10. The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

    Science.gov (United States)

    An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

    2008-09-05

    Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

  11. Active surveillance for clinically localized prostate cancer

    DEFF Research Database (Denmark)

    Thomsen, Frederik B; Brasso, Klaus; Klotz, Laurence H

    2014-01-01

    Active surveillance (AS) has been introduced as an observational strategy to delay or avoid curative treatment without compromising long-term cancer-specific survival. The 10 studies included in this review, published between 2008 and 2013, generally agreed upon patients selection for the AS stra......Active surveillance (AS) has been introduced as an observational strategy to delay or avoid curative treatment without compromising long-term cancer-specific survival. The 10 studies included in this review, published between 2008 and 2013, generally agreed upon patients selection...

  12. Localization of cortical areas activated by thinking

    DEFF Research Database (Denmark)

    Roland, P E; Friberg, L

    1985-01-01

    midtemporal cortex exclusively during jingle thinking. The intermediate and remote visual association areas, the superior occipital, posterior inferior temporal, and posterior superior parietal cortex, increased their rCBF exclusively during route-finding thinking. We observed no decreases in rCBF. All r......These experiments were undertaken to demonstrate that pure mental activity, thinking, increases the cerebral blood flow and that different types of thinking increase the regional cerebral blood flow (rCBF) in different cortical areas. As a first approach, thinking was defined as brain work...... communication with the outside world. In 50-3 thinking, the subjects started with 50 and then, in their minds only, continuously subtracted 3 from the result. In jingle thinking the subjects internally jumped every second word in a nine-word circular jingle. In route-finding thinking the subjects imagined...

  13. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Science.gov (United States)

    2012-01-01

    Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

  14. Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

    Science.gov (United States)

    Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

    2004-07-01

    Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

  15. Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

    Science.gov (United States)

    Okubo, Shinya; Uto, Takuhiro; Goto, Aya; Tanaka, Hiroyuki; Nishioku, Tsuyoshi; Yamada, Katsushi; Shoyama, Yukihiro

    2017-01-01

    Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

  16. Effect of. gamma. radiation in relatively low dose on the activity of glutaminase in subcellular fraction of brain and liver cells. [Rats

    Energy Technology Data Exchange (ETDEWEB)

    Tkach, V M

    1973-01-01

    The effect of ..gamma..-irradiation at a dose of 40 rads was studied on the exchange of glutamine in rats. It has been shown that the irradiation leads to a significant lowering of the activity of glutaminamidohydrolase (I) in brain mitochondria and in the liver after 1, 3, 7, 15, and 30 days post exposure. In the fractions containing nuclei, fraction of myofibrillae and connective tissue, a slow down of the deamidation of glutamine also takes place, and only after 7 days the ammonium separation from glutamine increases and returns to normal. At the 15 and 30 days a second wave of the lower rate of the activity of I takes place. The type of the changes of I is the same in both organs, but in the liver it is expressed to a lesser degree. (JPRS)

  17. Activity and immunohistochemical localization of porphobilinogen deaminase in rat tissues

    DEFF Research Database (Denmark)

    Jørgensen, P E; Erlandsen, E J; Poulsen, Steen Seier

    2000-01-01

    the activity and the immunohistochemical localization of PBGD in the following tissues of wistar female rats: brain, heart, submandibular gland, liver, kidney, pancreas, ovary, stomach, duodenum, jejunum, ileum, colon and musculature. The PBGD activity varied considerably among the tissues. It was highest...

  18. Activity Recognition and Localization on a Truck Parking Lot

    NARCIS (Netherlands)

    Andersson, M.; Patino, L.; Burghouts, G.J.; Flizikowski, A.; Evans, M.; Gustafsson, D.; Petersson, H.; Schutte, K.; Ferryman, J.

    2013-01-01

    In this paper we present a set of activity recognition and localization algorithms that together assemble a large amount of information about activities on a parking lot. The aim is to detect and recognize events that may pose a threat to truck drivers and trucks. The algorithms perform zone-based

  19. Quantification of local morphodynamics and local GTPase activity by edge evolution tracking.

    Directory of Open Access Journals (Sweden)

    Yuki Tsukada

    2008-11-01

    Full Text Available Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function.

  20. Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

    Science.gov (United States)

    Zurbuchen, Mark A.; Lake, Michael P.; Kohan, Sirus A.; Leung, Belinda; Bouchard, Louis-S.

    2013-01-01

    There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

  1. Subcellular sites for bacterial protein export

    NARCIS (Netherlands)

    Campo, Nathalie; Tjalsma, Harold; Buist, Girbe; Stepniak, Dariusz; Meijer, Michel; Veenhuis, Marten; Westermann, Martin; Müller, Jörg P.; Bron, Sierd; Kok, Jan; Kuipers, Oscar P.; Jongbloed, Jan D.H.

    2004-01-01

    Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the

  2. Subcellular sites for bacterial protein export.

    NARCIS (Netherlands)

    Campo, N.; Tjalsma, H.; Buist, G.; Stepniak, D.; Meijer, M.; Veenhuis, M.; Westermann, M.; Muller, J.P.; Bron, S.; Kok, J.; Kuipers, O.P.; Jongbloed, J.D.

    2004-01-01

    Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the

  3. Localization of urease activity in ureaplasma urealyticum cells

    International Nuclear Information System (INIS)

    Vinther, O.

    1976-01-01

    Measurements of the urease activity of various cell fractions of U. urealyticum showed that this activity was confined to the soluble fraction of the cytoplasm. An attempt was made to devise a method for electron microscopic detection of the sites of urease activity based on precipitation of electron-dense MnO 2 at the alkaline pH created by the hydrolysis of urea. The results obtained supported the previous results indicating a cytoplasmatic localization of the urease activity in the cells. Helical ribosome patterns were observed when glutaraldehyde-fixed cells were treated with cytochemical test solutions. (author)

  4. Localization of urease activity in Ureaplasma urealyticum cells

    Energy Technology Data Exchange (ETDEWEB)

    Vinther, O [Aarhus Univ. (Denmark)

    1976-01-01

    Measurements of the urease activity of various cell fractions of U. urealyticum showed that this activity was confined to the soluble fraction of the cytoplasm. An attempt was made to devise a method for electron microscopic detection of the sites of urease activity based on precipitation of electron-dense MnO/sub 2/ at the alkaline pH created by the hydrolysis of urea. The results obtained supported the previous results indicating a cytoplasmatic localization of the urease activity in the cells. Helical ribosome patterns were observed when glutaraldehyde-fixed cells were treated with cytochemical test solutions.

  5. 21 CFR 346.10 - Local anesthetic active ingredients.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Local anesthetic active ingredients. 346.10 Section 346.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... ingredient: (a) Benzocaine 5 to 20 percent. (b) Benzyl alcohol 1 to 4 percent. (c) Dibucaine 0.25 to 1...

  6. Antimicrobial Activity of Sabulun Salo a Local Traditional Medicated ...

    African Journals Online (AJOL)

    The antimicrobial activity of Sabulun salo; a local traditional medicated soap widely used by different tribes in Nigeria such as Hausa, Yoruba and Nupe against skin infections was examined against some clinical isolates of pathogenic microorganisms (Staphylococcus aureus, Escherichia coli and Candida albicans) using ...

  7. Antihypertensive Activity of Residue From “Gebto Arekei”. Locally ...

    African Journals Online (AJOL)

    Antihypertensive Activity of Residue From “Gebto Arekei”. Locally Distilled Medicinal Spirit From a Brew Containing Lupinus albus Seeds in Renovascular Hypertensive Guines-Pigs. Cherinet Ambaye, Tesfaye Tolessa, Abebe Abera, Hassen Taha Sherief, Dawit Abebe, Kelbessa Urga ...

  8. Finer discrimination of brain activation with local multivariate distance

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The organization of human brain function is diverse on different spatial scales.Various cognitive states are alwavs represented as distinct activity patterns across the specific brain region on fine scales.Conventional univariate analysis of functional MRI data seeks to determine how a particular cognitive state is encoded in brain activity by analyzing each voxel separately without considering the fine-scale patterns information contained in the local brain regions.In this paper,a local multivariate distance mapping(LMDM)technique is proposed to detect the brain activation and to map the fine-scale brain activity patterns.LMDM directly represents the local brain activity with the patterns across multiple voxels rather than individual voxels,and it employs the multivariate distance between different patterns to discriminate the brain state on fine scales.Experiments with simulated and real fMRI data demonstrate that LMDM technique can dramatically increase the sensitivity of the detection for the fine-scale brain activity pettems which contain the subtle information of the experimental conditions.

  9. Teollisuuden Voima Oy - Industrial Power Company Ltd. Local information activity

    International Nuclear Information System (INIS)

    Engros, Taina

    1989-01-01

    There are two nuclear power producers in Finland - the state-owned power company Imatran Voima Oy which operates two 440 MW Soviet-made PWR units in southern Finland, east of Helsinki and the Teollisuuden Voima Oy - Industrial Power Company Ltd, or TVO, owned by Finnish industrial companies. TVO operates two 710 MW ABB ATOM BWR units producing about one fifth of the country's electricity consumption. Operating experiences are extremely good from all Finnish nuclear power plants. The Finns' attitude towards nuclear power has changed into a positive direction in recent times. This can probably be noted as an international trend now that the Chernobyl accident is becoming an incident of the past. The Finnish citizens and politicians are facing two questions; first, what is their attitude towards nuclear power as a source of energy, in other words, do they approve of the plants currently in operation. The second question is how they stand on the building of new plants. It is probably another universal phenomenon that the attitudes of people living in the vicinity of nuclear power plants are less critical than the attitudes of those living farther away. This does not, by any means, result in local information activity being easier or less important than nationwide information activity. On the contrary, local decision-makers, local media and inhabitants are those who can, and through whom we can, influence also wider circles. The Nuclear Energy Act, which became effective in Finland last year, defines that the final decision on whether nuclear power plants can be built inside a municipality, is made at local level. As far as TVO is concerned one factor making local information activity easier is the small size of the locality. The difficulty TVO has to face is the people's suspicion of information activity. All information is considered propaganda, regardless of its form, and only negative news are considered information. Also, a large proportion of people are passive

  10. Teollisuuden Voima Oy - Industrial Power Company Ltd. Local information activity

    Energy Technology Data Exchange (ETDEWEB)

    Engros, Taina [Department of Information, TVO, Olkiluoto FIN-27160 (Finland)

    1989-07-01

    There are two nuclear power producers in Finland - the state-owned power company Imatran Voima Oy which operates two 440 MW Soviet-made PWR units in southern Finland, east of Helsinki and the Teollisuuden Voima Oy - Industrial Power Company Ltd, or TVO, owned by Finnish industrial companies. TVO operates two 710 MW ABB ATOM BWR units producing about one fifth of the country's electricity consumption. Operating experiences are extremely good from all Finnish nuclear power plants. The Finns' attitude towards nuclear power has changed into a positive direction in recent times. This can probably be noted as an international trend now that the Chernobyl accident is becoming an incident of the past. The Finnish citizens and politicians are facing two questions; first, what is their attitude towards nuclear power as a source of energy, in other words, do they approve of the plants currently in operation. The second question is how they stand on the building of new plants. It is probably another universal phenomenon that the attitudes of people living in the vicinity of nuclear power plants are less critical than the attitudes of those living farther away. This does not, by any means, result in local information activity being easier or less important than nationwide information activity. On the contrary, local decision-makers, local media and inhabitants are those who can, and through whom we can, influence also wider circles. The Nuclear Energy Act, which became effective in Finland last year, defines that the final decision on whether nuclear power plants can be built inside a municipality, is made at local level. As far as TVO is concerned one factor making local information activity easier is the small size of the locality. The difficulty TVO has to face is the people's suspicion of information activity. All information is considered propaganda, regardless of its form, and only negative news are considered information. Also, a large proportion of people are passive

  11. Differential subcellular membrane recruitment of Src may specify its downstream signalling

    International Nuclear Information System (INIS)

    Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah; D'Auria, Ludovic; Van Der Smissen, Patrick; Platek, Anna; Mettlen, Marcel; Caplanusi, Adrian; Hove, Marie-France van den; Tyteca, Donatienne; Courtoy, Pierre J.

    2008-01-01

    Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined

  12. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Directory of Open Access Journals (Sweden)

    Violeta Calle-Guisado

    2017-01-01

    Full Text Available AMP-activated kinase (AMPK, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work′s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS in the presence or absence of the AMPK inhibitor compound C (CC. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  13. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2017-01-01

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

  14. Reinforcement active learning in the vibrissae system: optimal object localization.

    Science.gov (United States)

    Gordon, Goren; Dorfman, Nimrod; Ahissar, Ehud

    2013-01-01

    Rats move their whiskers to acquire information about their environment. It has been observed that they palpate novel objects and objects they are required to localize in space. We analyze whisker-based object localization using two complementary paradigms, namely, active learning and intrinsic-reward reinforcement learning. Active learning algorithms select the next training samples according to the hypothesized solution in order to better discriminate between correct and incorrect labels. Intrinsic-reward reinforcement learning uses prediction errors as the reward to an actor-critic design, such that behavior converges to the one that optimizes the learning process. We show that in the context of object localization, the two paradigms result in palpation whisking as their respective optimal solution. These results suggest that rats may employ principles of active learning and/or intrinsic reward in tactile exploration and can guide future research to seek the underlying neuronal mechanisms that implement them. Furthermore, these paradigms are easily transferable to biomimetic whisker-based artificial sensors and can improve the active exploration of their environment. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Subcellular Nanoparticle Distribution from Light Transmission Spectroscopy

    Science.gov (United States)

    Deatsch, Alison; Sun, Nan; Johnson, Jeffrey; Stack, Sharon; Tanner, Carol; Ruggiero, Steven

    We have measured the particle-size distribution (PSD) of subcellular structures in plant and animal cells. We have employed a new technique developed by our group, Light Transmission Spectroscopy-combined with cell fractionation-to accurately measure PSDs over a wide size range: from 10 nm to 3000nm, which includes objects from the size of individual proteins to organelles. To date our experiments have included cultured human oral cells and spinach cells. These results show a power-law dependence of particle density with particle diameter, implying a universality of the packing distribution. We discuss modeling the cell as a self-similar (fractal) body comprised of spheres on all size scales. This goal of this work is to obtain a better understanding of the fundamental nature of particle packing within cells in order to enrich our knowledge of the structure, function, and interactions of sub-cellular nanostructures across cell types.

  16. Drug Trafficking Organizations and Local Economic Activity in Mexico.

    Science.gov (United States)

    González, Felipe

    2015-01-01

    Little is known about the relationship between illegal firms and local economic activity. In this paper I study changes in satellite night lights across Mexican municipalities after the arrival of large drug trafficking organizations in the period 2000-2010. After accounting for state trends and differences in political regimes, results indicate no significant change in night lights after the arrival of these illegal firms. Estimated coefficients are precise, robust, and similar across different drug trafficking organizations.

  17. Pseudomonas oleovorans strain KBPF-004 culture supernatants reduced seed transmission of Cucumber green mottle mosaic virus and Pepper mild mottle virus, and remodeled aggregation of 126 kDa and subcellular localization......

    Science.gov (United States)

    Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts from a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against pl...

  18. Local Helioseismology of Emerging Active Regions: A Case Study

    Science.gov (United States)

    Kosovichev, Alexander G.; Zhao, Junwei; Ilonidis, Stathis

    2018-04-01

    Local helioseismology provides a unique opportunity to investigate the subsurface structure and dynamics of active regions and their effect on the large-scale flows and global circulation of the Sun. We use measurements of plasma flows in the upper convection zone, provided by the Time-Distance Helioseismology Pipeline developed for analysis of solar oscillation data obtained by Helioseismic and Magnetic Imager (HMI) on Solar Dynamics Observatory (SDO), to investigate the subsurface dynamics of emerging active region NOAA 11726. The active region emergence was detected in deep layers of the convection zone about 12 hours before the first bipolar magnetic structure appeared on the surface, and 2 days before the emergence of most of the magnetic flux. The speed of emergence determined by tracking the flow divergence with depth is about 1.4 km/s, very close to the emergence speed in the deep layers. As the emerging magnetic flux becomes concentrated in sunspots local converging flows are observed beneath the forming sunspots. These flows are most prominent in the depth range 1-3 Mm, and remain converging after the formation process is completed. On the larger scale converging flows around active region appear as a diversion of the zonal shearing flows towards the active region, accompanied by formation of a large-scale vortex structure. This process occurs when a substantial amount of the magnetic flux emerged on the surface, and the converging flow pattern remains stable during the following evolution of the active region. The Carrington synoptic flow maps show that the large-scale subsurface inflows are typical for active regions. In the deeper layers (10-13 Mm) the flows become diverging, and surprisingly strong beneath some active regions. In addition, the synoptic maps reveal a complex evolving pattern of large-scale flows on the scale much larger than supergranulation

  19. Cellular and subcellular distribution of BSH in human glioblastoma multiforme

    International Nuclear Information System (INIS)

    Neumann, M.; Gabel, D.

    2000-01-01

    The cellular and subcellular distribution of mercaptoundecahydrododecaborate (BSH) in seven glioblastoma multiforme tissue sections of six patients having received BSH prior to surgery was investigated by light, fluorescence and electron microscopy. With use of specific antibodies against BSH its localization could be found in tissue sections predominantly (approx. 90%) in the cytoplasm of GFAP-positive cells of all but one patient. The latter was significantly younger (33 years in contrast of 46-71 (mean 60) years). In none of the tissue sections BSH could be found to a significant amount in the cell nuclei. In contrast, electron microscopy studies show BSH as well associated with the cell membrane as with the chromatin in the nucleus. (author)

  20. AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Jarrod S Johnson

    2011-05-01

    Full Text Available Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR, we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus

  1. LocTree3 prediction of localization

    DEFF Research Database (Denmark)

    Goldberg, T.; Hecht, M.; Hamp, T.

    2014-01-01

    The prediction of protein sub-cellular localization is an important step toward elucidating protein function. For each query protein sequence, LocTree2 applies machine learning (profile kernel SVM) to predict the native sub-cellular localization in 18 classes for eukaryotes, in six for bacteria a...

  2. Local seismic activity monitored at King Sejong Station, Antarctica

    OpenAIRE

    Lee,Duk Kee; Kim,Yea Dong; Nam,Sang Heon; Jin,Young Keun

    1998-01-01

    Source location estimation from single station earthquake data collected at King Sejong Station (62°13'3l"N, 58°47'07"W) from 1995 to 1996 provides seismic activity around King Sejong Station. Analysis of local events, less than 1.5°in angular epicentral distance, finds epicenters located near the Shackleton Fracture Zone, the South Shetland Platform, Deception Island, and North Bransfield Basin. Estimated magnitudes range from 2.2 to 4.5 on the Richter scale, averaging 4.0 in North Bransfiel...

  3. Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

    Science.gov (United States)

    Peng, ChiehFu Jeff; Wikramanayake, Athula H.

    2013-01-01

    Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved

  4. Subcellular distribution of curium in beagle liver

    International Nuclear Information System (INIS)

    Bruenger, F.W.; Grube, B.J.; Atherton, D.R.; Taylor, G.N.; Stevens, W.

    1976-01-01

    The subcellular distribution of curium ( 243 244 Cm) was studied in canine liver from 2 hr to 47 days after injection of 3 μCi 243 244 Cm/kg of body weight. The pattern of distribution for Cm was similar to other trivalent actinide elements studied previously (Am, Cf). Initially (2 hr), most of the nuclide was found in the cytosol and at least 90 percent was protein bound. About 70 percent of the Cm was bound to ferritin, approximately 5 percent was associated with a protein of MW approximately 200,000, and approximately 25 percent was found in the low-molecular-weight region (approximately 5000). The decrease in the Cm content of cytosol, nuclei, and microsomes coincided with an increase in the amount associated with mitochondria and lysosomes. The concentration of the Cm in the mitochondrial fraction was higher than it was in the lysosomal fraction at each time studied. In the mitochondrial fraction approximately 30 percent of the Cm was bound to membranous or granular material, and 70 percent was found in the soluble fraction. The Cm concentration initially associated with cell nuclei was high but had diminished to 20 percent of the 2 hr concentration by 20 days post injection (PI). The subcellular distribution of Cm in the liver of a dog which had received the same dose and was terminated because of severe liver damage was studied at 384 days PI. The liver weighed 130 g and contained approximately 30 percent of the injected Cm. In contrast, a normal liver weighs 280 g and at 2 hr PI contains approximately 40 percent of the injected dose. The subcellular distribution of Cm in this severely damaged liver differed from the pattern observed at earlier times after injection. The relative concentration of Cm in the cytosol was doubled; it was higher in the nuclei-debris fraction; and it was lower in the mitochondrial and lysosomal fractions when compared to earlier times

  5. Spherical loudspeaker array for local active control of sound.

    Science.gov (United States)

    Rafaely, Boaz

    2009-05-01

    Active control of sound has been employed to reduce noise levels around listeners' head using destructive interference from noise-canceling sound sources. Recently, spherical loudspeaker arrays have been studied as multiple-channel sound sources, capable of generating sound fields with high complexity. In this paper, the potential use of a spherical loudspeaker array for local active control of sound is investigated. A theoretical analysis of the primary and secondary sound fields around a spherical sound source reveals that the natural quiet zones for the spherical source have a shell-shape. Using numerical optimization, quiet zones with other shapes are designed, showing potential for quiet zones with extents that are significantly larger than the well-known limit of a tenth of a wavelength for monopole sources. The paper presents several simulation examples showing quiet zones in various configurations.

  6. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

  7. Accurate prediction of subcellular location of apoptosis proteins combining Chou’s PseAAC and PsePSSM based on wavelet denoising

    Science.gov (United States)

    Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

    2017-01-01

    Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research. PMID:29296195

  8. Dynamic full field OCT: metabolic contrast at subcellular level (Conference Presentation)

    Science.gov (United States)

    Apelian, Clement; Harms, Fabrice; Thouvenin, Olivier; Boccara, Claude A.

    2016-03-01

    Cells shape or density is an important marker of tissues pathology. However, individual cells are difficult to observe in thick tissues frequently presenting highly scattering structures such as collagen fibers. Endogenous techniques struggle to image cells in these conditions. Moreover, exogenous contrast agents like dyes, fluorophores or nanoparticles cannot always be used, especially if non-invasive imaging is required. Scatterers motion happening down to the millisecond scale, much faster than the still and highly scattering structures (global motion of the tissue), allowed us to develop a new approach based on the time dependence of the FF-OCT signals. This method reveals hidden cells after a spatiotemporal analysis based on singular value decomposition and wavelet analysis concepts. It does also give us access to local dynamics of imaged scatterers. This dynamic information is linked with the local metabolic activity that drives these scatterers. Our technique can explore subcellular scales with micrometric resolution and dynamics ranging from the millisecond to seconds. By this mean we studied a wide range of tissues, animal and human in both normal and pathological conditions (cancer, ischemia, osmotic shock…) in different organs such as liver, kidney, and brain among others. Different cells, undetectable with FF-OCT, were identified (erythrocytes, hepatocytes…). Different scatterers clusters express different characteristic times and thus can be related to different mechanisms that we identify with metabolic functions. We are confident that the D-FFOCT, by accessing to a new spatiotemporal metabolic contrast, will be a leading technique on tissue imaging and for better medical diagnosis.

  9. Design parameters for measurements of local catalytic activity on surfaces

    DEFF Research Database (Denmark)

    Johansson, Martin; Johannessen, Tue; Jørgensen, Jan Hoffmann

    2006-01-01

    Computational fluid dynamics in combination with experiments is used to characterize a gas sampling device for measurements of the local catalytic activity on surfaces. The device basically consists of a quartz capillary mounted concentrically inside an aluminum tube. Reactant gas is blown toward......, the limits of the range in reaction rate, which can be Studied are estimated. (c) 2005 Elsevier B.V. All rights reserved.......Computational fluid dynamics in combination with experiments is used to characterize a gas sampling device for measurements of the local catalytic activity on surfaces. The device basically consists of a quartz capillary mounted concentrically inside an aluminum tube. Reactant gas is blown toward...... limit for the lateral resolution of the measurement, and that a flow rate of the order of 240 (ml/min)(n) is sufficient to achieve this resolution. The sensitivity is reasonable also with high flow rates, due to the presence of a pocket of stagnant gas under the tip of the capillary. Furthermore...

  10. Subcellular localization and expression analysis of the BmDSCLP ...

    African Journals Online (AJOL)

    In addition, real-time fluorescence quantification polymerase chain reaction studies were conducted to investigate BmDSCLP transcription at different developmental stages and in different tissues of the fifth instar larva. The results indicated that, BmDSCLP is widely transcribed in different stages and tissues of the silkworm.

  11. Convolutional LSTM Networks for Subcellular Localization of Proteins

    DEFF Research Database (Denmark)

    Sønderby, Søren Kaae; Sønderby, Casper Kaae; Nielsen, Henrik

    2015-01-01

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model...

  12. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Subcellular localization and expression analysis of the BmDSCLP ...

    African Journals Online (AJOL)

    user

    2011-04-04

    Apr 4, 2011 ... The three-dimensional (3D) structure of the LRR present in a complex of ... expasy.org/) and viewed in the Swiss PDB viewer (Schwede et al.,. 2003). ... containing 0.1% trifluoroacetic acid/water) and injected into a. POROS 10 ...

  14. Accumulation of fission fragment 147Pm in subcellular level studied by electron microscopic autoradiography

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Wang Yuanchang

    1990-11-01

    The subcellular localization of fission fragment 147 Pm in tissue cells by electron microscopic autoradiography was investigated. The early harm of internal contaminated accumulation of 147 Pm appeared in blood cells and endothelium cells, obviously in erythrocytes. Then 147 Pm was selectively deposited in ultrastructure of liver cells. Autoradiographic study demonstrated that dense tracks appeared in mitochondria and lysosome of podal cells within renal corpuscle. In nucleus as well as in mitochondria and microbodies of epicyte of kidney near-convoluted tubule, there are numerous radioactive 149 Pm accumulated. With the prolongation of observing time, 149 Pm was selectively and steadily deposited in subcellular level of organic component bone. The radionuclides could be accumulated in nucleus of osteoclasts and osteoblasts. In organelles, the radionuclides was mainly accumulated in rough endoplasmic reticulum and mitochondria. Autoradiographic tracks of 149 Pm was obviously found to be localized in combined point between Golgi complex and transitive vesicle of rough endoplasmic reticulum

  15. Forms of Supporting Local Innovative Business Activity in European Countries

    Directory of Open Access Journals (Sweden)

    Oleksandr Fedirko

    2017-04-01

    Full Text Available The article investigates contemporary trends of innovation policy of European countries, describes the essence of contemporary mechanisms and tools for supporting local innovative development. The following most powerful tools for facilitating scientific and technical and innovative business activity are discovered: direct support of private R&D, financing of innovative enterprises, governmental and private cooperative scientific and research projects. A trend is identified for decreasing the share of institutional financing of R&D, and increasing of weight of competitive financing of academic institutions. A conclusion is made as for spreading of technologies commercialization processes support, especially on final stages thereof; the share of these has increased in respect of governmental programs focused on early stages of scientific and research projects. An insight is that within the last two decades the tools for facilitating local innovative business activities have been diversified in the EU: alongside with long-term collaborative governmental and private R&D and initiatives for developing innovative science intensive clusters, short-term tools have been significantly spread, such as innovation projects vouchers and science intensive start-ups support. Given that, it is established that traditionally developed toolkit for supporting small and medium enterprises is being complimented with scaled programs of large companies direct financing. A general trend is identified for increasing the weight of collaborative programs, while the share of individual subsidies and grants for R&D and that of companies innovative activity has substantially decreased. Higher effectiveness of start-ups facilitation measures is concluded, as well as that of venture investments, in comparison with individual subsidies. The leading role of start-ups in EU economy is determined by a range of advantages originating from dynamic process of formation thereof

  16. Calculation of neutron radiation energy deposition distribution in subcellular parts of tissue using recombination chamber microdosimetry

    International Nuclear Information System (INIS)

    Golnik, N.; Zielczynski, M.

    1999-01-01

    Recombination chamber microdosimetry was used as an instrument for determination of local neutron radiation energy deposition distribution. The method allows to simulate of subcellular regions of tissue of the order of 70 nm in size. The results obtained qualitatively correspond to relationship between biological efficiency and neutron energy, and show regular differences of distributions achieved by the recombination method and distributions measured using tissue equivalent proportional counters (TEPC), which simulates greater tissue regions of 1 μm in size

  17. Optically-controlled platforms for transfection and single- and sub-cellular surgery

    DEFF Research Database (Denmark)

    Villangca, Mark Jayson; Casey, Duncan; Glückstad, Jesper

    2015-01-01

    and specificity of optical trapping in conjunction with other modalities to perform single and sub-cellular surgery. These tools form highly tuneable platforms for the delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic...... structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics research....

  18. Localizing Brain Activity from Multiple Distinct Sources via EEG

    Directory of Open Access Journals (Sweden)

    George Dassios

    2014-01-01

    Full Text Available An important question arousing in the framework of electroencephalography (EEG is the possibility to recognize, by means of a recorded surface potential, the number of activated areas in the brain. In the present paper, employing a homogeneous spherical conductor serving as an approximation of the brain, we provide a criterion which determines whether the measured surface potential is evoked by a single or multiple localized neuronal excitations. We show that the uniqueness of the inverse problem for a single dipole is closely connected with attaining certain relations connecting the measured data. Further, we present the necessary and sufficient conditions which decide whether the collected data originates from a single dipole or from numerous dipoles. In the case where the EEG data arouses from multiple parallel dipoles, an isolation of the source is, in general, not possible.

  19. The effects of the local environment on active galactic nuclei

    International Nuclear Information System (INIS)

    Manzer, L. H.; De Robertis, M. M.

    2014-01-01

    There continues to be significant controversy regarding the mechanism(s) responsible for the initiation and maintenance of activity in galactic nuclei. In this paper we will investigate possible environmental triggers of nuclear activity through a statistical analysis of a large sample of galaxy groups. The focus of this paper is to identify active galactic nuclei (AGNs) and other emission-line galaxies in these groups and to compare their frequency with a sample of over 260,000 isolated galaxies from the same catalog. The galaxy groups are taken from the catalog of Yang et al., in which over 20,000 virialized groups of galaxies (2 ≤ N ≤ 20) with redshifts between 0.01 and 0.20 are from the Sloan Digital Sky Survey. We first investigate the completeness of our data set and find, though biases are a concern particularly at higher redshift, that our data provide a fair representation of the local universe. After correcting emission-line equivalent widths for extinction and underlying Balmer stellar absorption, we classify galaxies in the sample using traditional emission-line ratios, while incorporating measurement uncertainties. We find a significantly higher fraction of AGNs in groups compared with the isolated sample. Likewise, a significantly higher fraction of absorption-line galaxies are found in groups, while a higher fraction of star-forming galaxies prefer isolated environments. Within grouped environments, AGNs and star-forming galaxies are found more frequently in small- to medium-richness groups, while absorption-line galaxies prefer groups with larger richnesses. Groups containing only emission-line galaxies have smaller virial radii, velocity dispersions, and masses compared with those containing only absorption-line galaxies. Furthermore, the AGN fraction increases with decreasing distance to the group centroid, independent of galaxy morphology. Using properties obtained from Galaxy Zoo, there is an increased fraction of AGNs within merging systems

  20. The effects of the local environment on active galactic nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Manzer, L. H.; De Robertis, M. M., E-mail: liannemanzer@gmail.com, E-mail: mmdr@yorku.ca [Department of Physics and Astronomy, York University, Toronto, ON M3J 1P3 (Canada)

    2014-06-20

    There continues to be significant controversy regarding the mechanism(s) responsible for the initiation and maintenance of activity in galactic nuclei. In this paper we will investigate possible environmental triggers of nuclear activity through a statistical analysis of a large sample of galaxy groups. The focus of this paper is to identify active galactic nuclei (AGNs) and other emission-line galaxies in these groups and to compare their frequency with a sample of over 260,000 isolated galaxies from the same catalog. The galaxy groups are taken from the catalog of Yang et al., in which over 20,000 virialized groups of galaxies (2 ≤ N ≤ 20) with redshifts between 0.01 and 0.20 are from the Sloan Digital Sky Survey. We first investigate the completeness of our data set and find, though biases are a concern particularly at higher redshift, that our data provide a fair representation of the local universe. After correcting emission-line equivalent widths for extinction and underlying Balmer stellar absorption, we classify galaxies in the sample using traditional emission-line ratios, while incorporating measurement uncertainties. We find a significantly higher fraction of AGNs in groups compared with the isolated sample. Likewise, a significantly higher fraction of absorption-line galaxies are found in groups, while a higher fraction of star-forming galaxies prefer isolated environments. Within grouped environments, AGNs and star-forming galaxies are found more frequently in small- to medium-richness groups, while absorption-line galaxies prefer groups with larger richnesses. Groups containing only emission-line galaxies have smaller virial radii, velocity dispersions, and masses compared with those containing only absorption-line galaxies. Furthermore, the AGN fraction increases with decreasing distance to the group centroid, independent of galaxy morphology. Using properties obtained from Galaxy Zoo, there is an increased fraction of AGNs within merging systems

  1. Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss)

    International Nuclear Information System (INIS)

    Kamunde, Collins; MacPhail, Ruth

    2011-01-01

    Highlights: Interactions of Cu, Cd and Zn were studied at the subcellular level in rainbow trout. Metals accumulated in the liver were predominantly metabolically active. Cd, Cu and Zn exhibited both competitive and cooperative interactions. The metal–metal interactions altered subcellular metals partitioning. - Abstract: Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing (μg/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67–83%; Cu, 68–79% and Zn, 60–76

  2. Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss)

    Energy Technology Data Exchange (ETDEWEB)

    Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada); MacPhail, Ruth [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada)

    2011-10-15

    Highlights: Interactions of Cu, Cd and Zn were studied at the subcellular level in rainbow trout. Metals accumulated in the liver were predominantly metabolically active. Cd, Cu and Zn exhibited both competitive and cooperative interactions. The metal-metal interactions altered subcellular metals partitioning. - Abstract: Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing ({mu}g/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67-83%; Cu, 68-79% and Zn, 60-76%. Taken

  3. The Effects of the Local Environment on Active Galactic Nuclei

    Science.gov (United States)

    Manzer, L. H.; De Robertis, M. M.

    2014-06-01

    There continues to be significant controversy regarding the mechanism(s) responsible for the initiation and maintenance of activity in galactic nuclei. In this paper we will investigate possible environmental triggers of nuclear activity through a statistical analysis of a large sample of galaxy groups. The focus of this paper is to identify active galactic nuclei (AGNs) and other emission-line galaxies in these groups and to compare their frequency with a sample of over 260,000 isolated galaxies from the same catalog. The galaxy groups are taken from the catalog of Yang et al., in which over 20,000 virialized groups of galaxies (2 universe. After correcting emission-line equivalent widths for extinction and underlying Balmer stellar absorption, we classify galaxies in the sample using traditional emission-line ratios, while incorporating measurement uncertainties. We find a significantly higher fraction of AGNs in groups compared with the isolated sample. Likewise, a significantly higher fraction of absorption-line galaxies are found in groups, while a higher fraction of star-forming galaxies prefer isolated environments. Within grouped environments, AGNs and star-forming galaxies are found more frequently in small- to medium-richness groups, while absorption-line galaxies prefer groups with larger richnesses. Groups containing only emission-line galaxies have smaller virial radii, velocity dispersions, and masses compared with those containing only absorption-line galaxies. Furthermore, the AGN fraction increases with decreasing distance to the group centroid, independent of galaxy morphology. Using properties obtained from Galaxy Zoo, there is an increased fraction of AGNs within merging systems, unlike star-forming galaxies. These results provide some indication that the local environment does play a role in initiating activity in galactic nuclei, but it is by no means simple or straightforward.

  4. Human Activity Recognition Supported on Indoor Localization: A Systematic Review.

    Science.gov (United States)

    Cerón, Jesús; López, Diego M

    2018-01-01

    The number of older adults is growing worldwide. This has a social and economic impact in all countries because of the increased number of older adults affected by chronic diseases, health emergencies, and disabilities, representing at the end high cost for the health system. To face this problem, the Ambient Assisted Living (AAL) domain has emerged. Its main objective is to extend the time that older adults can live independently in their homes. AAL is supported by different fields and technologies, being Human Activity Recognition (HAR), control of vital signs and location tracking the three of most interest during the last years. To perform a systematic review about Human Activity Recognition (HAR) approaches supported on Indoor Localization (IL) and vice versa, describing the methods they have used, the accuracy they have obtained and whether they have been directed towards the AAL domain or not. A systematic review of six databases was carried out (ACM, IEEE Xplore, PubMed, Science Direct and Springer). 27 papers were found. They were categorised into three groups according their approach: paper focus on 1. HAR, 2. IL, 3. HAR and IL. A detailed analysis of the following factors was performed: type of methods and technologies used for HAR, IL and data fusion, as well as the precision obtained for them. This systematic review shows that the relationship between HAR and IL has been very little studied, therefore providing insights of its potential mutual support to provide AAL solutions.

  5. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  6. Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution.

    Science.gov (United States)

    Porras, Pablo; McDonagh, Brian; Pedrajas, Jose Rafael; Bárcena, J Antonio; Padilla, C Alicia

    2010-04-01

    We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide. Copyright 2009 Elsevier B.V. All rights reserved.

  7. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthe...

  8. Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

    Science.gov (United States)

    Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

    1999-04-01

    4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

  9. Machine learning of molecular properties: Locality and active learning

    Science.gov (United States)

    Gubaev, Konstantin; Podryabinkin, Evgeny V.; Shapeev, Alexander V.

    2018-06-01

    In recent years, the machine learning techniques have shown great potent1ial in various problems from a multitude of disciplines, including materials design and drug discovery. The high computational speed on the one hand and the accuracy comparable to that of density functional theory on another hand make machine learning algorithms efficient for high-throughput screening through chemical and configurational space. However, the machine learning algorithms available in the literature require large training datasets to reach the chemical accuracy and also show large errors for the so-called outliers—the out-of-sample molecules, not well-represented in the training set. In the present paper, we propose a new machine learning algorithm for predicting molecular properties that addresses these two issues: it is based on a local model of interatomic interactions providing high accuracy when trained on relatively small training sets and an active learning algorithm of optimally choosing the training set that significantly reduces the errors for the outliers. We compare our model to the other state-of-the-art algorithms from the literature on the widely used benchmark tests.

  10. Differential subcellular distribution of ion channels and the diversity of neuronal function.

    Science.gov (United States)

    Nusser, Zoltan

    2012-06-01

    Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity, and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent, subcellular site and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissues followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. No evidence was obtained for the production of volatile Cd complexes in tobacco

  12. FACTORES FINANCIEROS LOCALES INFLUYEN LAS ACTIVIDADES DE LOS BANCOS COOPERATIVOS GRIEGOS / LOCAL FINANCIAL FACTORS INFLUENCING ACTIVITIES OF GREEK COOPERATIVE BANKS

    Directory of Open Access Journals (Sweden)

    GEORGE KONTEOS

    2010-09-01

    Full Text Available Este artículo examina la influencia de las condiciones locales en las actividades bancarias centrandose en el caso de los 14 bancos cooperativos en Grecia durante el período 1999-2007. Estos bancos han sido seleccionados por su carácter local y en consecuencia, están más influenciadas por las condiciones locales, en comparación con los bancos con red nacional e incluso internacional. Las condiciones locales pueden ser endógenas y exógenas al banco. Las endógenas al banco es la red bancaria que se desarrolla a nivel local o regional asi como a los miembros de los bancos que son la parte principal de la clientela de estas. Una macroeconómica y exógena condición del banco es la cantidad de ahorros en la localidad donde opera. Las actividades bancarias que han sido examinadas incluyen los depósitos y los préstamos que constituyen la parte principal de las actividades bancarias de los bancos cooperativos en Grecia. El crecimiento de la red bancaria es el principal factor de aumento de las actividades del banco; el crecimiento de los miembros de las cooperativas y el aumento de las economías locales tienen una influencia positiva, pero menos importante en las actividades de los bancos cooperativos./This paper examines the influence of local conditions on banking activities focusing on the case of 14 cooperative banks in Greece for the period 1999-2007. These banks have been chosen because of their local character and consequently they are more influenced by local conditions in comparison to banks having a national and even international network. Local conditions could be endogenous and exogenous to the bank. The endogenous to the bank are the banking network developed locally or regionally, and the members of the banks who are the main part of the banks’ clientele. A macroeconomic and exogenous to the bank condition is the amount of savings in the prefecture in which the cooperative bank operates. The banking activities which have

  13. Earthquake precursory events around epicenters and local active faults

    Science.gov (United States)

    Valizadeh Alvan, H.; Mansor, S. B.; Haydari Azad, F.

    2013-05-01

    shakes, mapping foreshocks and aftershocks, and following changes in the above-mentioned precursors prior to past earthquake instances all over the globe. Our analyses also encompass the geographical location and extents of local and regional faults which are considered as important factors during earthquakes. The co-analysis of direct and indirect observation for precursory events is considered as a promising method for possible future successful earthquake predictions. With proper and thorough knowledge about the geological setting, atmospheric factors and geodynamics of the earthquake-prone regions we will be able to identify anomalies due to seismic activity in the earth's crust.

  14. Diversity and subcellular distribution of archaeal secreted proteins.

    Science.gov (United States)

    Szabo, Zalan; Pohlschroder, Mechthild

    2012-01-01

    Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis, and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat) pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as phyla-specific pathways among the archaea. A more comprehensive understanding of the transport pathways used and post-translational modifications of secreted archaeal proteins will also facilitate the identification and heterologous expression of commercially valuable archaeal enzymes.

  15. Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

    OpenAIRE

    Zinser, E; Paltauf, F; Daum, G

    1993-01-01

    Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergostero...

  16. local

    Directory of Open Access Journals (Sweden)

    Abílio Amiguinho

    2005-01-01

    Full Text Available The process of socio-educational territorialisation in rural contexts is the topic of this text. The theme corresponds to a challenge to address it having as main axis of discussion either the problem of social exclusion or that of local development. The reasons to locate the discussion in this last field of analysis are discussed in the first part of the text. Theoretical and political reasons are there articulated because the question is about projects whose intentions and practices call for the political both in the theoretical debate and in the choices that anticipate intervention. From research conducted for several years, I use contributions that aim at discuss and enlighten how school can be a potential locus of local development. Its identification and recognition as local institution (either because of those that work and live in it or because of those that act in the surrounding context are crucial steps to progressively constitute school as a partner for development. The promotion of the local values and roots, the reconstruction of socio-personal and local identities, the production of sociabilities and the equation and solution of shared problems were the dimensions of a socio-educative intervention, markedly globalising. This scenario, as it is argued, was also, intentionally, one of transformation and of deliberate change of school and of the administration of the educative territoires.

  17. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    Science.gov (United States)

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  18. LOCAL LUMINOUS INFRARED GALAXIES. II. ACTIVE GALACTIC NUCLEUS ACTIVITY FROM SPITZER/INFRARED SPECTROGRAPH SPECTRA

    Energy Technology Data Exchange (ETDEWEB)

    Alonso-Herrero, Almudena; Pereira-Santaella, Miguel [Centro de Astrobiologia, INTA-CSIC, E-28850 Torrejon de Ardoz, Madrid (Spain); Rieke, George H. [Steward Observatory, University of Arizona, Tucson, AZ 85721 (United States); Rigopoulou, Dimitra [Astrophysics Department, University of Oxford, Oxford OX1 3RH (United Kingdom)

    2012-01-01

    We quantify the active galactic nucleus (AGN) contribution to the mid-infrared (mid-IR) and the total infrared (IR, 8-1000 {mu}m) emission in a complete volume-limited sample of 53 local luminous infrared galaxies (LIRGs, L{sub IR} = 10{sup 11}-10{sup 12} L{sub Sun }). We decompose the Spitzer Infrared Spectrograph low-resolution 5-38 {mu}m spectra of the LIRGs into AGN and starburst components using clumpy torus models and star-forming galaxy templates, respectively. We find that 50% (25/50) of local LIRGs have an AGN component detected with this method. There is good agreement between these AGN detections through mid-IR spectral decomposition and other AGN indicators, such as the optical spectral class, mid-IR spectral features, and X-ray properties. Taking all the AGN indicators together, the AGN detection rate in the individual nuclei of LIRGs is {approx}62%. The derived AGN bolometric luminosities are in the range L{sub bol}(AGN) = (0.4-50) Multiplication-Sign 10{sup 43} erg s{sup -1}. The AGN bolometric contribution to the IR luminosities of the galaxies is generally small, with 70% of LIRGs having L{sub bol}[AGN]/L{sub IR} {<=} 0.05. Only {approx_equal} 8% of local LIRGs have a significant AGN bolometric contribution L{sub bol}[AGN]/L{sub IR} > 0.25. From the comparison of our results with literature results of ultraluminous infrared galaxies (L{sub IR} = 10{sup 12}-10{sup 13} L{sub Sun }), we confirm that in the local universe the AGN bolometric contribution to the IR luminosity increases with the IR luminosity of the galaxy/system. If we add up the AGN bolometric luminosities we find that AGNs only account for 5%{sub -3%}{sup +8%} of the total IR luminosity produced by local LIRGs (with and without AGN detections). This proves that the bulk of the IR luminosity of local LIRGs is due to star formation activity. Taking the newly determined IR luminosity density of LIRGs in the local universe, we then estimate an AGN IR luminosity density of {Omega}{sup AGN

  19. Subcellular controls of mercury trophic transfer to a marine fish

    International Nuclear Information System (INIS)

    Dang Fei; Wang Wenxiong

    2010-01-01

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  20. Subcellular controls of mercury trophic transfer to a marine fish

    Energy Technology Data Exchange (ETDEWEB)

    Dang Fei [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2010-09-15

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  1. Subcellular metabolic contrast in living tissue using dynamic full field OCT (D-FFOCT) (Conference Presentation)

    Science.gov (United States)

    Apelian, Clement; Harms, Fabrice; Thouvenin, Olivier; Boccara, Claude A.

    2016-03-01

    Cells shape or density is an important marker of tissues pathology. However, individual cells are difficult to observe in thick tissues frequently presenting highly scattering structures such as collagen fibers. Endogenous techniques struggle to image cells in these conditions. Moreover, exogenous contrast agents like dyes, fluorophores or nanoparticles cannot always be used, especially if non-invasive imaging is required. Scatterers motion happening down to the millisecond scale, much faster than the fix and highly scattering structures (global motion of the tissue), allowed us to develop a new approach based on the time dependence of the FF-OCT signals. This method reveals hidden cells after a spatiotemporal analysis based on singular value decomposition and wavelet analysis concepts. It does also give us access to local dynamics of imaged scatterers. This dynamic information is linked with the local metabolic activity that drives these scatterers. Our technique can explore subcellular scales with micrometric resolution and dynamics ranging from the millisecond to seconds. By this mean we studied a wide range of tissues, animal and human in both normal and pathological conditions (cancer, ischemia, osmotic shock…) in different organs such as liver, kidney, and brain among others. Different cells, undetectable with FF-OCT, were identified (erythrocytes, hepatocytes…). Different scatterer clusters express different characteristic times and thus can be related to different mechanisms that we identify with metabolic functions. We are confident that the D-FFOCT, by accessing to a new spatiotemporal metabolic contrast, will be a leading technique on tissue imaging and could lead to better medical diagnosis.

  2. Study of subcellular distribution of /sup 67/Ga in tumor and liver

    Energy Technology Data Exchange (ETDEWEB)

    Ando, A; Takeshita, M; Hiraki, T [Kanazawa Univ. (Japan). School of Paramedicine; Ando, T; Hisada, K

    1977-02-01

    The following animals and transplanted tumors were used: rats implanted with Yoshida sarcoma and hepatoma AH109A, and mice implanted with Ehrlich tumor. /sup 67/Ga-citrate was injected into the rats intravenously and into the mice intraperitoneally. Ten minutes to 48 hours after the administration of /sup 67/Ga-citrate, the animals were sacrificed, and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and livers was carried out according to the method of Hogeboom and Schneider. Radioactivity of each fraction was counted with a well type scintillation counter, and the protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and a small amount of radioactivity was localized in the mitochondrial fraction (lysosome contains in this fraction). But in the liver, most of the radioactivity was concentrated in the mitochondrial fraction, and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of the total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction was increased with the passage of time after administration, and about 30% of total activity was concentrated in this fraction at 24 hours after administration. From these results it is concluded that the lysosome does not play an important role in the concentration of /sup 67/Ga in the tumor, but that the lysosome plays an important role in the concentration of /sup 67/Ga in the liver. In the case of hepatoma AH109A, it is presumed that the lysosome plays a very important role in the concentration of /sup 67/Ga in the tumor, hepatoma AH109A having some nature of liver.

  3. Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL).

    Science.gov (United States)

    Hooper, Cornelia M; Castleden, Ian R; Aryamanesh, Nader; Jacoby, Richard P; Millar, A Harvey

    2016-01-01

    Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Cytotoxic activity and apoptotic induction of some edible Thai local ...

    African Journals Online (AJOL)

    Purpose: To evaluate eight edible Thai local plant extracts (Camellia sinensis, Careya sphaerica, Cratoxylum formosum, Eleutherococcus trifoliatus, Ficus auriculata, Persicaria odorata, Schima wallichii, and Vaccinium sprengelii) against colon and liver cancer cell lines. Methods: The 80 % ethanol plant extracts were ...

  5. Women fishing activities in rural communities of Epe Local ...

    African Journals Online (AJOL)

    Women are not aware of fishery extension activities in the area and the fisherwomen faced different types of problems in their fishery activities. However, to improve fishery activities by women in the area, extension agents should organize training workshops for the women on how to construct and mend fishing nets and ...

  6. Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus

    Science.gov (United States)

    Somogyi, Peter; Katona, Linda; Klausberger, Thomas; Lasztóczi, Bálint; Viney, Tim J.

    2014-01-01

    The behaviour-contingent rhythmic synchronization of neuronal activity is reported by local field potential oscillations in the theta, gamma and sharp wave-related ripple (SWR) frequency ranges. In the hippocampus, pyramidal cell assemblies representing temporal sequences are coordinated by GABAergic interneurons selectively innervating specific postsynaptic domains, and discharging phase locked to network oscillations. We compare the cellular network dynamics in the CA1 and CA3 areas recorded with or without anaesthesia. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. The axon initial segment is exclusively innervated by axo-axonic cells, preferentially firing after the peak of the pyramidal layer theta cycle, when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs, when many pyramidal cells fire synchronously. This dual inverse correlation demonstrates the key inhibitory role of axo-axonic cells. Parvalbumin-expressing basket cells fire phase locked to field gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs, together with dendrite-innervating bistratified cells, phasing pyramidal cell discharge. Subcellular domain-specific GABAergic innervation probably developed for the coordination of multiple glutamatergic inputs on different parts of pyramidal cells through the temporally distinct activity of GABAergic interneurons, which differentially change their firing during different network states. PMID:24366131

  7. Local jurisdictions and active shooters : building networks, building capacities

    OpenAIRE

    Frazzano, Tracy L.

    2010-01-01

    CHDS State/Local Approved for public release; distribution is unlimited States incidents (Columbine High School shooting (April 20, 1999) and North Hollywood Bank shoot out (February 28, 1997) were studied. Individuals from the U.S. cases were interviewed to explore information not necessarily documented. Data from the case studies and interviews were collated and reviewed for common themes. These themes were analyzed to draw conclusions on how smaller jurisdictions should proceed in bu...

  8. Capacity-building activities for local leadership in Egypt from the local to the national and regional levels.

    Science.gov (United States)

    El Sioufi, M

    1996-03-01

    In order to make the Sustainable Ismailia Project institutionally sustainable, the National Project Manager identified the need to build the capacity of local leadership. The Training Section, in UNCHS, proposed a capacity-building program designed to respond to this demand focusing on the skill-training and attitude formation needed for effective local leadership. The proposed program is adapted from the "Training for Elected Leadership" series widely tested and used in many regions. It combines direct training for local officials from villages in Ismailia, and indirect training for trainers from the Ministry of Local Administration (MLA). After the course, the MLA trainers would design a training program which they will implement nationally. The Minister for Local Administration decided to finance the translation and production of the manuals, while the Governor of Ismailia offered to cover the trainees' expenses. The UNCHS Training and Capacity-Building Section would provide technical guidance and coordinate and implement the program. Once the Arabic version of the training materials would be available, UNCHS would use it regionally to reach out to other Arab States. This example illustrates how concerned stakeholders cooperate to address capacity-building needs. The UNCHS Training Section assumes the role of facilitator and acts as a catalyst for the formulation of such activities. The feeling of ownership of the locally produced/adapted training materials enhances the propensity of their effective and extensive use. This approach has succeeded across regions, cultures, and languages with out-reaching multiplier effects. full text

  9. Bioaccumulation and subcellular partitioning of zinc in rainbow trout (Oncorhynchus mykiss): Cross-talk between waterborne and dietary uptake

    International Nuclear Information System (INIS)

    Sappal, Ravinder; Burka, John; Dawson, Susan; Kamunde, Collins

    2009-01-01

    Zinc homeostasis was studied at the tissue and gill subcellular levels in rainbow trout (Oncorhynchus mykiss) following waterborne and dietary exposures, singly and in combination. Juvenile rainbow trout were exposed to 150 or 600 μg l -1 waterborne Zn, 1500 or 4500 μg g -1 dietary Zn, and a combination of 150 μg l -1 waterborne and 1500 μg g -1 dietary Zn for 40 days. Accumulation of Zn in tissues and gill subcellular fractions was measured. At the tissue level, the carcass acted as the main Zn depot containing 84-90% of whole body Zn burden whereas the gill held 4-6%. At the subcellular level, the majority of gill Zn was bioavailable with the estimated metabolically active pool being 81-90%. Interestingly, the nuclei-cellular debris fraction bound the highest amount (40%) of the gill Zn burden. There was low partitioning of Zn into the detoxified pool (10-19%) suggesting that sequestration and chelation are not major mechanisms of cellular Zn homeostasis in rainbow trout. Further, the subcellular partitioning of Zn did not conform to the spill-over model of metal toxicity because Zn binding was indiscriminate irrespective of exposure concentration and duration. The contribution of the branchial and gastrointestinal uptake pathways to Zn accumulation depended on the tissue. Specifically, in plasma, blood cells, and gill, uptake from water was dominant whereas both pathways appeared to contribute equally to Zn accumulation in the carcass. Subcellularly, additive uptake from the two pathways was observed in the heat-stable proteins (HSP) fraction. Toxicologically, Zn exposure caused minimal adverse effects manifested by a transitory inhibition of protein synthesis in gills in the waterborne exposure. Overall, subcellular fractionation appears to have value in the quest for a better understanding of Zn homeostasis and interactions between branchial and gastrointestinal uptake pathways

  10. Bioaccumulation and subcellular partitioning of zinc in rainbow trout (Oncorhynchus mykiss): Cross-talk between waterborne and dietary uptake

    Energy Technology Data Exchange (ETDEWEB)

    Sappal, Ravinder; Burka, John; Dawson, Susan [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Kamunde, Collins [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada)], E-mail: ckamunde@upei.ca

    2009-03-09

    Zinc homeostasis was studied at the tissue and gill subcellular levels in rainbow trout (Oncorhynchus mykiss) following waterborne and dietary exposures, singly and in combination. Juvenile rainbow trout were exposed to 150 or 600 {mu}g l{sup -1} waterborne Zn, 1500 or 4500 {mu}g g{sup -1} dietary Zn, and a combination of 150 {mu}g l{sup -1} waterborne and 1500 {mu}g g{sup -1} dietary Zn for 40 days. Accumulation of Zn in tissues and gill subcellular fractions was measured. At the tissue level, the carcass acted as the main Zn depot containing 84-90% of whole body Zn burden whereas the gill held 4-6%. At the subcellular level, the majority of gill Zn was bioavailable with the estimated metabolically active pool being 81-90%. Interestingly, the nuclei-cellular debris fraction bound the highest amount (40%) of the gill Zn burden. There was low partitioning of Zn into the detoxified pool (10-19%) suggesting that sequestration and chelation are not major mechanisms of cellular Zn homeostasis in rainbow trout. Further, the subcellular partitioning of Zn did not conform to the spill-over model of metal toxicity because Zn binding was indiscriminate irrespective of exposure concentration and duration. The contribution of the branchial and gastrointestinal uptake pathways to Zn accumulation depended on the tissue. Specifically, in plasma, blood cells, and gill, uptake from water was dominant whereas both pathways appeared to contribute equally to Zn accumulation in the carcass. Subcellularly, additive uptake from the two pathways was observed in the heat-stable proteins (HSP) fraction. Toxicologically, Zn exposure caused minimal adverse effects manifested by a transitory inhibition of protein synthesis in gills in the waterborne exposure. Overall, subcellular fractionation appears to have value in the quest for a better understanding of Zn homeostasis and interactions between branchial and gastrointestinal uptake pathways.

  11. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

    Science.gov (United States)

    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-12-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  12. Implicit Active Contours Driven by Local and Global Image Fitting Energy for Image Segmentation and Target Localization

    Directory of Open Access Journals (Sweden)

    Xiaosheng Yu

    2013-01-01

    Full Text Available We propose a novel active contour model in a variational level set formulation for image segmentation and target localization. We combine a local image fitting term and a global image fitting term to drive the contour evolution. Our model can efficiently segment the images with intensity inhomogeneity with the contour starting anywhere in the image. In its numerical implementation, an efficient numerical schema is used to ensure sufficient numerical accuracy. We validated its effectiveness in numerous synthetic images and real images, and the promising experimental results show its advantages in terms of accuracy, efficiency, and robustness.

  13. Sub-cellular distribution and translocation of TRP channels.

    Science.gov (United States)

    Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

    2011-01-01

    Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

  14. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  15. 78 FR 19632 - Special Local Regulations; St. Thomas Carnival Watersport Activities, Charlotte Amalie Harbor; St...

    Science.gov (United States)

    2013-04-02

    ...-AA08 Special Local Regulations; St. Thomas Carnival Watersport Activities, Charlotte Amalie Harbor; St... proposes to establish a special local regulation on the waters of Charlotte Amalie Harbor in St Thomas, USVI during the St. Thomas Carnival Watersport Activities, a high speed boat race. The event is...

  16. Localization-Aware Active Learning for Object Detection

    OpenAIRE

    Kao, Chieh-Chi; Lee, Teng-Yok; Sen, Pradeep; Liu, Ming-Yu

    2018-01-01

    Active learning - a class of algorithms that iteratively searches for the most informative samples to include in a training dataset - has been shown to be effective at annotating data for image classification. However, the use of active learning for object detection is still largely unexplored as determining informativeness of an object-location hypothesis is more difficult. In this paper, we address this issue and present two metrics for measuring the informativeness of an object hypothesis,...

  17. Novel active contour model based on multi-variate local Gaussian distribution for local segmentation of MR brain images

    Science.gov (United States)

    Zheng, Qiang; Li, Honglun; Fan, Baode; Wu, Shuanhu; Xu, Jindong

    2017-12-01

    Active contour model (ACM) has been one of the most widely utilized methods in magnetic resonance (MR) brain image segmentation because of its ability of capturing topology changes. However, most of the existing ACMs only consider single-slice information in MR brain image data, i.e., the information used in ACMs based segmentation method is extracted only from one slice of MR brain image, which cannot take full advantage of the adjacent slice images' information, and cannot satisfy the local segmentation of MR brain images. In this paper, a novel ACM is proposed to solve the problem discussed above, which is based on multi-variate local Gaussian distribution and combines the adjacent slice images' information in MR brain image data to satisfy segmentation. The segmentation is finally achieved through maximizing the likelihood estimation. Experiments demonstrate the advantages of the proposed ACM over the single-slice ACM in local segmentation of MR brain image series.

  18. Diversity and subcellular distribution of archaeal secreted proteins

    Directory of Open Access Journals (Sweden)

    Mechthild ePohlschroder

    2012-07-01

    Full Text Available Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell-wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as archaeal kingdom-specific pathways. A more comprehensive understanding of the transport pathways and post-translational modifications of secreted archaeal proteins will also generate invaluable insights that will facilitate the identification of commercially valuable archaeal enzymes and the development of heterologous systems in which to efficiently express them.

  19. Organ accumulation and subcellular location of Cicer arietinum ST1 protein.

    Science.gov (United States)

    Albornos, Lucía; Cabrera, Javier; Hernández-Nistal, Josefina; Martín, Ignacio; Labrador, Emilia; Dopico, Berta

    2014-07-01

    The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent of, subcellular site of and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissue followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. Particulate materials containing other cell components were also labeled. Of the 109 Cd supplied to plants, 2 to 10% was recovered in both cytosol preparations and in particulate materials. Cytosol contained proteinaceous--Cd complexes, free metal and low molecular weight Cd complexes. Labeling of protoplasts gave similar results. No evidence was obtained for the production of volatile Cd complexes in tobacco

  1. Cytotoxic activity and apoptotic induction of some edible Thai local ...

    African Journals Online (AJOL)

    inverted microscopy and DNA fragmentation using agarose gel electrophoresis. Results: P. ... However, further studies are needed to isolate the active compounds responsible for the cytotoxic ..... D-E: TL at 500 and 4,000 μg/mL. Arrows ...

  2. Globalization of rheumatology: activities of ILAR. Think global - act local

    NARCIS (Netherlands)

    Dequeker, Jan; Rasker, Johannes J.; El-Hadidi, Tahsin

    2001-01-01

    In 1997 a distinguished EULAR rheumatologist involved in the development of biologics asked somewhat ironically, “What is ILAR [International League of Associations for Rheumatology] doing?” Now, 3 years later, we are in a position to review ILAR’s activities in recent years and its plans for the

  3. Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

    OpenAIRE

    Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

    2008-01-01

    The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

  4. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    Science.gov (United States)

    Antonacci, Giuseppe; Braakman, Sietse

    2016-11-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p biomechanics and its role in pathophysiology.

  5. Neptunium 237 behaviour in subcellular fractions of rat kidneys

    International Nuclear Information System (INIS)

    Kreslov, V.V.; Maksutova, A.Ya.; Mushkacheva, G.S.

    1978-01-01

    Subcellular distribution of intravenously injected (1 and 0.5 μCi/rat) neptunium nitrate (5- and 6-valent) in kidneys of rat males and females has been investigated. It has been shown that the radionuclide was unevenly distributed within the cell. As early as 24 hours after administration, about 50 per cent of neptunium were concentrated in the mitochondrial fraction. The data are presented on variations in neptunium behaviour within subcellular fractions of rat kidneys depending on the sex of animals, valency and dose of the isotope

  6. Weighted Local Active Pixel Pattern (WLAPP for Face Recognition in Parallel Computation Environment

    Directory of Open Access Journals (Sweden)

    Gundavarapu Mallikarjuna Rao

    2013-10-01

    Full Text Available Abstract  - The availability of multi-core technology resulted totally new computational era. Researchers are keen to explore available potential in state of art-machines for breaking the bearer imposed by serial computation. Face Recognition is one of the challenging applications on so ever computational environment. The main difficulty of traditional Face Recognition algorithms is lack of the scalability. In this paper Weighted Local Active Pixel Pattern (WLAPP, a new scalable Face Recognition Algorithm suitable for parallel environment is proposed.  Local Active Pixel Pattern (LAPP is found to be simple and computational inexpensive compare to Local Binary Patterns (LBP. WLAPP is developed based on concept of LAPP. The experimentation is performed on FG-Net Aging Database with deliberately introduced 20% distortion and the results are encouraging. Keywords — Active pixels, Face Recognition, Local Binary Pattern (LBP, Local Active Pixel Pattern (LAPP, Pattern computing, parallel workers, template, weight computation.  

  7. The active titration method for measuring local hydroxyl radical concentration

    Science.gov (United States)

    Sprengnether, Michele; Prinn, Ronald G.

    1994-01-01

    We are developing a method for measuring ambient OH by monitoring its rate of reaction with a chemical species. Our technique involves the local, instantaneous release of a mixture of saturated cyclic hydrocarbons (titrants) and perfluorocarbons (dispersants). These species must not normally be present in ambient air above the part per trillion concentration. We then track the mixture downwind using a real-time portable ECD tracer instrument. We collect air samples in canisters every few minutes for roughly one hour. We then return to the laboratory and analyze our air samples to determine the ratios of the titrant to dispersant concentrations. The trends in these ratios give us the ambient OH concentration from the relation: dlnR/dt = -k(OH). A successful measurement of OH requires that the trends in these ratios be measureable. We must not perturb ambient OH concentrations. The titrant to dispersant ratio must be spatially invariant. Finally, heterogeneous reactions of our titrant and dispersant species must be negligible relative to the titrant reaction with OH. We have conducted laboratory studies of our ability to measure the titrant to dispersant ratios as a function of concentration down to the few part per trillion concentration. We have subsequently used these results in a gaussian puff model to estimate our expected uncertainty in a field measurement of OH. Our results indicate that under a range of atmospheric conditions we expect to be able to measure OH with a sensitivity of 3x10(exp 5) cm(exp -3). In our most optimistic scenarios, we obtain a sensitivity of 1x10(exp 5) cm(exp -3). These sensitivity values reflect our anticipated ability to measure the ratio trends. However, because we are also using a rate constant to obtain our (OH) from this ratio trend, our accuracy cannot be better than that of the rate constant, which we expect to be about 20 percent.

  8. HERSCHEL/SPIRE SUBMILLIMETER SPECTRA OF LOCAL ACTIVE GALAXIES {sup ,}

    Energy Technology Data Exchange (ETDEWEB)

    Pereira-Santaella, Miguel; Spinoglio, Luigi; Busquet, Gemma [Istituto di Astrofisica e Planetologia Spaziali, INAF, Via Fosso del Cavaliere 100, I-00133 Roma (Italy); Wilson, Christine D.; Schirm, Maximilien R. P. [Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, L8S 4M1 (Canada); Glenn, Jason; Kamenetzky, Julia; Rangwala, Naseem [Center for Astrophysics and Space Astronomy, 389-UCB, University of Colorado, Boulder, CO 80303 (United States); Isaak, Kate G. [ESA Astrophysics Missions Division, ESTEC, P.O. Box 299, 2200 AG Noordwijk (Netherlands); Baes, Maarten [Sterrenkundig Observatorium, Universiteit Gent, Krijgslaan 281 S9, B-9000 Gent (Belgium); Barlow, Michael J. [Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT (United Kingdom); Boselli, Alessandro [Laboratoire d' Astrophysique de Marseille (LAM), Universite d' Aix-Marseille and CNRS, UMR7326, 38 rue F. Joliot-Curie, F-13388 Marseille Cedex 13 (France); Cooray, Asantha [Center for Cosmology, Department of Physics and Astronomy, University of California, Irvine, CA 92697 (United States); Cormier, Diane, E-mail: miguel.pereira@ifsi-roma.inaf.it [Laboratoire AIM, CEA/DSM-CNRS-Universite Paris Diderot, Irfu/Service d' Astrophysique, CEA Saclay, F-91191 Gif-sur-Yvette (France)

    2013-05-01

    We present the submillimeter spectra from 450 to 1550 GHz of 11 nearby active galaxies observed with the SPIRE Fourier Transform Spectrometer (SPIRE/FTS) on board Herschel. We detect CO transitions from J{sub up} = 4 to 12, as well as the two [C I] fine structure lines at 492 and 809 GHz and the [N II]1461 GHz line. We used radiative transfer models to analyze the observed CO spectral line energy distributions. The FTS CO data were complemented with ground-based observations of the low-J CO lines. We found that the warm molecular gas traced by the mid-J CO transitions has similar physical conditions (n{sub H{sub 2}}{approx} 10{sup 3.2}-10{sup 3.9} cm{sup -3} and T{sub kin} {approx} 300-800 K) in most of our galaxies. Furthermore, we found that this warm gas is likely producing the mid-IR rotational H{sub 2} emission. We could not determine the specific heating mechanism of the warm gas, however, it is possibly related to the star formation activity in these galaxies. Our modeling of the [C I] emission suggests that it is produced in cold (T{sub kin} < 30 K) and dense (n{sub H{sub 2}}>10{sup 3} cm{sup -3}) molecular gas. Transitions of other molecules are often detected in our SPIRE/FTS spectra. The HF J = 1-0 transition at 1232 GHz is detected in absorption in UGC 05101 and in emission in NGC 7130. In the latter, near-infrared pumping, chemical pumping, or collisional excitation with electrons are plausible excitation mechanisms likely related to the active galactic nucleus of this galaxy. In some galaxies, few H{sub 2}O emission lines are present. Additionally, three OH{sup +} lines at 909, 971, and 1033 GHz are identified in NGC 7130.

  9. LOCAL DEVELOPMENT IN NORTHEST REGION THROUGH ACTIVITIES IN ITC DOMAIN

    Directory of Open Access Journals (Sweden)

    Daniela\tENACHESCU

    2015-06-01

    Full Text Available Economic areas with high technology are key drivers in sustainable regional development, including unemployment and consequently decreasing population migration in the region. Northeast Region is the largest development region of Romania in terms of number of inhabitants and the owned area. On 01/01/2014, according to balance employment, labor resources of the region were numbered 2,428,700, which represent 49.6% of employed population. The registered unemployment rate at 31 August 2014 was 6.5%, with 82 thousand unemployed registered. In terms of participation in the main economic activities, civilian employment in agriculture, forestry and fishing is predominant (40.1% while in service, civilian employment is 37.1%, while industry and construction is 22.8%. The paper aims to analyze the situation that the potential employment and development opportunities for the Northeast region through activities in the field of ITC domain. Unfortunately, this area was the worst in most indicators, the use of computers and the internet to the turnover of companies and investments in the IT & C and unfortunately in terms of employment population that is under 50%

  10. Analysis of potato virus X replicase and TGBp3 subcellular locations

    International Nuclear Information System (INIS)

    Bamunusinghe, Devinka; Hemenway, Cynthia L.; Nelson, Richard S.; Sanderfoot, Anton A.; Ye, Chang M.; Silva, Muniwarage A.T.; Payton, M.; Verchot-Lubicz, Jeanmarie

    2009-01-01

    Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

  11. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

  12. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: Subcellular distribution, chemical forms and thiol pools

    International Nuclear Information System (INIS)

    Weng Bosen; Xie Xiangyu; Weiss, Dominik J.; Liu Jingchun; Lu Haoliang; Yan Chongling

    2012-01-01

    Highlights: ► Cadmium tolerance mechanisms of Kandelia obovata was investigated systematacially. ► Thiol pool can play roles in cadmium detoxification mechanisms. ► Increasing cadmium treatment strength caused proportional increase of cadmium uptake. ► More than half of cadmium was localized in cell walls, and lowest in membranes. ► Sodium chloride and acetic acid extractable fractions were dominant. - Abstract: In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  13. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Science.gov (United States)

    Chandra, Subhash

    2008-12-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  14. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    International Nuclear Information System (INIS)

    Chandra, Subhash

    2008-01-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2 + ) revealed local secondary ion signal enhancements correlated with the water image signals of 19 (H 3 O) + . A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  15. Evaluation on subcellular partitioning and biodynamics of pulse copper toxicity in tilapia reveals impacts of a major environmental disturbance.

    Science.gov (United States)

    Ju, Yun-Ru; Yang, Ying-Fei; Tsai, Jeng-Wei; Cheng, Yi-Hsien; Chen, Wei-Yu; Liao, Chung-Min

    2017-07-01

    Fluctuation exposure of trace metal copper (Cu) is ubiquitous in aquatic environments. The purpose of this study was to investigate the impacts of chronically pulsed exposure on biodynamics and subcellular partitioning of Cu in freshwater tilapia (Oreochromis mossambicus). Long-term 28-day pulsed Cu exposure experiments were performed to explore subcellular partitioning and toxicokinetics/toxicodynamics of Cu in tilapia. Subcellular partitioning linking with a metal influx scheme was used to estimate detoxification and elimination rates. A biotic ligand model-based damage assessment model was used to take into account environmental effects and biological mechanisms of Cu toxicity. We demonstrated that the probability causing 50% of susceptibility risk in response to pulse Cu exposure in generic Taiwan aquaculture ponds was ~33% of Cu in adverse physiologically associated, metabolically active pool, implicating no significant susceptibility risk for tilapia. We suggest that our integrated ecotoxicological models linking chronic exposure measurements with subcellular partitioning can facilitate a risk assessment framework that provides a predictive tool for preventive susceptibility reduction strategies for freshwater fish exposed to pulse metal stressors.

  16. Nuclear localization of SNARK; its impact on gene expression

    International Nuclear Information System (INIS)

    Kuga, Wataru; Tsuchihara, Katsuya; Ogura, Tsutomu; Kanehara, Sakyo; Saito, Marie; Suzuki, Atsushi; Esumi, Hiroyasu

    2008-01-01

    SNARK, a member of the AMPK-related kinases, has been involved in the cellular stress responses but its precise mechanisms remain unclear. Subcellular localization of SNARK protein was identified. Unlike cytoplasmic localizing AMPKα, SNARK was predominantly localized in the nucleus. SNARK was constitutively distributed in the nucleus even when SNARK was activated by metabolic stimuli such as AICAR and glucose-deprivation. Conserved nuclear localization signal (NLS) was identified at the N-terminal portion ( 68 KKAR 71 ). Deletion and point mutation of this part resulted in the cytoplasmic translocation of mutant proteins. Furthermore, GFP fused with the SNARK fragment containing 68 KKAR 71 translocated to the nucleus. A microarray analysis revealed that the nuclear localizing SNARK altered transcriptome profiles and a considerable part of these alterations were canceled by the mutation of NLS, suggesting the ability of SNARK to modulate gene expression dependent on its nuclear localization.

  17. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    Science.gov (United States)

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-04-02

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

  18. Tip chip : Subcellular sampling from single cancer cells

    NARCIS (Netherlands)

    Quist, Jos; Sarajlic, Edin; Lai, Stanley C.S.; Lemay, Serge G.

    2016-01-01

    To analyze the molecular content of single cells, cell lysis is typically required, yielding a snapshot of cell behavior only. To follow complex molecular profiles over time, subcellular sampling methods potentially can be used, but to date these methods involve laborious offline analysis. Here we

  19. Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

    Science.gov (United States)

    Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

    2014-12-14

    Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

  20. Sexual function with localized prostate cancer: active surveillance vs radical therapy

    NARCIS (Netherlands)

    van den Bergh, Roderick C. N.; Korfage, Ida J.; Roobol, Monique J.; Bangma, Chris H.; de Koning, Harry J.; Steyerberg, Ewout W.; Essink-Bot, Marie-Louise

    2012-01-01

    OBJECTIVE To compare sexual function of men with localized prostate cancer (PCa) on active surveillance (AS) with similar patients who received radical therapy. PATIENTS AND METHODS Two groups of men with screening-detected localized PCa were compared. The first were men on AS within the prospective

  1. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  2. Subcellular neuropharmacology: the importance of intracellular targeting.

    Science.gov (United States)

    Miyashiro, Kevin Y; Bell, Thomas J; Sul, Jai-Yoon; Eberwine, James

    2009-04-01

    Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.

  3. Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

    Directory of Open Access Journals (Sweden)

    Laura C Simone

    Full Text Available The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4 play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy, and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

  4. Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

    Science.gov (United States)

    Avaritt, Brittany R; Swaan, Peter W

    2015-06-01

    Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

  5. Local climate activities in co-operation between municipality, civil society and science shop

    DEFF Research Database (Denmark)

    Jørgensen, Michael Søgaard

    The Science Shop at DTU co-operates with the local municipal administration and the local branch of an environmental NGO about climate change. The co-operation was initiated by a proposal to the Science Shop from the municipal administration. Since the Science Shop requests civil society...... involvement in projects it was proposed to involve the local branch of the environmental NGO. The starting point was topics developed by the administration and the NGO together and announced to students as part of the Science Shop project supply. The focus is climate impact of local activities and strategies...... are initiated and co-ordinated by a group with members from municipal administration, the local NGO and the Science Shop. All projects have involved student projects, but most projects have also contributed to ongoing research activities. The projects up till now have focused on the municipal food supply...

  6. Endoplasmic Reticulum Export, Subcellular Distribution, and Fibril Formation by Pmel17 Require an Intact N-terminal Domain Junction*

    Science.gov (United States)

    Leonhardt, Ralf M.; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J.; Cresswell, Peter

    2010-01-01

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes. PMID:20231267

  7. fMRI activation patterns in an analytic reasoning task: consistency with EEG source localization

    Science.gov (United States)

    Li, Bian; Vasanta, Kalyana C.; O'Boyle, Michael; Baker, Mary C.; Nutter, Brian; Mitra, Sunanda

    2010-03-01

    Functional magnetic resonance imaging (fMRI) is used to model brain activation patterns associated with various perceptual and cognitive processes as reflected by the hemodynamic (BOLD) response. While many sensory and motor tasks are associated with relatively simple activation patterns in localized regions, higher-order cognitive tasks may produce activity in many different brain areas involving complex neural circuitry. We applied a recently proposed probabilistic independent component analysis technique (PICA) to determine the true dimensionality of the fMRI data and used EEG localization to identify the common activated patterns (mapped as Brodmann areas) associated with a complex cognitive task like analytic reasoning. Our preliminary study suggests that a hybrid GLM/PICA analysis may reveal additional regions of activation (beyond simple GLM) that are consistent with electroencephalography (EEG) source localization patterns.

  8. Localized, Non-Harmonic Active Flap Motions for Low Frequency In-Plane Rotor Noise Reduction

    Science.gov (United States)

    Sim, Ben W.; Potsdam, Mark; Kitaplioglu, Cahit; LeMasurier, Philip; Lorber, Peter; Andrews, Joseph

    2012-01-01

    A first-of-its-kind demonstration of the use of localized, non-harmonic active flap motions, for suppressing low frequency, in-plane rotor noise, is reported in this paper. Operational feasibility is verified via testing of the full-scale AATD/Sikorsky/UTRC active flap demonstration rotor in the NFAC's 40- by 80-Foot anechoic wind tunnel. Effectiveness of using localized, non-harmonic active flap motions are compared to conventional four-per-rev harmonic flap motions, and also active flap motions derived from closed-loop acoustics implementations. All three approaches resulted in approximately the same noise reductions over an in-plane three-by-three microphone array installed forward and near in-plane of the rotor in the nearfield. It is also reported that using an active flap in this localized, non-harmonic manner, resulted in no more that 2% rotor performance penalty, but had the tendency to incur higher hub vibration levels.

  9. Hit by waves-living with local advanced or localized prostate cancer treated with endocrine therapy or under active surveillance.

    Science.gov (United States)

    Ervik, Bente; Nordøy, Tone; Asplund, Kenneth

    2010-01-01

    Previous studies of living with prostate cancer have shown that the illness and the treatment cause physical as well as psychosocial problems. The aim of this study was to illuminate men's experiences living with localized or local advanced prostate cancer when curative treatment such as surgery or radiation therapy is not an option at the time of diagnosis. The study was conducted via qualitative interviews, using a phenomenological hermeneutic approach. Ten men treated with endocrine therapy or under active surveillance were interviewed. Being diagnosed with prostate cancer was described as a shock, with different aspects of the illness revealed gradually. The limited amount of time available for meeting with health care providers contributed to patients' feelings of being left alone with difficulty getting information and help. Sexual and urinary problems were perceived as a threat to their manhood. The spouses provided the closest everyday support. The life situation of these patients can be understood as living in a "state of readiness," expecting something to happen regarding their illness, and not always knowing where to get help. The results confirm existing knowledge of patient's experiences in living with prostate cancer regarding the initial shock perceived by the patients, the bodily alterations, and the important role of their spouses. Nurses, as well as general practitioners, must play a more active role in follow-up to ensure that the men and their spouses receive better help and support.

  10. Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma.

    Science.gov (United States)

    Higashi-Kuwata, Nobuyo; Makino, Takamitsu; Inoue, Yuji; Takeya, Motohiro; Ihn, Hironobu

    2009-08-01

    Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor beta. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.

  11. Local adaptation of the National Physical Activity Plan: creation of the Active Living Plan for a Healthier San Antonio.

    Science.gov (United States)

    Esparza, Laura A; Velasquez, Katherine S; Zaharoff, Annette M

    2014-03-01

    Physical inactivity and related health consequences are serious public health threats. Effective strategies to facilitate and support active-living opportunities must be implemented at national, state, and local levels. San Antonio, Texas, health department officials launched the Active Living Council of San Antonio (ALCSA) to engage the community in developing a 3- to 5-year plan to promote active living. A steering committee set preliminary ALCSA aims and established a multisector membership structure modeled after the US National Physical Activity Plan (NPAP). ALCSA adopted governance standards, increased knowledge of physical activity and health, and engaged in an 18-month collaborative master plan writing process. ALCSA selected overarching strategies and evidence-based strategies for each societal sector and adapted strategies to the local context, including tactics, measures of success, and timelines. Community and expert engagement led to a localized plan reflecting national recommendations, the Active Living Plan for a Healthier San Antonio. Multisector collaborations among governmental agencies and community organizations, which were successfully developed in this case to produce the first-ever local adaptation of the NPAP, require clearly defined expectations. Lessons learned in ALCSA's organizational and plan development can serve as a model for future community-driven efforts to increase active living.

  12. Prediction of protein subcellular locations by GO-FunD-PseAA predictor.

    Science.gov (United States)

    Chou, Kuo-Chen; Cai, Yu-Dong

    2004-08-06

    The localization of a protein in a cell is closely correlated with its biological function. With the explosion of protein sequences entering into DataBanks, it is highly desired to develop an automated method that can fast identify their subcellular location. This will expedite the annotation process, providing timely useful information for both basic research and industrial application. In view of this, a powerful predictor has been developed by hybridizing the gene ontology approach [Nat. Genet. 25 (2000) 25], functional domain composition approach [J. Biol. Chem. 277 (2002) 45765], and the pseudo-amino acid composition approach [Proteins Struct. Funct. Genet. 43 (2001) 246; Erratum: ibid. 44 (2001) 60]. As a showcase, the recently constructed dataset [Bioinformatics 19 (2003) 1656] was used for demonstration. The dataset contains 7589 proteins classified into 12 subcellular locations: chloroplast, cytoplasmic, cytoskeleton, endoplasmic reticulum, extracellular, Golgi apparatus, lysosomal, mitochondrial, nuclear, peroxisomal, plasma membrane, and vacuolar. The overall success rate of prediction obtained by the jackknife cross-validation was 92%. This is so far the highest success rate performed on this dataset by following an objective and rigorous cross-validation procedure.

  13. HPSLPred: An Ensemble Multi-Label Classifier for Human Protein Subcellular Location Prediction with Imbalanced Source.

    Science.gov (United States)

    Wan, Shixiang; Duan, Yucong; Zou, Quan

    2017-09-01

    Predicting the subcellular localization of proteins is an important and challenging problem. Traditional experimental approaches are often expensive and time-consuming. Consequently, a growing number of research efforts employ a series of machine learning approaches to predict the subcellular location of proteins. There are two main challenges among the state-of-the-art prediction methods. First, most of the existing techniques are designed to deal with multi-class rather than multi-label classification, which ignores connections between multiple labels. In reality, multiple locations of particular proteins imply that there are vital and unique biological significances that deserve special focus and cannot be ignored. Second, techniques for handling imbalanced data in multi-label classification problems are necessary, but never employed. For solving these two issues, we have developed an ensemble multi-label classifier called HPSLPred, which can be applied for multi-label classification with an imbalanced protein source. For convenience, a user-friendly webserver has been established at http://server.malab.cn/HPSLPred. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Participation of the Neighborhood Representatives (Mukhtars into Managerial Activities in Local Level: Burdur Case

    Directory of Open Access Journals (Sweden)

    Mustafa LAMBA

    2017-05-01

    Full Text Available Neighborhood representatives (mukhtars are the nearest administrative units to citizens in local level. It is required to take advantage of that the neighborhood representatives know their districts and residents better than central and local government authorities. In addition, it will be appropriate to the principles of subsidiarity and the European Charter of Local Self Government to determine problems and demands of neighborhoods by their representatives. It is necessary to maintain communication channels and keep close relations with neighborhood representatives by local governments to use that advantage efficiently. It is aimed in this study to determine the participation level of neighborhood representatives into managerial activities in local level. For that purpose, the neighborhood representatives in the center of Burdur province are included in the research. According to the findings, it has been determined that the neighborhood representatives have participated into various activities without experiencing any difficulty, and they have been informed about the services regarding to their neighborhoods, and they have kept close relationship with central and local administrations, and most of them could communicate with local authorities directly. Therefore, it has been concluded that the neighborhood representatives have been influential enough in decision-making mechanisms in local level in Burdur.

  15. An implicit spatiotemporal shape model for human activity localization and recognition

    NARCIS (Netherlands)

    Oikonomopoulos, A.; Patras, I.; Pantic, Maja

    2009-01-01

    In this paper we address the problem of localisation and recognition of human activities in unsegmented image sequences. The main contribution of the proposed method is the use of an implicit representation of the spatiotemporal shape of the activity which relies on the spatiotemporal localization

  16. Response of eddy activities to localized diabatic heating in Held-Suarez simulations

    Science.gov (United States)

    Lin, Yanluan; Zhang, Jishi; Li, Xingrui; Deng, Yi

    2018-01-01

    Widespread air pollutions, such as black carbon over East Asia in recent years, could induce a localized diabatic heating, and thus lead to localized static stability and meridional temperature gradient (MTG) changes. Although effect of static stability and MTG on eddies has been addressed by the linear baroclinic instability theory, impacts of a localized heating on mid-latitude eddy activities have not been well explored and quantified. Via a series of idealized global Held-Suarez simulations with different magnitudes of localized heating at different altitudes and latitudes, responses of mid-latitude eddy activity and circulation to these temperature perturbations are systematically investigated. Climatologically, the localized heating in the lower atmosphere induces a wave-like response of eddy activity near the mid-latitude jet stream. Over the heating region, eddy activity tends to be weakening due to the increased static stability. However, there are cyclonic anomalies over the upstream and downstream of the heating region. The zonal mean eddy activity weakens along the baroclinic zone due to reduced MTG and increased static stability. Furthermore, the response of eddy activity increased as the heating magnitude is increased and moved to higher altitudes. The influence of the heating decreases as the heating is prescribed further away from the climatological mid-latitude jet. This implies that the localized heating is most effective over the region with the maximum baroclinicity. Besides, enhanced storm track downstream of the localized heating area found here suggests that increased aerosols over East Asia might strengthen the North Pacific storm track.

  17. Subcellular distribution of styrene oxide in rat liver

    International Nuclear Information System (INIS)

    Pacifici, G.M.; Cuoci, L.; Rane, A.

    1984-01-01

    The subcellular distribution of ( 3 H)-styrene-7,8-oxide was studied in the rat liver. The compound was added to liver homogenate to give a final concentration of 2 X 10(-5); 2 X 10(-4) and 2 X 10(-3) M. Subcellular fractions were obtained by differential centrifugation. Most of styrene oxide (59-88%) was associated with the cytosolic fraction. Less than 15 percent of the compound was retrieved in each of the nuclear, mitochondrial and microsomal fractions. A considerable percentage of radioactivity was found unextractable with the organic solvents, suggesting that styrene oxide reacted with the endogenous compounds. The intracellular distribution of this epoxide was also studied in the perfused rat liver. Comparable results with those previously described were obtained. The binding of styrene oxide to the cytosolic protein was investigated by equilibrium dialysis and ultrafiltration. Only a small percentage of the compound was bound to protein

  18. Brain MRI Tumor Detection using Active Contour Model and Local Image Fitting Energy

    Science.gov (United States)

    Nabizadeh, Nooshin; John, Nigel

    2014-03-01

    Automatic abnormality detection in Magnetic Resonance Imaging (MRI) is an important issue in many diagnostic and therapeutic applications. Here an automatic brain tumor detection method is introduced that uses T1-weighted images and K. Zhang et. al.'s active contour model driven by local image fitting (LIF) energy. Local image fitting energy obtains the local image information, which enables the algorithm to segment images with intensity inhomogeneities. Advantage of this method is that the LIF energy functional has less computational complexity than the local binary fitting (LBF) energy functional; moreover, it maintains the sub-pixel accuracy and boundary regularization properties. In Zhang's algorithm, a new level set method based on Gaussian filtering is used to implement the variational formulation, which is not only vigorous to prevent the energy functional from being trapped into local minimum, but also effective in keeping the level set function regular. Experiments show that the proposed method achieves high accuracy brain tumor segmentation results.

  19. Source localization of rhythmic ictal EEG activity: a study of diagnostic accuracy following STARD criteria.

    Science.gov (United States)

    Beniczky, Sándor; Lantz, Göran; Rosenzweig, Ivana; Åkeson, Per; Pedersen, Birthe; Pinborg, Lars H; Ziebell, Morten; Jespersen, Bo; Fuglsang-Frederiksen, Anders

    2013-10-01

    Although precise identification of the seizure-onset zone is an essential element of presurgical evaluation, source localization of ictal electroencephalography (EEG) signals has received little attention. The aim of our study was to estimate the accuracy of source localization of rhythmic ictal EEG activity using a distributed source model. Source localization of rhythmic ictal scalp EEG activity was performed in 42 consecutive cases fulfilling inclusion criteria. The study was designed according to recommendations for studies on diagnostic accuracy (STARD). The initial ictal EEG signals were selected using a standardized method, based on frequency analysis and voltage distribution of the ictal activity. A distributed source model-local autoregressive average (LAURA)-was used for the source localization. Sensitivity, specificity, and measurement of agreement (kappa) were determined based on the reference standard-the consensus conclusion of the multidisciplinary epilepsy surgery team. Predictive values were calculated from the surgical outcome of the operated patients. To estimate the clinical value of the ictal source analysis, we compared the likelihood ratios of concordant and discordant results. Source localization was performed blinded to the clinical data, and before the surgical decision. Reference standard was available for 33 patients. The ictal source localization had a sensitivity of 70% and a specificity of 76%. The mean measurement of agreement (kappa) was 0.61, corresponding to substantial agreement (95% confidence interval (CI) 0.38-0.84). Twenty patients underwent resective surgery. The positive predictive value (PPV) for seizure freedom was 92% and the negative predictive value (NPV) was 43%. The likelihood ratio was nine times higher for the concordant results, as compared with the discordant ones. Source localization of rhythmic ictal activity using a distributed source model (LAURA) for the ictal EEG signals selected with a standardized method

  20. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    International Nuclear Information System (INIS)

    Chandra, Subhash

    2004-01-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13 C and 15 N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13 C 15 N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39 K, 23 Na and 40 Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors

  1. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash

    2004-06-15

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  2. The role of water flow into subcellular organella in cell death

    International Nuclear Information System (INIS)

    Chiba-Kamoshida, Kaori

    2008-01-01

    Mitochondrion is a subcellular organella producing most of the energy necessary for living cells. The structure consisting of double membrane, inner and outer membranes, has a close relationship with activity and diseases. Its accurate regulation of the membrane permeability plays an important role in the homeostatic energy production. Abnormal membrane permeability has a potential to lead to cell depth. Although, even transportation of water molecule is regulated by a specific membrane protein, aquapoline, there has not been reported any method to monitor the water flow through the membrane. Neutron small-angle scattering allows us to perform measurements with biological materials and subcellular organella such as mitochondria in solution under the experimental condition maintaining the activity of the biological samples. Outstanding advantage of neutron spectroscopy is its ability to distinguish hydrogen spread over biomolecules from deuterium. In order to explore a new method to monitor conformational change inside mitochondria, wide-range neutron small angle scattering data introducing two neutron spectrometers in JAEA JRR-3, SANS-J and PNO covering not only the size for the thickness of the double membrane but also that for isolated whole mitochondria particle, ∼1 μm was employed. Utilizing the excess protein content, 70%, in the inner membrane of mitochondria, a new attempt was began to figure out the structure change in inner membrane caused by the change such as in oxygen and in the substrate concentration, and to examine the relationship between the structure change and water flow through the mitochondria membrane. (author)

  3. MEG source localization of spatially extended generators of epileptic activity: comparing entropic and hierarchical bayesian approaches.

    Science.gov (United States)

    Chowdhury, Rasheda Arman; Lina, Jean Marc; Kobayashi, Eliane; Grova, Christophe

    2013-01-01

    Localizing the generators of epileptic activity in the brain using Electro-EncephaloGraphy (EEG) or Magneto-EncephaloGraphy (MEG) signals is of particular interest during the pre-surgical investigation of epilepsy. Epileptic discharges can be detectable from background brain activity, provided they are associated with spatially extended generators. Using realistic simulations of epileptic activity, this study evaluates the ability of distributed source localization methods to accurately estimate the location of the generators and their sensitivity to the spatial extent of such generators when using MEG data. Source localization methods based on two types of realistic models have been investigated: (i) brain activity may be modeled using cortical parcels and (ii) brain activity is assumed to be locally smooth within each parcel. A Data Driven Parcellization (DDP) method was used to segment the cortical surface into non-overlapping parcels and diffusion-based spatial priors were used to model local spatial smoothness within parcels. These models were implemented within the Maximum Entropy on the Mean (MEM) and the Hierarchical Bayesian (HB) source localization frameworks. We proposed new methods in this context and compared them with other standard ones using Monte Carlo simulations of realistic MEG data involving sources of several spatial extents and depths. Detection accuracy of each method was quantified using Receiver Operating Characteristic (ROC) analysis and localization error metrics. Our results showed that methods implemented within the MEM framework were sensitive to all spatial extents of the sources ranging from 3 cm(2) to 30 cm(2), whatever were the number and size of the parcels defining the model. To reach a similar level of accuracy within the HB framework, a model using parcels larger than the size of the sources should be considered.

  4. MEG source localization of spatially extended generators of epileptic activity: comparing entropic and hierarchical bayesian approaches.

    Directory of Open Access Journals (Sweden)

    Rasheda Arman Chowdhury

    Full Text Available Localizing the generators of epileptic activity in the brain using Electro-EncephaloGraphy (EEG or Magneto-EncephaloGraphy (MEG signals is of particular interest during the pre-surgical investigation of epilepsy. Epileptic discharges can be detectable from background brain activity, provided they are associated with spatially extended generators. Using realistic simulations of epileptic activity, this study evaluates the ability of distributed source localization methods to accurately estimate the location of the generators and their sensitivity to the spatial extent of such generators when using MEG data. Source localization methods based on two types of realistic models have been investigated: (i brain activity may be modeled using cortical parcels and (ii brain activity is assumed to be locally smooth within each parcel. A Data Driven Parcellization (DDP method was used to segment the cortical surface into non-overlapping parcels and diffusion-based spatial priors were used to model local spatial smoothness within parcels. These models were implemented within the Maximum Entropy on the Mean (MEM and the Hierarchical Bayesian (HB source localization frameworks. We proposed new methods in this context and compared them with other standard ones using Monte Carlo simulations of realistic MEG data involving sources of several spatial extents and depths. Detection accuracy of each method was quantified using Receiver Operating Characteristic (ROC analysis and localization error metrics. Our results showed that methods implemented within the MEM framework were sensitive to all spatial extents of the sources ranging from 3 cm(2 to 30 cm(2, whatever were the number and size of the parcels defining the model. To reach a similar level of accuracy within the HB framework, a model using parcels larger than the size of the sources should be considered.

  5. OPPORTUNITIES OF LOCAL DEVELOPMENT USING NATURAL AND ANTHROPOGENIC RESOURCES IN TOURISM ACTIVITIES. CASE STUDY: ULMENI, MARAMUREŞ

    Directory of Open Access Journals (Sweden)

    Diana Mihaela MOJOLIC

    2013-06-01

    Full Text Available Ulmeni locality, that became town less than a decade ago, still shows, to a high degree, the general aspects of a rural locality, where agriculture is the main economic component. As capital city of the administrative-territorial division with the same name, Ulmeni town directs the activities of the entire territory. Possessing natural resources marked by the presence of Somes River and the existence of well-wooded areas, as well as anthropogenic resources embodied in values of the national heritage: museums, monuments, religious structures, folk activities, there is the possibility of providing the impulse of local development by means of tourism activities and the awareness of the need to revitalize the entire community.

  6. Muscle activation described with a differential equation model for large ensembles of locally coupled molecular motors.

    Science.gov (United States)

    Walcott, Sam

    2014-10-01

    Molecular motors, by turning chemical energy into mechanical work, are responsible for active cellular processes. Often groups of these motors work together to perform their biological role. Motors in an ensemble are coupled and exhibit complex emergent behavior. Although large motor ensembles can be modeled with partial differential equations (PDEs) by assuming that molecules function independently of their neighbors, this assumption is violated when motors are coupled locally. It is therefore unclear how to describe the ensemble behavior of the locally coupled motors responsible for biological processes such as calcium-dependent skeletal muscle activation. Here we develop a theory to describe locally coupled motor ensembles and apply the theory to skeletal muscle activation. The central idea is that a muscle filament can be divided into two phases: an active and an inactive phase. Dynamic changes in the relative size of these phases are described by a set of linear ordinary differential equations (ODEs). As the dynamics of the active phase are described by PDEs, muscle activation is governed by a set of coupled ODEs and PDEs, building on previous PDE models. With comparison to Monte Carlo simulations, we demonstrate that the theory captures the behavior of locally coupled ensembles. The theory also plausibly describes and predicts muscle experiments from molecular to whole muscle scales, suggesting that a micro- to macroscale muscle model is within reach.

  7. Changes of natural killer activity following local 60Co irradiation in intracranial tumor-bearing mice

    International Nuclear Information System (INIS)

    Otsuka, Shin-ichi; Suda, Kinya; Yamashita, Junkoh; Takeuchi, Juji; Handa, Hajime

    1982-01-01

    Changes of natural killer activity (NK activity) by local 60 Co irradiation in intracranial tumor-bearing mice were studied by the method of 51 Cr release assay. Local irradiation was administered 10 days after intracranial transplantation of 203-Glioma which had been originally induced by 20-methylcholanthrene in C57BL mice. Irradiation suppressed the growth of tumor and prolonged the mean survival time. The 50% survival time of untreated mice was about 2.5 weeks but that of mice treated by a single dose of 1000 rad and 1500 rad of irradiation was about 4.5 weeks and 6.5 weeks respectively. NK activity of spleen cells in these mice was serially examined. NK activity was gradually increased in mice treated by local irradiation, while it was gradually decreased in mice without treatment. On the other hand, NK activity remained unchanged in non-tumor-bearing control mice. Mice treated with 1000 rad and 1500 rad of irradiation showed 44.0% and 47.6% of % specific 51 Cr release respectively 11 days after irradiation while normal mice showed 18.0%. The increased NK activity after local irradiation suggested that local irradiation might have enhanced the immunological defence mechanisms against the tumor in the tumor-bearing hosts. Some characteristics of effector cells in this assay system were examined. The cytotoxicity of spleen cells was removed by the treatment of anti-BAT serum and complement but was not removed by the treatment of anti-Thy-1.2 serum and complement. Since NK activity reflects the immunological resistance to tumors to some extent, it is felt important to clarify the significance of changes of NK activity in patients with brain tumors in relation to various treatments including surgery, radiotherapy, chemotherapy and immunotherapy in the next step. (author)

  8. Redox competition mode of scanning electrochemical microscopy (RC-SECM) for visualisation of local catalytic activity.

    Science.gov (United States)

    Eckhard, Kathrin; Chen, Xingxing; Turcu, Florin; Schuhmann, Wolfgang

    2006-12-07

    In order to locally analyse catalytic activity on modified surfaces a transient redox competition mode of scanning electrochemical microscopy (SECM) has been developed. In a bi-potentiostatic experiment the SECM tip competes with the sample for the very same analyte. This leads to a current decrease at the SECM tip, if it is positioned in close proximity to an active catalyst site on the surface. Specifically, local catalytic activity of a Pt-catalyst modified sample with respect to the catalytic reduction of molecular oxygen was investigated. At higher local catalytic activity the local 02 partial pressure within the gap between accurately positioned SECM tip and sample is depleted, leading to a noticeable tip current decrease over active sites. A flexible software module has been implemented into the SECM to adapt the competition conditions by proper definition of tip and sample potentials. A potential pulse profile enables the localised electrochemically induced generation of molecular oxygen prior to the competition detection. The current decay curves are recorded over the entire duration of the applied reduction pulse. Hence, a time resolved processing of the acquired current values provides movies of the local oxygen concentration against x,y-position. The SECM redox competition mode was verified with a macroscopic Pt-disk electrode as a test sample to demonstrate the feasibility of the approach. Moreover, highly dispersed electro-deposited spots of gold and platinum on glassy carbon were visualised using the redox competition mode of SECM. Catalyst spots of different nature as well as activity inhomogeneities within one spot caused by local variations in Pt-loading were visualised successfully.

  9. Subcellular distribution of [3H]-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    International Nuclear Information System (INIS)

    Stalker, A.; Hermo, L.; Antakly, T.

    1991-01-01

    The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively

  10. Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy

    International Nuclear Information System (INIS)

    Bacquart, Thomas; Deves, Guillaume; Ortega, Richard

    2010-01-01

    Identification of arsenic chemical species at a sub-cellular level is a key to understanding the mechanisms involved in arsenic toxicology and antitumor pharmacology. When performed with a microbeam, X-ray absorption near-edge structure (μ-XANES) enables the direct speciation analysis of arsenic in sub-cellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. This methodology couples tracking of cellular organelles in a single cell by confocal or epifluorescence microscopy with local analysis of chemical species by μ-XANES. Here we report the results obtained with a μ-XANES experimental setup based on Kirkpatrick-Baez X-ray focusing optics that maintains high flux of incoming radiation (>10 11 ph/s) at micrometric spatial resolution (1.5x4.0 μm 2 ). This original experimental setup enabled the direct speciation analysis of arsenic in sub-cellular organelles with a 10 -15 g detection limit. μ-XANES shows that inorganic arsenite, As(OH) 3 , is the main form of arsenic in the cytosol, nucleus, and mitochondrial network of cultured cancer cells exposed to As 2 O 3 . On the other hand, a predominance of As(III) species is observed in HepG2 cells exposed to As(OH) 3 with, in some cases, oxidation to a pentavalent form in nuclear structures of HepG2 cells. The observation of intra-nuclear mixed redox states suggests an inter-individual variability in a cell population that can only be evidenced with direct sub-cellular speciation analysis.

  11. Preliminary study of selenium and mercury distribution in some porcine tissues and their subcellular fractions by NAA and HG-AFS

    International Nuclear Information System (INIS)

    Jiujiang Zhao; Chunying Chen; Peiqun Zhang; Zhifang Chai

    2004-01-01

    Selenium and mercury distribution in porcine tissues and their subcellular fractions from a mercury-polluted area of Guizhou Province and from a not mercury-exposed area of Beijing in China have been studied with neutron activation analysis and hydride generation-atomic fluorescence spectrometry. Both the selenium and mercury levels are higher in Guizhou porcine tissues and their subcellular fractions than those in Beijing. These two elements are highly enriched in kidney and liver of Guizhou pig, while selenium is only enriched in the kidney of Beijing pig. Exposure of mercury may result in redistribution of Se and Hg in vivo. The Hg/Se molar ratio of the subcellular fractions is very low in the case of relatively low mercury level and gradually reaches to a high constant value with increasing level of mercury, which implies that selenium and mercury may form some special complexes in the organisms. (author)

  12. Local Environmental Grassroots Activism: Contributions from Environmental Psychology, Sociology and Politics

    Science.gov (United States)

    Mihaylov, Nikolay L.; Perkins, Douglas D.

    2015-01-01

    Local environmental grassroots activism is robust and globally ubiquitous despite the ebbs and flows of the general environmental movement. In this review we synthesize social movement, environmental politics, and environmental psychology literatures to answer the following questions: How does the environment emerge as a topic for community action and how a particular environmental discourse (preservation, conservation, public health, Deep Ecology, justice, localism and other responses to modernization and development) becomes dominant? How does a community coalesce around the environmental issue and its particular framing? What is the relationship between local and supralocal (regional, national, global) activism? We contrast “Not in My Back Yard” (NIMBY) activism and environmental liberation and discuss the significance of local knowledge and scale, nature as an issue for activism, place attachment and its disruption, and place-based power inequalities. Environmental psychology contributions to established scholarship on environmental activism are proposed: the components of place attachment are conceptualized in novel ways and a continuous dweller and activist place attachment is elaborated. PMID:25806672

  13. Local environmental grassroots activism: contributions from environmental psychology, sociology and politics.

    Science.gov (United States)

    Mihaylov, Nikolay L; Perkins, Douglas D

    2015-03-23

    Local environmental grassroots activism is robust and globally ubiquitous despite the ebbs and flows of the general environmental movement. In this review we synthesize social movement, environmental politics, and environmental psychology literatures to answer the following questions: How does the environment emerge as a topic for community action and how a particular environmental discourse (preservation, conservation, public health, Deep Ecology, justice, localism and other responses to modernization and development) becomes dominant? How does a community coalesce around the environmental issue and its particular framing? What is the relationship between local and supralocal (regional, national, global) activism? We contrast "Not in My Back Yard" (NIMBY) activism and environmental liberation and discuss the significance of local knowledge and scale, nature as an issue for activism, place attachment and its disruption, and place-based power inequalities. Environmental psychology contributions to established scholarship on environmental activism are proposed: the components of place attachment are conceptualized in novel ways and a continuous dweller and activist place attachment is elaborated.

  14. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

    Science.gov (United States)

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

  15. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Science.gov (United States)

    Lake, Michael P; Bouchard, Louis-S

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  16. Travel determinants and multi-scale transferability of national activity patterns to local populations

    Energy Technology Data Exchange (ETDEWEB)

    Henson, Kriste M [Los Alamos National Laboratory; Gou; ias, Konstadinos G [UCSB

    2010-11-30

    The ability to transfer national travel patterns to a local population is of interest when attempting to model megaregions or areas that exceed metropolitan planning organization (MPO) boundaries. At the core of this research are questions about the connection between travel behavior and land use, urban form, and accessibility. As a part of this process, a group of land use variables have been identified to define activity and travel patterns for individuals and households. The 2001 National Household Travel Survey (NHTS) participants are divided into categories comprised of a set of latent cluster models representing persons, travel, and land use. These are compared to two sets of cluster models constructed for two local travel surveys. Comparison of means statistical tests are used to assess differences among sociodemographic groups residing in localities with similar land uses. The results show that the NHTS and the local surveys share mean population activity and travel characteristics. However, these similarities mask behavioral heterogeneity that are shown when distributions of activity and travel behavior are examined. Therefore, data from a national household travel survey cannot be used to model local population travel characteristics if the goal to model the actual distributions and not mean travel behavior characteristics.

  17. Localized MHD activity near transport barriers in JT-60U and TFTR

    International Nuclear Information System (INIS)

    Manickam, J.

    2001-01-01

    Localized MHD activity observed in JT-60U and TFTR near transport barriers with their associated large pressure gradients is investigated. Stability analysis of equilibria modeling the experiments supports an identification of this MHD as being due to an ideal MHD n=1 instability. The appearance of the instability depends on the local pressure gradient, local shear in the q profile and the proximity of rational surfaces where q∼m/n and m and n are the poloidal and toroidal mode numbers respectively. The mode width is shown to depend on the local value of q, and is larger when q is smaller. In addition the role of the edge current density in coupling the internal mode to the plasma edge and of the energetic particles which can drive fishbone like modes is investigated. (author)

  18. Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

    Science.gov (United States)

    Vardi, Assaf; Haramaty, Liti; Van Mooy, Benjamin A. S.; Fredricks, Helen F.; Kimmance, Susan A.; Larsen, Aud; Bidle, Kay D.

    2012-01-01

    Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host–virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the “arms race” between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi–EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host–virus interactions in natural systems. PMID:23134731

  19. In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

    2010-01-01

    A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

  20. In situ spatiotemporal mapping of flow fields around seeded stem cells at the subcellular length scale.

    Directory of Open Access Journals (Sweden)

    Min Jae Song

    2010-09-01

    Full Text Available A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms.

  1. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  2. Colonic localization of indium-111 labeled leukocytes in active Behcet's disease

    International Nuclear Information System (INIS)

    Harre, R.G.; Conrad, G.R.; Seabold, J.E.

    1988-01-01

    A patient with known Behcet's disease demonstrated intense colonic localization of In-111 labeled leukocytes. Gastrointestinal involvement had not been previously manifested, but extensive colonic inflammation was documented by endoscopy. This case illustrates the utility of In-111 labeled leukocyte imaging for detecting active bowel disease in a debilitated patient with documented Behcet's vasculitis

  3. Local contexts as activation mechanisms of market development: contemporary art in emerging markets

    NARCIS (Netherlands)

    Komarova, N.; Velthuis, O.

    2018-01-01

    The paper studies how local contexts contribute to the emergence of markets. In particular, it explains how potential entrepreneurs are motivated to become active in establishing new markets. Empirically, the focus is on contemporary art markets in two emerging countries: India and Russia. The paper

  4. Localization of Ca2+ -activated big-conductance K+ channels in rabbit distal colon

    DEFF Research Database (Denmark)

    Hay-Schmidt, Anders; Grunnet, Morten; Abrahamse, Salomon L

    2003-01-01

    Big-conductance Ca(2+)-activated K(+) channels (BK channels) may play an important role in the regulation of epithelial salt and water transport, but little is known about the expression level and the precise localization of BK channels in epithelia. The aim of the present study was to quantify a...

  5. 77 FR 26659 - Political Activity-Federal Employees Residing in Designated Localities

    Science.gov (United States)

    2012-05-07

    ... following activities: (1) Run as independent candidates for election to partisan political office in... contribution as, or on behalf of, an independent candidate for partisan political office in elections for local...) Solicit, accept, or receive uncompensated volunteer services as an independent candidate, or on behalf of...

  6. Young Children as Active Citizens in Local Government: Possibilities and Challenges from an Australian Perspective

    Science.gov (United States)

    Bartholomaeus, Clare; Gregoric, Carolyn; Krieg, Susan

    2016-01-01

    Despite considerable research and discussion regarding children and young people's rights and citizenship, the participation of young children in community decision-making is still limited. In this exploratory research, a case study is reported on how ideas about young children as active citizens are interpreted within one local government…

  7. Local elastic expansion model for viscous-flow activation energies of glass-forming molecular liquids

    DEFF Research Database (Denmark)

    Dyre, Jeppe; Olsen, Niels Boye; Christensen, Tage Emil

    1996-01-01

    A model for the viscosity of glass-forming molecular liquids is proposed in which a "flow event" requires a local volume increase. The activation energy for a flow event is identified with the work done in shoving aside the surrounding liquid; this work is proportional to the high-frequency shear...

  8. Cytochemical Localization of Catalase Activity in Methanol-Grown Hansenula polymorpha

    NARCIS (Netherlands)

    Dijken, J.P. van; Veenhuis, M.; Vermeulen, C.A.; Harder, W.

    1975-01-01

    The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorpha has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop during growth on methanol,

  9. Contribution of the local and referred pain from active myofascial trigger points in fibromyalgia syndrome

    DEFF Research Database (Denmark)

    Ge, Hong-You; Nie, Hongling; Madeleine, Pascal

    2009-01-01

    The generalized hypersensitivity associated with fibromyalgia syndrome (FMS) may in part be driven by peripheral nociceptive sources. The aim of the study was to investigate whether local and referred pain from active myofascial trigger points (MTrPs) contributes to fibromyalgia pain. FMS patients...

  10. Participation in development activities at the local level : case studies from a Sri Lankan village

    NARCIS (Netherlands)

    Frerks, G.E.

    1991-01-01

    This study is a sociological analysis of popular participation in local level development activities in Tegashena village in the Matara District, Sri Lanka. Social, economic, political and administrative factors that influence this process are identified.

    The study discusses how the

  11. Localization of endocardial ectopic activity by means of noninvasive endocardial surface current density reconstruction

    Energy Technology Data Exchange (ETDEWEB)

    Lai Dakun; Liu Chenguang; Eggen, Michael D; He Bin [Department of Biomedical Engineering, University of Minnesota, MN (United States); Iaizzo, Paul A, E-mail: binhe@umn.edu [Department of Surgery, University of Minnesota, MN (United States)

    2011-07-07

    Localization of the source of cardiac ectopic activity has direct clinical benefits for determining the location of the corresponding ectopic focus. In this study, a recently developed current-density (CD)-based localization approach was experimentally evaluated in noninvasively localizing the origin of the cardiac ectopic activity from body-surface potential maps (BSPMs) in a well-controlled experimental setting. The cardiac ectopic activities were induced in four well-controlled intact pigs by single-site pacing at various sites within the left ventricle (LV). In each pacing study, the origin of the induced ectopic activity was localized by reconstructing the CD distribution on the endocardial surface of the LV from the measured BSPMs and compared with the estimated single moving dipole (SMD) solution and precise pacing site (PS). Over the 60 analyzed beats corresponding to ten pacing sites (six for each), the mean and standard deviation of the distance between the locations of maximum CD value and the corresponding PSs were 16.9 mm and 4.6 mm, respectively. In comparison, the averaged distance between the SMD locations and the corresponding PSs was slightly larger (18.4 {+-} 3.4 mm). The obtained CD distribution of activated sources extending from the stimulus site also showed high consistency with the endocardial potential maps estimated by a minimally invasive endocardial mapping system. The present experimental results suggest that the CD method is able to locate the approximate site of the origin of a cardiac ectopic activity, and that the distribution of the CD can portray the propagation of early activation of an ectopic beat.

  12. Early diagnostic value of Bcl-3 localization in colorectal cancer

    International Nuclear Information System (INIS)

    Saamarthy, Karunakar; Björner, Sofie; Johansson, Martin; Landberg, Göran; Massoumi, Ramin; Jirström, Karin; Masoumi, Katarzyna Chmielarska

    2015-01-01

    B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. Elevated expression, sustained nuclear accumulation, and uncontrolled activation of Bcl-3 causes increased cellular proliferation or survival, dependent on the tissue and type of stimuli. We retrospectively reviewed patients who were diagnosed with colorectal cancer at Skåne University Hospital in Malmö between 1st of January 1990 and 31st of December 1991. Bcl-3 localization in colorectal cancer was assessed by immunohistochemistry on tissue microarray and freshly isolated colon from patients. Correlation between Bcl-3 localization and clinicopathological parameters of the cohort were evaluated using the Spearman rank-order correlation coefficient. In addition, Bcl-3 expression and localization in colon adenocarcinoma cells were analysed by western blot, immunohistochemistry and subcellular fractionation separately. We found that Bcl-3 was mainly localized in the cytoplasm in the tumour tissue isolated from colon cancer patients. Normal colon samples from the same patients showed Bcl-3 localization in the nucleus. In three out of six colon cancer cell lines, we detected elevated levels of Bcl-3. In these cell lines Bcl-3 was accumulated in the cytosol. We confirmed these findings by analysing Bcl-3 localization in a colon tissue micro array consisting of 270 cases. In these samples Bcl-3 localization correlated with the proliferation marker Ki-67, but not with the apoptotic marker Caspase 3. These findings indicate that analysis of the subcellular localization of Bcl-3 could be a potential-early diagnostic marker in colon cancer

  13. The Effect of Local Events to Rural Tourism as a Recreational Activity

    Directory of Open Access Journals (Sweden)

    Elif Zeynep ÖZER

    2014-08-01

    Full Text Available Recreation is the activities that person attends voluntarily in his/her spare time to refreshing, relaxing and motivation. Activities that are made in rural area are option for recreational activities. There is an increase in consumer demand for rural tour ism as an alternative tourism option. Participants get a chance to know different cultural structures and chance to see natural beauties by attending rural activities. Events that are performed with attendees form different destinations are support area fr om economy, development and advertising point of view. Objective of this work is making contribution to development rural tourism and recreational activities by defining the effect of local events to rural tourism as a recreational activity. In this work, the effect of participation of recreational tourism activities to rural tourism is investigated. Data that is required is gathered by semi structured interview technique. The result of this work has a potential to use a resource to lead event managers. Thi s work also has a potential to use a resource for studies that are related to recreation tourism, local activities and rural tourism.

  14. Subcellular partitioning kinetics, metallothionein response and oxidative damage in the marine mussel Mytilus galloprovincialis exposed to cadmium-based quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, Thiago Lopes [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Gomes, Tânia [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo (Norway); Durigon, Emerson Giuliani [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Bebianno, Maria João, E-mail: mbebian@ualg.pt [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)

    2016-06-01

    The environmental health impact of metal-based nanomaterials is of emerging concern, but their metabolism and detoxification pathways in marine bioindicator species remain unclear. This study investigated the role of subcellular partitioning kinetics, metallothioneins (MTs) response and oxidative damage (lipid peroxidation – LPO) in the marine mussel Mytilus galloprovincialis exposed to CdTe quantum dots (QDs) in comparison with its dissolved counterpart. Mussels were exposed to QDs and dissolved Cd for 21 days at 10 μg Cd L{sup −1} followed by a 50 days depuration. Higher Cd concentrations were detected in fractions containing mitochondria, nucleus and lysosomes, suggesting potential subcellular targets of QDs toxicity in mussel tissues. Tissue specific metabolism patterns were observed in mussels exposed to both Cd forms. Although MT levels were directly associated with Cd in both forms, QDs subcellular partitioning is linked to biologically active metal (BAM), but no increase in LPO occurred, while in the case of dissolved Cd levels are in the biologically detoxified metal (BDM) form, indicating nano-specific effects. Mussel gills showed lower detoxification capability of QDs, while the digestive gland is the major tissue for storage and detoxification of both Cd forms. Both mussel tissues were unable to completely eliminate the Cd accumulated in the QDs form (estimated half-life time > 50 days), highlighting the potential source of Cd and QDs toxicity for human and environmental health. Results indicate tissue specific metabolism patterns and nano-specific effects in marine mussel exposed to QDs. - Highlights: • Subcellular partitioning and MT response are Cd form, tissue and time dependent. • Tissue specific metabolism of Cd-based quantum dots (QDs) in marine mussels. • QDs are slower biologically detoxified when compared to dissolved Cd. • Subcellular partitioning and biomarker responses indicate nano-specific effects. • Subcellular

  15. Dynamic neuroanatomy at subcellular resolution in the zebrafish.

    Science.gov (United States)

    Faucherre, Adèle; López-Schier, Hernán

    2014-01-01

    Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

  16. MECHANISMS OF DAMAGING EFFECT OF MANGENESE IN TOXIC CONCENTRATIONS ON CELLULAR AND SUBCELLULAR LEVELS

    Directory of Open Access Journals (Sweden)

    Goncharenko A. V.

    2012-11-01

    Full Text Available Influence of subtoxic concentration of manganese chloride in dose equal to LD 50 on condition of plasmatic membranes (model: erythrocytes and functional activity of cell power (model: the isolated liver mitochondrion of rats was studied. It was established that manganese chloride in fixed concentration caused authentic augmentation of sorption capacity of erythrocytes towards alcian blue, influenced increasing of their spontaneous haemolysis and activation of peroxide oxidation of lipids. In experiment on the isolated mitochondrion it was proved that manganese chloride caused dissociation of an oxidizing phosphorusling and complete inhibition of respiration in concentrations of 3 and 4,5mM. These dependences testify that subtoxic concentration of manganese can damage the cell energy. Thus, this pilot research indicated damaging effect of manganese on cellular (erythrocytes and subcellular (mitochondrion levels which are realized through external functioning of membrane structures and deprived them from restoration.

  17. Local and Landscape Correlates of Spider Activity Density and Species Richness in Urban Gardens.

    Science.gov (United States)

    Otoshi, Michelle D; Bichier, Peter; Philpott, Stacy M

    2015-08-01

    Urbanization is a major threat to arthropod biodiversity and abundance due to reduction and loss of suitable natural habitat. Green spaces and small-scale agricultural areas may provide habitat and resources for arthropods within densely developed cities. We studied spider activity density (a measure of both abundance and degree of movement) and diversity in urban gardens in Santa Cruz, Santa Clara, and Monterey counties in central California, USA. We sampled for spiders with pitfall traps and sampled 38 local site characteristics for 5 mo in 19 garden sites to determine the relative importance of individual local factors. We also analyzed 16 landscape variables at 500-m and 1-km buffers surrounding each garden to determine the significance of landscape factors. We identified individuals from the most common families to species and identified individuals from other families to morphospecies. Species from the families Lycosidae and Gnaphosidae composed 81% of total adult spider individuals. Most of the significant factors that correlated with spider activity density and richness were local rather than landscape factors. Spider activity density and richness increased with mulch cover and flowering plant species, and decreased with bare soil. Thus, changes in local garden management have the potential to promote diversity of functionally important spiders in urban environments. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Children's active free play in local neighborhoods: a behavioral mapping study.

    Science.gov (United States)

    Veitch, J; Salmon, J; Ball, K

    2008-10-01

    Many Australian children are more sedentary than they should be, and almost one in five are currently overweight or obese. Some children may face difficulties finding opportunities to be active, having poor access to safe public open spaces or having low independent mobility limiting their access to places to play. This study aimed to examine children's access to places in their neighborhood for active free play and how these vary by age, sex and socioeconomic status (SES). Behavioral maps of the local neighborhood were completed by children (8-12 years) from five primary schools across different areas of Melbourne. Children living in low SES outer-urban neighborhoods had to travel greater distances to access local parks compared with those in inner-urban mid and high SES areas. One-third (32%) of children reported an independent mobility range of <100 m from home. In conclusion, for some children opportunities to engage in active free play in the local neighborhood may be limited due to lack of parks in close proximity to home and restricted independent mobility. It is important to collaborate with local governments, urban planners and community groups to improve access to neighborhood parks and to promote a sense of neighborhood safety.

  19. Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

    Science.gov (United States)

    Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

    2018-01-01

    Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

  20. A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

    Science.gov (United States)

    Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

    2011-01-12

    We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

  1. Subcellular Location of PKA Controls Striatal Plasticity: Stochastic Simulations in Spiny Dendrites

    Science.gov (United States)

    Oliveira, Rodrigo F.; Kim, MyungSook; Blackwell, Kim T.

    2012-01-01

    Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity. PMID:22346744

  2. Recurrently connected and localized neuronal communities initiate coordinated spontaneous activity in neuronal networks

    Science.gov (United States)

    Amin, Hayder; Maccione, Alessandro; Nieus, Thierry

    2017-01-01

    Developing neuronal systems intrinsically generate coordinated spontaneous activity that propagates by involving a large number of synchronously firing neurons. In vivo, waves of spikes transiently characterize the activity of developing brain circuits and are fundamental for activity-dependent circuit formation. In vitro, coordinated spontaneous spiking activity, or network bursts (NBs), interleaved within periods of asynchronous spikes emerge during the development of 2D and 3D neuronal cultures. Several studies have investigated this type of activity and its dynamics, but how a neuronal system generates these coordinated events remains unclear. Here, we investigate at a cellular level the generation of network bursts in spontaneously active neuronal cultures by exploiting high-resolution multielectrode array recordings and computational network modelling. Our analysis reveals that NBs are generated in specialized regions of the network (functional neuronal communities) that feature neuronal links with high cross-correlation peak values, sub-millisecond lags and that share very similar structural connectivity motifs providing recurrent interactions. We show that the particular properties of these local structures enable locally amplifying spontaneous asynchronous spikes and that this mechanism can lead to the initiation of NBs. Through the analysis of simulated and experimental data, we also show that AMPA currents drive the coordinated activity, while NMDA and GABA currents are only involved in shaping the dynamics of NBs. Overall, our results suggest that the presence of functional neuronal communities with recurrent local connections allows a neuronal system to generate spontaneous coordinated spiking activity events. As suggested by the rules used for implementing our computational model, such functional communities might naturally emerge during network development by following simple constraints on distance-based connectivity. PMID:28749937

  3. Recurrently connected and localized neuronal communities initiate coordinated spontaneous activity in neuronal networks.

    Directory of Open Access Journals (Sweden)

    Davide Lonardoni

    2017-07-01

    Full Text Available Developing neuronal systems intrinsically generate coordinated spontaneous activity that propagates by involving a large number of synchronously firing neurons. In vivo, waves of spikes transiently characterize the activity of developing brain circuits and are fundamental for activity-dependent circuit formation. In vitro, coordinated spontaneous spiking activity, or network bursts (NBs, interleaved within periods of asynchronous spikes emerge during the development of 2D and 3D neuronal cultures. Several studies have investigated this type of activity and its dynamics, but how a neuronal system generates these coordinated events remains unclear. Here, we investigate at a cellular level the generation of network bursts in spontaneously active neuronal cultures by exploiting high-resolution multielectrode array recordings and computational network modelling. Our analysis reveals that NBs are generated in specialized regions of the network (functional neuronal communities that feature neuronal links with high cross-correlation peak values, sub-millisecond lags and that share very similar structural connectivity motifs providing recurrent interactions. We show that the particular properties of these local structures enable locally amplifying spontaneous asynchronous spikes and that this mechanism can lead to the initiation of NBs. Through the analysis of simulated and experimental data, we also show that AMPA currents drive the coordinated activity, while NMDA and GABA currents are only involved in shaping the dynamics of NBs. Overall, our results suggest that the presence of functional neuronal communities with recurrent local connections allows a neuronal system to generate spontaneous coordinated spiking activity events. As suggested by the rules used for implementing our computational model, such functional communities might naturally emerge during network development by following simple constraints on distance-based connectivity.

  4. A computational framework for the optimal design of morphing processes in locally activated smart material structures

    International Nuclear Information System (INIS)

    Wang, Shuang; Brigham, John C

    2012-01-01

    A proof-of-concept study is presented for a strategy to obtain maximally efficient and accurate morphing structures composed of active materials such as shape memory polymers (SMP) through synchronization of adaptable and localized activation and actuation. The work focuses on structures or structural components entirely composed of thermo-responsive SMP, and particularly utilizes the ability of such materials to display controllable variable stiffness. The study presents and employs a computational inverse mechanics approach that combines a computational representation of the SMP thermo-mechanical behavior with a nonlinear optimization algorithm to determine location, magnitude and sequencing of the activation and actuation to obtain a desired shape change subject to design objectives such as prevention of damage. Two numerical examples are presented in which the synchronization of the activation and actuation and the location of activation excitation were optimized with respect to the combined thermal and mechanical energy for design concepts in morphing skeletal structural components. In all cases the concept of localized activation along with the optimal design strategy were able to produce far more energy efficient morphing structures and more accurately reach the desired shape change in comparison to traditional methods that require complete structural activation prior to actuation. (paper)

  5. Piper betle LEAVES EXTRACT PATCH: EVALUATION OF ANTIBACTERIAL ACTIVITY, RELEASE PROFILE OF EUGENOL, AND LOCAL TOLERANCE

    Directory of Open Access Journals (Sweden)

    Mufrod Mufrod

    2016-08-01

    Full Text Available Piper betle leaf in extract form is more effective than crude drug. Eugenol is a component in the extract that has antibacterial activity but irritate. Patch of piper betle leaf extract was used on the mucosa to make oral cavity hygiene. Antibacterial activity was influenced by the release of eugenol from the patch. Release enhancer substances (RES such as glycerin, propylen glicol and tween 80 were added in patch formulation to increase the release of active substances. The aim of the research was to investigate the physicochemical properties, eugenol release profiles, and local tolerance test of the patch. Extract of piper betle leaf was made using infundation method. Patch was made according to the variation concentration of extract (1, 2 and 4% and RES (glycerine, propylen glycol and tween 80 using chitosan as vehicle. Patch produced solvent casting method. Patch obtained was tested for swelling index, folding endurance, surface pH, antibacterial activity, release of eugenol, and local tolerance. Data obtained were analyzed descriptively. The result showed that the addition of RES did not affect the surface pH but increase the water absorption with in inconsistent way except patch with tween 80. The flexibility (folding endurance value increased, and the highest amount of eugenol released was achieved by patch using propylen glicol. Patch with tween 80 and glycerin for all extract concentration and patch with 1% extract concentration using propylen glycol showed medium sensation (local tolerance, and patch with 2 and 4% extract using propylen glycol showed severe sensation.

  6. Altered medial temporal activation related to local glutamate levels in subjects with prodromal signs of psychosis.

    Science.gov (United States)

    Valli, Isabel; Stone, James; Mechelli, Andrea; Bhattacharyya, Sagnik; Raffin, Marie; Allen, Paul; Fusar-Poli, Paolo; Lythgoe, David; O'Gorman, Ruth; Seal, Marc; McGuire, Philip

    2011-01-01

    Both medial temporal cortical dysfunction and perturbed glutamatergic neurotransmission are regarded as fundamental pathophysiological features of psychosis. However, although animal models of psychosis suggest that these two abnormalities are interrelated, their relationship in humans has yet to be investigated. We used a combination of functional magnetic resonance imaging and magnetic resonance spectroscopy to investigate the relationship between medial temporal activation during an episodic memory task and local glutamate levels in 22 individuals with an at-risk mental state for psychosis and 14 healthy volunteers. We observed a significant between-group difference in the coupling of medial temporal activation with local glutamate levels. In control subjects, medial temporal activation during episodic encoding was positively associated with medial temporal glutamate. However, in the clinical population, medial temporal activation was reduced, and the relationship with glutamate was absent. In individuals at high risk of psychosis, medial temporal dysfunction seemed related to a loss of the normal relationship with local glutamate levels. This study provides the first evidence that links medial temporal dysfunction with the central glutamate system in humans and is consistent with evidence that drugs that modulate glutamatergic transmission might be useful in the treatment of psychosis. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  7. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA

    Directory of Open Access Journals (Sweden)

    N. Charles Harata

    2014-09-01

    Full Text Available Background: An in‐frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE‐torsinA results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown.Methods: We evaluated the literature regarding the subcellular localization of torsinA.Results: Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild‐type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A into host cells and overexpress the protein therein. In both neurons and non‐neuronal cells, exogenous wild‐type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE‐torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate.Discussion: As patients’ cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non‐overexpressed vs. exogenously introduced (overexpressed proteins.

  8. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA.

    Science.gov (United States)

    Harata, N Charles

    2014-01-01

    An in-frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE-torsinA) results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown. We evaluated the literature regarding the subcellular localization of torsinA. Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild-type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A) into host cells and overexpress the protein therein. In both neurons and non-neuronal cells, exogenous wild-type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE-torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate. As patients' cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non-overexpressed) vs. exogenously introduced (overexpressed) proteins.

  9. Local increase of anticyclonic wave activity over northern Eurasia under amplified Arctic warming: WAVE ACTIVITY RESPONSE TO ARCTIC MELTING

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Daokai [School of Atmospheric Sciences, Nanjing University, Nanjing China; Lu, Jian [Atmospheric Sciences and Global Change Division, Pacific Northwest National Laboratory, Richland Washington USA; Sun, Lantao [CIRES, University of Colorado Boulder, Boulder Colorado USA; PSD, ESRL, NOAA, Boulder Colorado USA; Chen, Gang [Department of Earth and Atmospheric Sciences, UCLA, Los Angeles California USA; Zhang, Yaocun [School of Atmospheric Sciences, Nanjing University, Nanjing China

    2017-04-10

    In an attempt to resolve the controversy as to whether Arctic sea ice loss leads to more mid-latitude extremes, a metric of finite-amplitude wave activity is adopted to quantify the midlatitude wave activity and its change during the observed period of the drastic Arctic sea ice decline in both ERA Interim reanalysis data and a set of AMIP-type of atmospheric model experiments. Neither the experiment with the trend in the SST or that with the declining trend of Arctic sea ice can simulate the sizable midlatitude-wide reduction in the total wave activity (Ae) observed in the reanalysis, leaving its explanation to the atmospheric internal variability. On the other hand, both the diagnostics of the flux of the local wave activity and the model experiments lend evidence to a possible linkage between the sea ice loss near the Barents and Kara seas and the increasing trend of anticyclonic local wave activity over the northern part of the central Eurasia and the associated impacts on the frequency of temperature extremes.

  10. Retention and subcellular distribution of 67Ga in normal organs

    International Nuclear Information System (INIS)

    Ando, A.; Ando, I.; Hiraki, T.

    1986-01-01

    Using normal rats, retention values and subcellular distribution of 67 Ga in each organ were investigated. At 10 min after administration of 67 Ga-citrate the retention value of 67 Ga in blood was 6.77% dose/g, and this value decreased with time. The values for skeletal muscle, lung, pancreas, adrenal, heart muscle, brain, small intestine, large intestine and spinal cord were the highest at 10 min after administration, and they decreased with time. Conversely this value in bone increased until 10 days after injection. But in the liver, kidney, and stomach, these values increased with time after administration and were highest 24 h or 48 h after injection. After that, they decreased with time. The value in spleen reached a plateau 48 h after administration, and hardly varied for 10 days. From the results of subcellular fractionation, it was deduced that lysosome plays quite an important role in the concentration of 67 Ga in small intestine, stomach, lung, kidney and pancreas; a lesser role in its concentration in heart muscle, and hardly any role in the 67 Ga accumulation in skeletal muscle. In spleen, the contents in nuclear, mitochrondrial, microsomal, and supernatant fractions all contributed to the accumulation of 67 Ga. (orig.) [de

  11. Signal peptides and protein localization prediction

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2005-01-01

    In 1999, the Nobel prize in Physiology or Medicine was awarded to Gunther Blobel “for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell”. Since the subcellular localization of a protein is an important clue to its function, the characteriz...

  12. Volumetric localization of epileptic activities in tuberous sclerosis using synthetic aperture magnetometry

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Zheng [Hospital for Sick Children, Research Institute, Toronto (Canada); Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Xiang, Jing [Hospital for Sick Children, Research Institute, Toronto (Canada); Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Holowka, Stephanie; Chuang, Sylvester [Hospital for Sick Children, Department of Diagnostic Imaging, Toronto (Canada); Hunjan, Amrita; Sharma, Rohit; Otsubo, Hiroshi [Hospital for Sick Children, Division of Neurology, Toronto (Canada)

    2006-01-01

    Magnetoencephalography (MEG) is a novel noninvasive technique for localizing epileptic zones. Tuberous sclerosis complex (TSC) is often associated with medically refractory epilepsy with multiple epileptic zones. Surgical treatment of TSC requires accurate localization of epileptogenic tubers. The objective of this study was to introduce a new MEG technique, synthetic aperture magnetometry (SAM), to volumetrically localize irritable zones and clarify the correlations between SAM, dipole modeling and anatomical tubers. Eight pediatric patients with TSC confirmed by clinical and neuroimaging findings were retrospectively studied. MEG data were recorded using a whole-cortex CTF OMEGA system. Sleep deprivation was employed to provoke epileptiform activity. Irritable zones were localized using both dipole modeling and SAM. MRI detected 42 tubers in the eight patients. Dipole modeling localized 28 irritable zones, and 19 out of the 28 zones were near tubers (19/42, 45%). SAM found 51 irritable zones, and 31 out of the 51 zones were near tubers (31/42, 74%). Among the 51 irritable zones determined by SAM, thirty-five zones were in 1-35 Hz, nine zones were in 35-60 Hz, and seven zones were in 60-120 Hz. The new method, SAM, yielded very plausible equivalent sources for patients who showed anatomical tubers on MRI. Compared to conventional dipole modeling, SAM appeared to offer increased detection of irritable zones and beneficial volumetric and frequency descriptions. (orig.)

  13. TMS-induced cortical potentiation during wakefulness locally increases slow wave activity during sleep.

    Directory of Open Access Journals (Sweden)

    Reto Huber

    2007-03-01

    Full Text Available Sleep slow wave activity (SWA is thought to reflect sleep need, increasing in proportion to the length of prior wakefulness and decreasing during sleep. However, the process responsible for SWA regulation is not known. We showed recently that SWA increases locally after a learning task involving a circumscribed brain region, suggesting that SWA may reflect plastic changes triggered by learning.To test this hypothesis directly, we used transcranial magnetic stimulation (TMS in conjunction with high-density EEG in humans. We show that 5-Hz TMS applied to motor cortex induces a localized potentiation of TMS-evoked cortical EEG responses. We then show that, in the sleep episode following 5-Hz TMS, SWA increases markedly (+39.1+/-17.4%, p<0.01, n = 10. Electrode coregistration with magnetic resonance images localized the increase in SWA to the same premotor site as the maximum TMS-induced potentiation during wakefulness. Moreover, the magnitude of potentiation during wakefulness predicts the local increase in SWA during sleep.These results provide direct evidence for a link between plastic changes and the local regulation of sleep need.

  14. Local and Widely Distributed EEG Activity in Schizophrenia With Prevalence of Negative Symptoms.

    Science.gov (United States)

    Grin-Yatsenko, Vera A; Ponomarev, Valery A; Pronina, Marina V; Poliakov, Yury I; Plotnikova, Irina V; Kropotov, Juri D

    2017-09-01

    We evaluated EEG frequency abnormalities in resting state (eyes closed and eyes open) EEG in a group of chronic schizophrenia patients as compared with healthy subjects. The study included 3 methods of analysis of deviation of EEG characteristics: genuine EEG, current source density (CSD), and group independent component (gIC). All 3 methods have shown that the EEG in schizophrenia patients is characterized by enhanced low-frequency (delta and theta) and high-frequency (beta) activity in comparison with the control group. However, the spatial pattern of differences was dependent on the type of method used. Comparative analysis has shown that increased EEG power in schizophrenia patients apparently concerns both widely spatially distributed components and local components of signal. Furthermore, the observed differences in the delta and theta range can be described mainly by the local components, and those in the beta range mostly by spatially widely distributed ones. The possible nature of the widely distributed activity is discussed.

  15. Novel p47phox-related organizers regulate NADPH oxidase 1 (Nox1) activity and localization

    Science.gov (United States)

    Gianni, Davide; Diaz, Begoña; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A.; Bokoch, Gary M.

    2010-01-01

    The mechanisms that determine localized formation of reactive oxygen species (ROS) via NADPH oxidases (Nox) in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 and Tks5 are functional members of a p47phox-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (vs. Nox2 and Nox4) activity in reconstituted cellular systems, and interact with the NoxA1 activator protein through an SH3-mediated interaction. Endogenous Tks4 is required for Rac GTPase-dependent ROS production by DLD1 colon cancer cells. Tks4 recruits Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent novel members of an organizer superfamily that link Nox to localized ROS formation. PMID:19755710

  16. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

    Science.gov (United States)

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber’s oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  17. Health promotion activities in annual reports of local governments: 'Health for All' targets as a tool for content analysis.

    Science.gov (United States)

    Andersson, Camilla M; Bjärås, Gunilla E M; Tillgren, Per; Ostenson, Claes-Göran

    2003-09-01

    This article presents an instrument to study the annual reporting of health promotion activities in local governments within the three intervention municipalities of the Stockholm Diabetes Prevention Program (SDPP). The content of health promotion activities are described and the strengths, weaknesses and relevance of the method to health promotion discussed. A content analysis of local governmental reports from 1995-2000 in three Swedish municipalities. A matrix with WHO's 38 'Health for All' (HFA) targets from 1991 was used when coding the local health promotion activities. There are many public health initiatives within the local governmental structure even if they are not always addressed as health promotion. The main focuses in the local governmental reports were environmental issues, unemployment, social care and welfare. Local governmental reports were found to be a useful source of information that could provide knowledge about the priorities and organizational capacities for health promotion within local authorities. Additionally the HFA targets were an effective tool to identify and categorize systematically local health promotion activities in the annual reports of local governments. Identifying local health promotion initiatives by local authorities may ease the development of a health perspective and joint actions within the existing political and administrative structure. This paper provides a complementary method of attaining and structuring information about the local community for developments in health promotion.

  18. [Local brain activity in different motor subtypes of Parkinson's disease with fMRI].

    Science.gov (United States)

    Hou, Ya'nan; Zhang, Jiarong; Chen, Biao; Wu, Tao

    2015-02-17

    To explore the changes of local brain activity in motor subtypes of Parkinson's disease (PD) with functional magnetic resonance imaging (fMRI). A total of 60 idiopathic PD and 30 age- and gender-matched normal controls were examined with resting-state fMRI from January 2013 to March 2014. All subjects gave their written informed consent for the study. The amplitude of low-frequency fluctuation (ALFF) was calculated to measure local brain activity. The PD patients were divided into two groups of tremor dominant (TD) and postural instability/gait difficulty (PIGD) (n = 30 each). All subjects gave their written in formed consent for the study.One-way ANOVA and post-hoc t-test were performed to detect the differences of local brain activity between PD and normal subjects. And the correlations were examined between ALFF, scores and levodopa dose. Compared with normal subjects, the TD group showed increased activity in bilateral cerebellums (-37, -47, -38), thalamus (-18, -17,0), pons (-3, -23, -37) and left precentral gyrus (-41, -30, 46) versus decreased activity in bilateral frontal lobes (-13, 69, 6), temporal lobes (-42, 18, -21), left insula (-32, 22, 2) and left anterior cingulated (-7, 32, -5). The PIGD group showed increased activity in right postcentral gyrus (63, -18, 39) and decreased activity in bilateral putamens (-24, 12, 3), pre-supplementary motor area (10, 10, 58), frontal lobes (15, -15, 57), temporal lobes (-39, 18, -3) and left insula (-29, 20, 11). Compared with PIGD, the TD group showed increased activity in temporal lobes, but decreased activity in frontal lobes. Additionally, ALFF in bilateral cerebellums and frontal lobes was positively correlated with TD scores while ALFF in left precentral gyrus, bilateral putamens and temporal lobes negatively correlated with TD scores. ALFF in bilateral frontal lobes and left temporal lobe was positively correlated with PIGD scores.However, in right postcentral gyrus and bilateral putamens, ALFF was

  19. Local activation time sampling density for atrial tachycardia contact mapping: how much is enough?

    Science.gov (United States)

    Williams, Steven E; Harrison, James L; Chubb, Henry; Whitaker, John; Kiedrowicz, Radek; Rinaldi, Christopher A; Cooklin, Michael; Wright, Matthew; Niederer, Steven; O'Neill, Mark D

    2018-02-01

    Local activation time (LAT) mapping forms the cornerstone of atrial tachycardia diagnosis. Although anatomic and positional accuracy of electroanatomic mapping (EAM) systems have been validated, the effect of electrode sampling density on LAT map reconstruction is not known. Here, we study the effect of chamber geometry and activation complexity on optimal LAT sampling density using a combined in silico and in vivo approach. In vivo 21 atrial tachycardia maps were studied in three groups: (1) focal activation, (2) macro-re-entry, and (3) localized re-entry. In silico activation was simulated on a 4×4cm atrial monolayer, sampled randomly at 0.25-10 points/cm2 and used to re-interpolate LAT maps. Activation patterns were studied in the geometrically simple porcine right atrium (RA) and complex human left atrium (LA). Activation complexity was introduced into the porcine RA by incomplete inter-caval linear ablation. In all cases, optimal sampling density was defined as the highest density resulting in minimal further error reduction in the re-interpolated maps. Optimal sampling densities for LA tachycardias were 0.67 ± 0.17 points/cm2 (focal activation), 1.05 ± 0.32 points/cm2 (macro-re-entry) and 1.23 ± 0.26 points/cm2 (localized re-entry), P = 0.0031. Increasing activation complexity was associated with increased optimal sampling density both in silico (focal activation 1.09 ± 0.14 points/cm2; re-entry 1.44 ± 0.49 points/cm2; spiral-wave 1.50 ± 0.34 points/cm2, P density (0.61 ± 0.22 points/cm2 vs. 1.0 ± 0.34 points/cm2, P = 0.0015). Optimal sampling densities can be identified to maximize diagnostic yield of LAT maps. Greater sampling density is required to correctly reveal complex activation and represent activation across complex geometries. Overall, the optimal sampling density for LAT map interpolation defined in this study was ∼1.0-1.5 points/cm2. Published on behalf of the European Society of

  20. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  1. Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

    Science.gov (United States)

    Csukai, M; Mochly-Rosen, D

    1999-04-01

    Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems. Copyright 1999 The Italian Pharmacological Society.

  2. Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-11

    Science.gov (United States)

    Hyun, Young-Min; Chung, Hung-Li; McGrath, James L.; Waugh, Richard E.; Kim, Minsoo

    2009-01-01

    Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. Very Late Antigen-4 (VLA-4, α4β1) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer (FRET) analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence (TIRF) microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells, and is critical for T cell migration on VCAM-1. PMID:19542447

  3. Stronger activation of SREBP-1a by nucleus-localized HBx

    International Nuclear Information System (INIS)

    Wu, Qi; Qiao, Ling; Yang, Jian; Zhou, Yan; Liu, Qiang

    2015-01-01

    We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of the nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication

  4. Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation.

    Science.gov (United States)

    Proudhon, Charlotte; Snetkova, Valentina; Raviram, Ramya; Lobry, Camille; Badri, Sana; Jiang, Tingting; Hao, Bingtao; Trimarchi, Thomas; Kluger, Yuval; Aifantis, Iannis; Bonneau, Richard; Skok, Jane A

    2016-06-07

    V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation

    Directory of Open Access Journals (Sweden)

    Charlotte Proudhon

    2016-06-01

    Full Text Available V(DJ recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.