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Sample records for kda regulates nucleocytoplasmic

  1. Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5.

    PubMed

    Tong, Edith H Y; Guo, Jin-Jun; Huang, Ai-Long; Liu, Han; Hu, Chang-Deng; Chung, Stephen S M; Ko, Ben C B

    2006-08-18

    The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity. PMID:16782704

  2. Hormone- and light-regulated nucleocytoplasmic transport in plants: current status.

    PubMed

    Lee, Yew; Lee, Hak-Soo; Lee, June-Seung; Kim, Seong-Ki; Kim, Soo-Hwan

    2008-01-01

    The gene regulation mechanisms underlying hormone- and light-induced signal transduction in plants rely not only on post-translational modification and protein degradation, but also on selective inclusion and exclusion of proteins from the nucleus. For example, plant cells treated with light or hormones actively transport many signalling regulatory proteins, transcription factors, and even photoreceptors and hormone receptors into the nucleus, while actively excluding other proteins. The nuclear envelope (NE) is the physical and functional barrier that mediates this selective partitioning, and nuclear transport regulators transduce hormone- or light-initiated signalling pathways across the membrane to mediate nuclear activities. Recent reports revealed that mutating the proteins regulating nuclear transport through the pores, such as nucleoporins, alters the plant's response to a stimulus. In this review, recent works are introduced that have revealed the importance of regulated nucleocytoplasmic partitioning. These important findings deepen our understanding about how co-ordinated plant hormone and light signal transduction pathways facilitate communication between the cytoplasm and the nucleus. The roles of nucleoporin components within the nuclear pore complex (NPC) are also emphasized, as well as nuclear transport cargo, such as Ran/TC4 and its binding proteins (RanBPs), in this process. Recent findings concerning these proteins may provide a possible direction by which to characterize the regulatory potential of hormone- or light-triggered nuclear transport.

  3. Functional activity of RLIM/Rnf12 is regulated by phosphorylation-dependent nucleocytoplasmic shuttling

    PubMed Central

    Jiao, Baowei; Taniguchi-Ishigaki, Naoko; Güngör, Cenap; Peters, Marvin A.; Chen, Ya-Wen; Riethdorf, Sabine; Drung, Alexander; Ahronian, Leanne G.; Shin, JongDae; Pagnis, Rachna; Pantel, Klaus; Tachibana, Taro; Lewis, Brian C.; Johnsen, Steven A.; Bach, Ingolf

    2013-01-01

    The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain–interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival. PMID:23904271

  4. Salt Stress and Ethylene Antagonistically Regulate Nucleocytoplasmic Partitioning of COP1 to Control Seed Germination.

    PubMed

    Yu, Yanwen; Wang, Juan; Shi, Hui; Gu, Juntao; Dong, Jingao; Deng, Xing Wang; Huang, Rongfeng

    2016-04-01

    Seed germination, a critical stage initiating the life cycle of a plant, is severely affected by salt stress. However, the underlying mechanism of salt inhibition of seed germination (SSG) is unclear. Here, we report that the Arabidopsis (Arabidopsis thaliana) CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) counteracts SSG Genetic assays provide evidence that SSG in loss of function of the COP1 mutant was stronger than this in the wild type. A GUS-COP1 fusion was constitutively localized to the nucleus in radicle cells. Salt treatment caused COP1 to be retained in the cytosol, but the addition of ethylene precursor 1-aminocyclopropane-1-carboxylate had the reverse effect on the translocation of COP1 to the nucleus, revealing that ethylene and salt exert opposite regulatory effects on the localization of COP1 in germinating seeds. However, loss of function of the ETHYLENE INSENSITIVE3 (EIN3) mutant impaired the ethylene-mediated rescue of the salt restriction of COP1 to the nucleus. Further research showed that the interaction between COP1 and LONG HYPOCOTYL5 (HY5) had a role in SSG Correspondingly, SSG in loss of function of HY5 was suppressed. Biochemical detection showed that salt promoted the stabilization of HY5, whereas ethylene restricted its accumulation. Furthermore, salt treatment stimulated and ethylene suppressed transcription of ABA INSENSITIVE5 (ABI5), which was directly transcriptionally regulated by HY5. Together, our results reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controlling Arabidopsis seed germination via the COP1-mediated down-regulation of HY5 and ABI5. These findings enhance our understanding of the stress response and have great potential for application in agricultural production.

  5. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    SciTech Connect

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae; Park, Sang Chul

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  6. The Q-rich/PST domain of the AHR regulates both ligand-induced nuclear transport and nucleocytoplasmic shuttling

    PubMed Central

    Tkachenko, Anna; Henkler, Frank; Brinkmann, Joep; Sowada, Juliane; Genkinger, Doris; Kern, Christian; Tralau, Tewes; Luch, Andreas

    2016-01-01

    The aryl hydrocarbon receptor (AHR) shuttles continuously between cytoplasm and nucleus, unless ligand-binding triggers association with the AHR nuclear translocator (ARNT) and subsequent binding to cognate DNA motifs. We have now identified Val 647 as mandatory residue for export from the nucleus and AHR-function. This residue prevents inactivation of the receptor as a consequence of nuclear sequestration via constitutive import. Concomitantly mutants lacking this residue are exclusively localised in the nucleus. Although ligands accelerate nuclear import transiently, stable nuclear transition depends on a motif adjacent to Val 647 that comprises residues 650–661. Together, this defined region within the Q-rich domain regulates intracellular trafficking of the AHR in context of both nucleocytoplasmic shuttling and receptor activation. Nuclear export therefore depends on the previously characterised N-terminal NES and the newly identified motif that includes V647. Nucleocytoplasmic distribution of full-length human AHR is further affected by a section of the PST domain that shows sequence similarities with nuclear export signals. In concert, these motifs maintain a predominant cytoplasmic compartmentalisation, receptive for ligand binding. PMID:27535013

  7. The Q-rich/PST domain of the AHR regulates both ligand-induced nuclear transport and nucleocytoplasmic shuttling.

    PubMed

    Tkachenko, Anna; Henkler, Frank; Brinkmann, Joep; Sowada, Juliane; Genkinger, Doris; Kern, Christian; Tralau, Tewes; Luch, Andreas

    2016-01-01

    The aryl hydrocarbon receptor (AHR) shuttles continuously between cytoplasm and nucleus, unless ligand-binding triggers association with the AHR nuclear translocator (ARNT) and subsequent binding to cognate DNA motifs. We have now identified Val 647 as mandatory residue for export from the nucleus and AHR-function. This residue prevents inactivation of the receptor as a consequence of nuclear sequestration via constitutive import. Concomitantly mutants lacking this residue are exclusively localised in the nucleus. Although ligands accelerate nuclear import transiently, stable nuclear transition depends on a motif adjacent to Val 647 that comprises residues 650-661. Together, this defined region within the Q-rich domain regulates intracellular trafficking of the AHR in context of both nucleocytoplasmic shuttling and receptor activation. Nuclear export therefore depends on the previously characterised N-terminal NES and the newly identified motif that includes V647. Nucleocytoplasmic distribution of full-length human AHR is further affected by a section of the PST domain that shows sequence similarities with nuclear export signals. In concert, these motifs maintain a predominant cytoplasmic compartmentalisation, receptive for ligand binding. PMID:27535013

  8. p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

    PubMed

    Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W Y; Li, Zhen; Fu, Amy K Y; Ip, Nancy Y

    2014-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

  9. Functional insights of nucleocytoplasmic transport in plants

    PubMed Central

    Tamura, Kentaro; Hara-Nishimura, Ikuko

    2014-01-01

    Plant nucleocytoplasmic transport beyond the nuclear envelope is important not only for basic cellular functions but also for growth, development, hormonal signaling, and responses to environmental stimuli. Key components of this transport system include nuclear transport receptors and nucleoporins. The functional and physical interactions between receptors and the nuclear pore in the nuclear membrane are indispensable for nucleocytoplasmic transport. Recently, several groups have reported various plant mutants that are deficient in factors involved in nucleocytoplasmic transport. Here, we summarize the current state of knowledge about nucleocytoplasmic transport in plants, and we review the plant-specific regulation and roles of this process in plants. PMID:24765097

  10. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

    SciTech Connect

    Finan, John D.; Leddy, Holly A.; Guilak, Farshid

    2011-05-06

    Highlights: {yields} The rate of nucleocytoplasmic transport increases under hyper-osmotic stress. {yields} The mechanism is a change in nuclear geometry, not a change in permeability of the nuclear envelope. {yields} Intracytoplasmic but not intranuclear diffusion is sensitive to osmotic stress. {yields} Pores in the chromatin of the nucleus enlarge under hyper-osmotic stress. -- Abstract: Osmotic stress is a potent regulator of biological function in many cell types, but its mechanism of action is only partially understood. In this study, we examined whether changes in extracellular osmolality can alter chromatin condensation and the rate of nucleocytoplasmic transport, as potential mechanisms by which osmotic stress can act. Transport of 10 kDa dextran was measured both within and between the nucleus and the cytoplasm using two different photobleaching methods. A mathematical model was developed to describe fluorescence recovery via nucleocytoplasmic transport. As osmolality increased, the diffusion coefficient of dextran decreased in the cytoplasm, but not the nucleus. Hyper-osmotic stress decreased nuclear size and increased nuclear lacunarity, indicating that while the nucleus was getting smaller, the pores and channels interdigitating the chromatin had expanded. The rate of nucleocytoplasmic transport was increased under hyper-osmotic stress but was insensitive to hypo-osmotic stress, consistent with the nonlinear osmotic properties of the nucleus. The mechanism of this osmotic sensitivity appears to be a change in the size and geometry of the nucleus, resulting in a shorter effective diffusion distance for the nucleus. These results may explain physical mechanisms by which osmotic stress can influence intracellular signaling pathways that rely on nucleocytoplasmic transport.

  11. GNL3L Is a Nucleo-Cytoplasmic Shuttling Protein: Role in Cell Cycle Regulation.

    PubMed

    Thoompumkal, Indu Jose; Subba Rao, Malireddi Rama Krishna; Kumaraswamy, Anbarasu; Krishnan, Rehna; Mahalingam, Sundarasamy

    2015-01-01

    GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501-582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the 'G2/M' phase of cell cycle whereas depletion of endogenous GNL3L results in 'G2/M' arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L∆NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L∆NES results in faster 'S' phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote 'S' phase progression during cell proliferation.

  12. Salt Stress and Ethylene Antagonistically Regulate Nucleocytoplasmic Partitioning of COP1 to Control Seed Germination1[OPEN

    PubMed Central

    Shi, Hui; Gu, Juntao; Dong, Jingao; Deng, Xing Wang

    2016-01-01

    Seed germination, a critical stage initiating the life cycle of a plant, is severely affected by salt stress. However, the underlying mechanism of salt inhibition of seed germination (SSG) is unclear. Here, we report that the Arabidopsis (Arabidopsis thaliana) CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) counteracts SSG. Genetic assays provide evidence that SSG in loss of function of the COP1 mutant was stronger than this in the wild type. A GUS-COP1 fusion was constitutively localized to the nucleus in radicle cells. Salt treatment caused COP1 to be retained in the cytosol, but the addition of ethylene precursor 1-aminocyclopropane-1-carboxylate had the reverse effect on the translocation of COP1 to the nucleus, revealing that ethylene and salt exert opposite regulatory effects on the localization of COP1 in germinating seeds. However, loss of function of the ETHYLENE INSENSITIVE3 (EIN3) mutant impaired the ethylene-mediated rescue of the salt restriction of COP1 to the nucleus. Further research showed that the interaction between COP1 and LONG HYPOCOTYL5 (HY5) had a role in SSG. Correspondingly, SSG in loss of function of HY5 was suppressed. Biochemical detection showed that salt promoted the stabilization of HY5, whereas ethylene restricted its accumulation. Furthermore, salt treatment stimulated and ethylene suppressed transcription of ABA INSENSITIVE5 (ABI5), which was directly transcriptionally regulated by HY5. Together, our results reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controlling Arabidopsis seed germination via the COP1-mediated down-regulation of HY5 and ABI5. These findings enhance our understanding of the stress response and have great potential for application in agricultural production. PMID:26850275

  13. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    SciTech Connect

    Kumari, Gita; Mahalingam, S.

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  14. Nucleocytoplasmic transport of macromolecules.

    PubMed Central

    Corbett, A H; Silver, P A

    1997-01-01

    Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae. PMID:9184010

  15. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    PubMed Central

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  16. Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

    PubMed Central

    Lichius, Alexander; Seidl-Seiboth, Verena; Seiboth, Bernhard; Kubicek, Christian P

    2014-01-01

    Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei. PMID:25302561

  17. Viral subversion of nucleocytoplasmic trafficking.

    PubMed

    Yarbrough, Melanie L; Mata, Miguel A; Sakthivel, Ramanavelan; Fontoura, Beatriz M A

    2014-02-01

    Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Because of its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, whereas viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. As viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. This review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.

  18. Viral Subversion of Nucleocytoplasmic Trafficking

    PubMed Central

    Yarbrough, Melanie L.; Mata, Miguel A.; Sakthivel, Ramanavelan; Fontoura, Beatriz M. A.

    2014-01-01

    Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Due to its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, while viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Since viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. In addition, this review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens. PMID:24289861

  19. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Health and Disease States

    PubMed Central

    Batarseh, Amani; Papadopoulos, Vassilios

    2010-01-01

    Translocator protein (TSPO) is an 18-kDa high affinity cholesterol- and drug-binding protein found primarily in the outer mitochondrial membrane. Although TSPO is found in many tissue types, it is expressed at the highest levels under normal conditions in tissues that synthesize steroids. TSPO has been associated with cholesterol import into mitochondria, a key function in steroidogenesis, and directly or indirectly with multiple other cellular functions including apoptosis, cell proliferation, differentiation, anion transport, porphyrin transport, heme synthesis, and regulation of mitochondrial function. Aberrant expression of TSPO has been linked to multiple diseases, including cancer, brain injury, neurodegeneration, and ischemia reperfusion injury. There has been an effort during the last decade to understand the mechanisms regulating tissue- and disease-specific TSPO expression and to identify pharmacological means to control its expression. This review focuses on the current knowledge regarding the chemicals, hormones, and molecular mechanisms regulating Tspo gene expression under physiological conditions in a tissue- and disease-specific manner. The results described here provide evidence that the PKCε-ERK1/2-AP1/Stat3 signal transduction pathway is the primary regulator of Tspo gene expression in normal and pathological tissues expressing high levels of TSPO. PMID:20600583

  20. Abl Interactor 1 (Abi-1) Wave-Binding and SNARE Domains Regulate Its Nucleocytoplasmic Shuttling, Lamellipodium Localization, and Wave-1 Levels

    PubMed Central

    Echarri, Asier; Lai, Margaret J.; Robinson, Matthew R.; Pendergast, Ann Marie

    2004-01-01

    The Abl interactor 1 (Abi-1) protein has been implicated in the regulation of actin dynamics and localizes to the tips of lamellipodia and filopodia. Here, we show that Abi-1 binds the actin nucleator protein Wave-1 through an amino-terminal Wave-binding (WAB) domain and that disruption of the Abi-1-Wave-1 interaction prevents Abi-1 from reaching the tip of the lamellipodium. Abi-1 binds to the Wave homology domain of Wave-1, a region that is required for translocation of Wave-1 to the lamellipodium. Mouse embryo fibroblasts that lack one allele of Abi-1 and are homozygous null for the related Abi-2 protein exhibit decreased Wave-1 protein levels. This phenotype is rescued by Abi-1 proteins that retain Wave-1 binding but not by Abi-1 mutants that cannot bind to Wave-1. Moreover, we uncovered an overlapping SNARE domain in the amino terminus of Abi-1 that interacts with Syntaxin-1, a SNARE family member. Further, we demonstrated that Abi-1 shuttles in and out of the nucleus in a leptomycin B (LMB)-dependent manner and that complete nuclear translocation of Abi-1 in the absence of LMB requires the combined inactivation of the SNARE, WAB, and SH3 domains of Abi-1. Thus, Abi-1 undergoes nucleocytoplasmic shuttling and functions at the leading edge to regulate Wave-1 localization and protein levels. PMID:15143189

  1. Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA.

    PubMed

    Alonso-Caplen, F V; Nemeroff, M E; Qiu, Y; Krug, R M

    1992-02-01

    Influenza virus unspliced NS1 mRNA, like retroviral pre-mRNAs, is efficiently exported from the nucleus and translated in the cytoplasm of infected cells. With human immunodeficiency virus (HIV), the transport of viral pre-mRNAs is facilitated by the viral Rev protein. We tested the possibility that the influenza virus NS1 protein, a nuclear protein that is encoded by unspliced NS1 mRNA, has the same function as the HIV Rev protein. Surprisingly, using transient transfection assays, we found that rather than facilitating the nucleocytoplasmic transport of unspliced NS1 mRNA, the NS1 protein inhibited the transport of NS2 mRNA, the spliced mRNA generated from NS1 mRNA. The efficient transport of NS2 mRNA from the nucleus to the cytoplasm occurred only when the synthesis of the NS1 protein was abrogated by amber mutations. The NS1 protein down-regulated the export of NS2 mRNA whether or not it was generated by splicing, indicating that the NS1 protein acted directly on transport. Actinomycin D chase experiments verified that the NS1 protein acted on the transport and not on the differential stability of NS2 mRNA in the nucleus as compared to the cytoplasm. In addition, the NS1 protein inhibited the transport of NS1 mRNA itself, which contains all of the sequences in NS2 mRNA, particularly when NS1 mRNA was released from the splicing machinery by mutating its 3'-splice site. Our results indicate that the NS1 protein-mediated inhibition of transport requires sequences in NS2 mRNA. The transport of the viral PB1 protein, nucleocapsid protein, hemagglutinin, membrane protein, and M2 mRNAs was not affected by the NS1 protein. When the NS2 mRNA sequence was covalently attached to the PB1 mRNA, the transport of the chimeric mRNA was inhibited by the NS1 protein. Our results identify a novel function of the influenza virus NS1 protein and demonstrate that post-transcriptional control of gene expression can also occur at the level of the nucleocytoplasmic transport of a

  2. Targeting nucleocytoplasmic transport in cancer therapy

    PubMed Central

    de Pedro, Nuria

    2014-01-01

    The intracellular location and regulation of proteins within each cell is critically important and is typically deregulated in disease especially cancer. The clinical hypothesis for inhibiting the nucleo-cytoplasmic transport is based on the dependence of certain key proteins within malignant cells. This includes a host of well-characterized tumor suppressor and oncoproteins that require specifc localization for their function. This aberrant localization of tumour suppressors and oncoproteins results in their their respective inactivation or over-activation. This incorrect localization occurs actively via the nuclear pore complex that spans the nuclear envelope and is mediated by transport receptors. Accordingly, given the signifcant need for novel, specifc disease treatments, the nuclear envelope and the nuclear transport machinery have emerged as a rational therapeutic target in oncology to restore physiological nucleus/cytoplasmic homeostasis. Recent evidence suggests that this approach might be of substantial therapeutic use. This review summarizes the mechanisms of nucleo-cytoplasmic transport, its role in cancer biology and the therapeutic potential of targeting this critical cellular process PMID:24429466

  3. Intracellular structure and nucleocytoplasmic transport.

    PubMed

    Agutter, P S

    1995-01-01

    Intracellular movement of any solute or particle accords with one of two general schemes: either it takes place predominantly in the solution phase or it occurs by dynamic interactions with solid-state structures. If nucleocytoplasmic exchanges of macromolecules and complexes are predominantly solution-phase processes, i.e., if the former ("diffusionist") perspective applies, then the only significant structures in nucleocytoplasmic transport are the pore complexes. However, if such exchanges accord with the latter ("solid-state") perspective, then the roles of the nucleoskeleton and cytoskeleton in nucleocytoplasmic transport are potentially, at least, as important as that of the pore complexes. The role of the nucleoskeleton in mRNA transport is more difficult to evaluate than that of the cytoskeleton because it is less well characterized, and current evidence does not exclude either perspective. However, the balance of evidence favors a solid-state scheme. It is argued that ribosomal subunits are also more likely to migrate by a solid-state rather than a diffusionist mechanism, though the opposite is true of proteins and tRNAs. Moreover, recent data on the effects of viral proteins on intranuclear RNA processing and migration accord with the solid-state perspective. In view of this balance of evidence, three possible solid-state mechanisms for nucleocytoplasmic mRNA transport are described and evaluated. The explanatory advantage of solid-state models is contrasted with the heuristic advantage of diffusion theory, but it is argued that diffusion theory itself, even aided by modern computational techniques and numerical and graphical approaches, cannot account for data describing the movements of materials within the cell. Therefore, the mechanisms envisaged in a diffusionist perspective cannot be confined to diffusion alone, but must include other processes such as bulk fluid flow.

  4. Cocaine treatment increases expression of a 40 kDa catecholamine-regulated protein in discrete brain regions.

    PubMed

    Sharan, Niki; Chong, Victor Z; Nair, Venugopalan D; Mishra, Ram K; Hayes, Robert J; Gardner, Eliot L

    2003-01-01

    Previous reports from our laboratory have described brain-specific catecholamine-regulated proteins, which bind dopamine and related catecholamines. Evidence from the molecular cloning of a 40 kDa catecholamine-regulated protein (CRP40) revealed that CRP40 is dopamine-inducible and has properties similar to those of the 70 kDa heat shock protein (HSP70) family. The present study investigates the effects of acute and chronic cocaine treatment on CRP40 expression in the striatum, nucleus accumbens, prefrontal cortex, and medulla. Acute treatment with cocaine increased CRP40 expression in the nucleus accumbens and striatum, whereas chronic treatment with cocaine increased CRP40 expression in the nucleus accumbens only. Neither of these treatments affected CRP40 levels in the prefrontal cortex or medulla. In addition, pretreatment with the spin-trapping agent alpha-phenyl-tert-butylnitrone did not attenuate cocaine-induced expression of CRP40, suggesting that the observed increases in CRP40 levels were not caused by free radicals. On the other hand, pretreatment with anisomycin, a protein synthesis inhibitor, blocked the cocaine-induced expression of CRP40. Thus, protein synthesis may be involved in the observed CRP40 level increases. Furthermore, neither acute nor chronic cocaine treatment affected levels of inducible or constitutively expressed HSP70, which indicates a specificity of cocaine's effects on CRP40. Since cocaine has been shown to increase extracellular dopamine levels, these findings suggest that increased expression of CRP40 is associated with high extracellular levels of dopamine (or its metabolites). Elevated levels of CRP40 could play a protective role for dopamine neurons in response to increased oxidative stress that has been shown to be induced by cocaine and that can lead to apoptosis and neurodegeneration. PMID:12422371

  5. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells

    PubMed Central

    Manku, Gurpreet; Culty, Martine

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology. PMID:27608010

  6. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-09-06

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

  7. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology. PMID:27608010

  8. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase*

    PubMed Central

    Cottom, Joshua; Salvador, Lisa M.; Maizels, Evelyn T.; Reierstad, Scott; Park, Youngkyu; Carr, Daniel W.; Davare, Monika A.; Hell, Johannes W.; Palmer, Stephen S.; Dent, Paul; Kawakatsu, Hisaaki; Ogata, Masato; Hunzicker-Dunn, Mary

    2006-01-01

    In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase. PMID:12493768

  9. Influence of heart failure on nucleocytoplasmic transport in human cardiomyocytes

    PubMed Central

    Cortés, Raquel; Roselló-Lletí, Esther; Rivera, Miguel; Martínez-Dolz, Luis; Salvador, Antonio; Azorín, Inmaculada; Portolés, Manuel

    2010-01-01

    Aims The role of the cell nucleus in the development of heart failure (HF) is unknown, so the objectives of this study were to analyse the effect of HF on nucleocytoplasmic transport and density of the nuclear pore complex (NPC). Methods and results A total of 51 human heart samples from ischaemic (ICM, n = 30) and dilated (DCM, n = 16) patients undergoing heart transplantation and control donors (CNT, n = 5) were analysed by western blotting. Subcellular distribution of proteins and NPC were analysed by fluorescence and electron microscopy, respectively. When we compared nucleocytoplasmic machinery protein levels according to aetiology of HF, ICM showed higher levels of importins [(IMP-β3) (150%, P < 0.0001), IMP-α2 (69%, P = 0.001)] and exportins [EXP-1 (178%, P < 0.0001), EXP-4 (81%, P = 0.006)] than those of the CNT group. Furthermore, DCM also showed significant differences for IMP-β3 (192%, P < 0.0001), IMP-α2 (52%, P = 0.025), and EXP-1 (228%, P < 0.0001). RanGTPase-activating proteins (RanGAP1 and RaGAP1u) were increased in ICM (76%, P = 0.005; 51%, P = 0.012) and DCM (41%, P = 0.042; 50%, P = 0.029). Furthermore, subcellular distribution of nucleocytoplasmic machinery was not altered in pathological hearts. Finally, nucleoporin (Nup) p62 was increased in ICM (80%) and DCM (109%) (P < 0.001 and P = 0.024). Nuclear pore density was comparable in pathological and CNT hearts, and ICM showed a low diameter (P = 0.005) and different structural configuration of NPC. Conclusion This study shows the effect of HF on nucleocytoplasmic trafficking machinery, evidenced by higher levels of importins, exportins, Ran regulators and Nup p62 in ischaemic and dilated human hearts than those in the controls, with NPCs acquiring a different configuration and morphology in ICM. PMID:19819881

  10. Malignant progressive tumor cell clone exhibits significant up-regulation of cofilin-2 and 27-kDa modified form of cofilin-1 compared to regressive clone.

    PubMed

    Kuramitsu, Yasuhiro; Wang, Yufeng; Okada, Futoshi; Baron, Byron; Tokuda, Kazuhiro; Kitagawa, Takao; Akada, Junko; Nakamura, Kazuyuki

    2013-09-01

    QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p<0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p<0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

  11. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    PubMed

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

  12. Nucleocytoplasmic Distribution of Budding Yeast Protein Kinase A Regulatory Subunit Bcy1 Requires Zds1 and Is Regulated by Yak1-Dependent Phosphorylation of Its Targeting Domain

    PubMed Central

    Griffioen, Gerard; Branduardi, Paola; Ballarini, Annalisa; Anghileri, Paola; Norbeck, Joakim; Baroni, Maurizio D.; Ruis, Helmut

    2001-01-01

    In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner. PMID:11134339

  13. Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

    PubMed

    Jensen, T H; Neville, M; Rain, J C; McCarthy, T; Legrain, P; Rosbash, M

    2000-11-01

    Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

  14. Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization.

    PubMed

    Kawai, Yumiko; Garduño, Lakisha; Theodore, Melanie; Yang, Jianqi; Arinze, Ifeanyi J

    2011-03-01

    Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys(588) and Lys(591) of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export. PMID:21196497

  15. The C9ORF72 repeat expansion disrupts nucleocytoplasmic transport

    PubMed Central

    Haeusler, Aaron R.; Grima, Jonathan C.; Machamer, James B.; Steinwald, Peter; Daley, Elizabeth L.; Miller, Sean J.; Cunningham, Kathleen M.; Vidensky, Svetlana; Gupta, Saksham; Thomas, Michael A.; Hong, Ingie; Chiu, Shu-Ling; Huganir, Richard L.; Ostrow, Lyle W.; Matunis, Michael J.; Wang, Jiou; Sattler, Rita

    2016-01-01

    A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila ortholog of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9ORF72 ALS patient-derived induced pluripotent stem cells (iPSNs), and in C9ORF72 patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9ORF72 iPSNs, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD amenable to pharmacotherapeutic intervention. PMID:26308891

  16. Kinetic models for nucleocytoplasmic transport of messenger RNA.

    PubMed

    Schröder, H C; Müller, W E; Agutter, P S

    1995-05-21

    Much is known about the mechanism by which mRNAs cross the nuclear envelope (the translocation stage of nucleocytoplasmic transport), but far less is known about the preceding (intranuclear migration/release) and succeeding (cytoplasmic binding) stages. Therefore, existing information suffices for articulating detailed kinetic models of translocation, but not models for the overall mRNA transport process. In this paper, we show that simple kinetic models of translocation can (i) accommodate data about nucleocytoplasmic distributions of endogenous transcripts; (ii) predict the overall effects on these distributions of effectors such as insulin and epidermal growth factor; (iii) throw some light on the mechanism(s) of action of the HIV-1 protein Rev and produce experimentally testable predictions about this mechanism; and (iv) account for the action of influenza virus NS1 protein. However, the simplest forms of translocation models apparently fail to account for some properties of viral regulators such as HIV Rev and adenovirus E1B-E4 complex. To elucidate these topics, less narrowly focused models of mRNA transport are required, describing intranuclear binding/release as well as translocation. On the basis of our examination of translocation models, we suggest some criteria that the requisite broadly based models must satisfy.

  17. Introduction to nucleocytoplasmic transport: molecules and mechanisms.

    PubMed

    Peters, Reiner

    2006-01-01

    Nucleocytoplasmic transport, the exchange of matter between nucleus and cytoplasm, plays a fundamental role in human and other eukaryotic cells, affecting almost every aspect of health and disease. The only gate for the transport of small and large molecules as well as supramolecular complexes between nucleus and cytoplasm is the nuclear pore complex (NPC). The NPC is not a normal membrane transport protein (transporter). Composed of 500 to 1000 peptide chains, the NPC features a mysterious functional duality. For most molecules, it constitutes a molecular sieve with a blurred cutoff at approx 10 nm, but for molecules binding to phenylalanine-glycine (FG) motifs, the NPC appears to be a channel of approx 50 nm diameter, permitting bidirectional translocation at high speed. To achieve this, the NPC cooperates with soluble factors, the nuclear transport receptors, which shuttle between nuclear contents and cytoplasm. Here, we provide a short introduction to nucleocytoplasmic transport by describing first the structure and composition of the nuclear pore complex. Then, mechanisms of nucleocytoplasmic transport are discussed. Finally, the still essentially unresolved mechanisms by which nuclear transport receptors and transport complexes are translocated through the nuclear pore complex are considered, and a novel translocation model is suggested.

  18. EhNCABP166: a nucleocytoplasmic actin-binding protein from Entamoeba histolytica.

    PubMed

    Campos-Parra, A D; Hernández-Cuevas, N A; Hernandez-Rivas, R; Vargas, M

    2010-07-01

    The actin cytoskeleton consists of multiple actin binding proteins (ABPs) that participate cooperatively in different cellular functions such as the maintenance of polarity and cell motility as well as the invasion of target cells and regulation of gene expression, among others. Due to the important role of ABPs in the pathogenesis of Entamoeba histolytica, the role of a new nucleocytoplasmic ABP from E. histolytica named EhNCABP166 was investigated. The EhNCABP166 gene encodes a protein with an estimated molecular weight of 166kDa. Structurally, this peptide is composed of two CH domains arranged in tandem at the N-terminus of the protein, followed by an alpha-helical region containing a number of different domains with a low level of homology. Two (Bin1/Amphiphysin/Rvs167) (BAR) domains, one GTPase-binding/formin 3 homology (GBD/FH3) domain, three Bcl2-associated athanogene (BAG) domains, one basic-leucine zipper (bZIP) domain and one poly(A)-binding protein C-terminal (PABC) domain were also present. Molecular and biochemical studies showed that the EhNCABP166 protein is transcribed and translated in trophozoites of E. histolytica. It was also shown that the CH domains are functional and bind to F-actin, whereas the BAR and GBD/FH3 domains interact in vitro and in vivo with different families of GTPases such as Rho and Ras, and with different phosphoinositides. These findings suggest that these domains have the conserved functional properties described in other eukaryotic systems. These domains also interacted with additional GTPase and lipid targets that have not been previously described. Finally, cellular studies showed that EhNCABP166 is localized to the cytoplasm and nucleus of E. histolytica and that it has an important role in phagocytosis, proliferation, and motility of E. histolytica.

  19. Subcellular localization and mechanisms of nucleocytoplasmic distribution of p202, an interferon-inducible candidate for lupus susceptibility.

    PubMed

    Choubey, Divaker; Pramanik, Rocky; Xin, Hong

    2003-10-23

    Increased expression of p202 (52 kDa), an interferon (IFN)-inducible murine protein, in splenic cells (B- and T-cells) derived from female mice of the lupus-prone strains is correlated with increased susceptibility to develop systemic lupus erythematosus. However, the molecular mechanisms remain unclear. Our previous studies have indicated that, in IFN-treated fibroblasts, p202 is detected both in the cytoplasm and in the nucleus. Moreover, in the cytoplasm, a fraction of p202 associates with a membranous organelle. Here we report that, in the cytoplasm, a fraction of p202 associated with mitochondria. Additionally, we found that the constitutive p202 is primarily detected in the cytoplasm. Remarkably, the IFN treatment of cells potentiated nuclear accumulation of p202. Our observations are consistent with the possibility that IFN signaling regulates p202 levels as well as its nucleocytoplasmic distribution. These observations will serve as a basis to elucidate the molecular mechanisms by which p202 contributes to lupus susceptibility.

  20. Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).

    PubMed

    Šileikytė, Justina; Blachly-Dyson, Elizabeth; Sewell, Randall; Carpi, Andrea; Menabò, Roberta; Di Lisa, Fabio; Ricchelli, Fernanda; Bernardi, Paolo; Forte, Michael

    2014-05-16

    Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

  1. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast.

    PubMed

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y; Strahl, Sabine; Clausen, Henrik

    2015-12-22

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  2. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    PubMed Central

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

    2015-01-01

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  3. Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage.

    PubMed

    Alvarez-Sánchez, María Elizbeth; Carvajal-Gamez, Bertha Isabel; Solano-González, Eduardo; Martínez-Benitez, Máximo; Garcia, Ana F; Alderete, John F; Arroyo, Rossana

    2008-01-01

    Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.

  4. Temporal and Spatial Regulation of Ezrin-Radixin-Moesin-Binding Phosphoprotein-50-kDa (EBP50) during Embryo Implantation in Mouse Uterus

    PubMed Central

    Li, Xing; Xu, Wang-Ming; Yin, Tai-Lang; Zhao, Qing-Hong; Peng, Liang-Yu; Yang, Jing

    2012-01-01

    Embryo implantation is a crucial process for successful pregnancy. To date, the mechanism of embryo implantation remains unclear. Ezrin-radixin-moesin-binding protein-50-kDa (EBP50) is a scaffold protein, which has been shown to play an important role in cancer development. Embryo implantation and cancer follow a similar progression. Thus, in this article, we utilized immunohistochemical staining and western blot analyses to examine the spatiotemporal expression and regulation of EBP50 both in the mouse uterus during embryo implantation as well as in other related models. We found that EBP50 was detected in epithelial cells in all of the groups used in our study. During the peri-implantation period, EBP50 mainly localized in apical membranes. At the implantation site (IS) on day 5 (D5) of pregnancy, EBP50 was mainly expressed in the nuclei of stroma cells, whereas from day 6 to day 8 (D6–D8) of pregnancy, the expression of EBP50 was noted in the cytoplasm of decidual cells. The expression of EBP50 was not significantly different in the pseudopregnant uterus and decreased in the uteri subjected to activation of delayed implantation. Artificial decidualization also decreased EBP50 expression. Thus, the expression levels and location were affected by active blastocysts and decidualization during the window of implantation. PMID:23208378

  5. Increased Serum Levels of Anti-Carbamylated 78-kDa Glucose-Regulated Protein Antibody in Patients with Rheumatoid Arthritis.

    PubMed

    Yu, Hui-Chun; Lai, Pei-Hsuan; Lai, Ning-Sheng; Huang, Hsien-Bin; Koo, Malcolm; Lu, Ming-Chi

    2016-01-01

    The objective of this study was to investigate the presence and titer of anti-carbamylated 78-kDa glucose-regulated protein (anti-CarGRP78) antibody in serum from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren syndrome (pSS). Thirty-three RA patients, 20 SLE patients, 20 pSS patients, and 20 controls were enrolled from our outpatient clinic. GRP78 was cloned and carbamylated. Serum titers of anti- cyclic citrullinated peptides (anti-CCP), anti-GRP78, and anti-CarGRP78 were measured with an enzyme-linked immunosorbent assay. No differences in serum titers of anti-GRP78 antibody in patients with RA, SLE, or pSS compared with the controls were observed. Serum levels of anti-carGRP78 antibody in patients with RA, but not SLE or pSS, were significantly higher compared with the controls (OD405 0.15 ± 0.08 versus 0.11 ± 0.03, p = 0.033). There was a positive correlation between the serum levels of anti-GRP78 antibody, but not anti-CarGRP78 antibody, with the levels of anti-CCP antibody in patients with RA. Both anti-GRP78 and anti-carGRP78 antibodies failed to correlate with C-reactive protein levels in patients with RA. In conclusion, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA. In addition, the serum titer of anti-CarGRP78 antibody was significantly elevated in patients with RA compared with the controls. Anti-CarGRP78 antibody could also be detected in patients with SLE or pSS. PMID:27618024

  6. Increased Serum Levels of Anti-Carbamylated 78-kDa Glucose-Regulated Protein Antibody in Patients with Rheumatoid Arthritis

    PubMed Central

    Yu, Hui-Chun; Lai, Pei-Hsuan; Lai, Ning-Sheng; Huang, Hsien-Bin; Koo, Malcolm; Lu, Ming-Chi

    2016-01-01

    The objective of this study was to investigate the presence and titer of anti-carbamylated 78-kDa glucose-regulated protein (anti-CarGRP78) antibody in serum from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren syndrome (pSS). Thirty-three RA patients, 20 SLE patients, 20 pSS patients, and 20 controls were enrolled from our outpatient clinic. GRP78 was cloned and carbamylated. Serum titers of anti- cyclic citrullinated peptides (anti-CCP), anti-GRP78, and anti-CarGRP78 were measured with an enzyme-linked immunosorbent assay. No differences in serum titers of anti-GRP78 antibody in patients with RA, SLE, or pSS compared with the controls were observed. Serum levels of anti-carGRP78 antibody in patients with RA, but not SLE or pSS, were significantly higher compared with the controls (OD405 0.15 ± 0.08 versus 0.11 ± 0.03, p = 0.033). There was a positive correlation between the serum levels of anti-GRP78 antibody, but not anti-CarGRP78 antibody, with the levels of anti-CCP antibody in patients with RA. Both anti-GRP78 and anti-carGRP78 antibodies failed to correlate with C-reactive protein levels in patients with RA. In conclusion, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA. In addition, the serum titer of anti-CarGRP78 antibody was significantly elevated in patients with RA compared with the controls. Anti-CarGRP78 antibody could also be detected in patients with SLE or pSS. PMID:27618024

  7. The expression of '150-kDa oxygen regulated protein (ORP-150)' in human brain and its relationship with duration time until death.

    PubMed

    Ikematsu, Kazuya; Tsuda, Ryouichi; Kondo, Toshikazu; Kondo, Hisayoshi; Ozawa, Kentaro; Ogawa, Satoshi; Nakasono, Ichiro

    2004-04-01

    The expression of oxygen regulated protein 150-kDa (ORP-150) was strongly induced in human brain under the hypoxic conditions. We examined the expression of ORP-150 in the brain samples, and discussed its significance in forensic practice. The cerebral cortexes of 31 cases (asphyxia: 9 cases, hypothermia: 4, exsanguinations: 5, CO intoxication (CO): 6, sudden cardiac death (SCD): 7) were used for this study. Each tissue section was incubated with anti-ORP-150 polyclonal antibody and the number of ORP-150 positive cells were counted. In the multiple linear regression method, the estimated regression coefficient of ORP-150 on age was significant (P=0.039) thus, we could find that the ORP-150 expression level depended on age. Using analysis of covariance, we compared the means of ORP-150, LSMEAN, which means hypothetic average value excluding influence of age, for each cause of death. The LSMEAN+/-SE was 84.74+/-9.03 in hypothermia, 57.52+/-6.34 in asphyxia, 46.68+/-6.70 in CO, 24.84+/-8.05 in exsanguinations, and 16.24+/-7.35 in SCD. As a result of the analysis, the LSMEAN of the ORP-150 expression level was related to the cause of death. There might be differences in the duration of brain ischemia before death. For example, SCD is presumed to be instant death, while brain ischemia continues for several minutes in asphyxia, CO and exsanguinations, and for several hours in hypothermia cases. Therefore, the immunohistochemical and morphometrical analysis of ORP-150 in the brain may be very useful to determine the duration of brain ischemia before death in forensic autopsy cases.

  8. High-content classification of nucleocytoplasmic import or export inhibitors.

    PubMed

    Kwon, Yong-Jun; Genovesio, Auguste; Youl Kim, Nam; Hi Chul Kim; Jung, Sungyong; David-Watine, Brigitte; Nehrbass, Ulf; Emans, Neil

    2007-08-01

    Transcription factors of the nuclear factor kappa B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC(50) for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z' >0.95), speed, and robustness in a screen of a compound collection. It identified the IkappaB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocytoplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor kappa B.

  9. Mechanism of Human Nucleocytoplasmic Hexosaminidase D

    PubMed Central

    2016-01-01

    Mammalian β-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these β-hexosaminidases, hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized β-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-order rate constant at pH 6.5–7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to improve our understanding of the physiological role played by this unusual enzyme. PMID:27149221

  10. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold.

    PubMed

    Chen, Yen-Shan; Racca, Joseph D; Phillips, Nelson B; Weiss, Michael A

    2013-09-17

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity. PMID:24003159

  11. Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

    PubMed

    Grover, M; Gaur, T; Kochhar, A; Maheshwari, S C; Tyagi, A K

    1999-06-30

    The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected. PMID:10470848

  12. PRMT1 promotes glucose toxicity-induced β cell dysfunction by regulating the nucleo-cytoplasmic trafficking of PDX-1 in a FOXO1-dependent manner in INS-1 cells.

    PubMed

    Lv, Lixia; Chen, Hewen; Sun, Jiaying; Lu, Di; Chen, Chen; Liu, Dongfang

    2015-08-01

    Protein N-arginine methyltransferase-1 (PRMT1), the major asymmetric arginine methyltransferase, plays important roles in various cellular processes. Previous reports have demonstrated that levels and activities of PRMT1 can vary in animals with type 2 diabetes mellitus. The aim of this study was to assess the expression and mechanism of action of PRMT1 during glucose toxicity-induced β cell dysfunction. Liposome-mediated gene transfection was used to transfect INS-1 cells with siPRMT1, which inhibits PRMT1 expression, and pALTER-FOXO1, which overexpresses forkhead box protein O1 (FOXO1). The cells were then cultured in media containing 5.6 or 25 mmol/L glucose with or without the small molecule PRMT1 inhibitor AMI-1 for 48 h. The protein levels of PRMT1, the arginine methylated protein α-metR, FOXO1, Phospho-FOXO1, pancreas duodenum homeobox-1 (PDX-1), and the intracellular localization of PDX-1 and FOXO1 were then measured by western blotting. FOXO1 methylation was detected by immunoprecipitated with anti-PRMT1 antibody and were immunoblotted with α-metR. The levels of insulin mRNA were measured by real-time fluorescence quantitative PCR. Glucose-stimulated insulin secretion (GSIS) and intracellular insulin content were measured using radioimmunoassays. Intracellular Ca(2+) ([Ca(2+)]i) was detected using Fura-2 AM. Intracellular cAMP levels were measured using ELISA. Chronic exposure to high glucose impaired insulin secretion, decreased insulin mRNA levels and insulin content, increased intracellular [Ca(2+)]i and cAMP levels, and abolishes their responses to glucose. Inhibiting PRMT1 expression improved insulin secretion, increased mRNA levels and insulin content by regulating the intracellular translocation of PDX-1 and FOXO1, decreasing the methylation of FOXO1, and reducing intracellular [Ca(2+)]i and cAMP concentrations. Transient overexpression of constitutively active FOXO1 in nuclear reversed the AMI-1-induced improvement of β cell function without

  13. PRMT1 promotes glucose toxicity-induced β cell dysfunction by regulating the nucleo-cytoplasmic trafficking of PDX-1 in a FOXO1-dependent manner in INS-1 cells.

    PubMed

    Lv, Lixia; Chen, Hewen; Sun, Jiaying; Lu, Di; Chen, Chen; Liu, Dongfang

    2015-08-01

    Protein N-arginine methyltransferase-1 (PRMT1), the major asymmetric arginine methyltransferase, plays important roles in various cellular processes. Previous reports have demonstrated that levels and activities of PRMT1 can vary in animals with type 2 diabetes mellitus. The aim of this study was to assess the expression and mechanism of action of PRMT1 during glucose toxicity-induced β cell dysfunction. Liposome-mediated gene transfection was used to transfect INS-1 cells with siPRMT1, which inhibits PRMT1 expression, and pALTER-FOXO1, which overexpresses forkhead box protein O1 (FOXO1). The cells were then cultured in media containing 5.6 or 25 mmol/L glucose with or without the small molecule PRMT1 inhibitor AMI-1 for 48 h. The protein levels of PRMT1, the arginine methylated protein α-metR, FOXO1, Phospho-FOXO1, pancreas duodenum homeobox-1 (PDX-1), and the intracellular localization of PDX-1 and FOXO1 were then measured by western blotting. FOXO1 methylation was detected by immunoprecipitated with anti-PRMT1 antibody and were immunoblotted with α-metR. The levels of insulin mRNA were measured by real-time fluorescence quantitative PCR. Glucose-stimulated insulin secretion (GSIS) and intracellular insulin content were measured using radioimmunoassays. Intracellular Ca(2+) ([Ca(2+)]i) was detected using Fura-2 AM. Intracellular cAMP levels were measured using ELISA. Chronic exposure to high glucose impaired insulin secretion, decreased insulin mRNA levels and insulin content, increased intracellular [Ca(2+)]i and cAMP levels, and abolishes their responses to glucose. Inhibiting PRMT1 expression improved insulin secretion, increased mRNA levels and insulin content by regulating the intracellular translocation of PDX-1 and FOXO1, decreasing the methylation of FOXO1, and reducing intracellular [Ca(2+)]i and cAMP concentrations. Transient overexpression of constitutively active FOXO1 in nuclear reversed the AMI-1-induced improvement of β cell function without

  14. Proteomic analysis of cell lines expressing small hepatitis B surface antigen revealed decreased glucose-regulated protein 78 kDa expression in association with higher susceptibility to apoptosis.

    PubMed

    Zhao, Chao; Zhang, Wei; Tian, Xiaochen; Fang, Caiyun; Lu, Haojie; Yuan, Zhenghong; Yang, Pengyuan; Wen, Yumei

    2010-01-01

    Accumulating evidence suggests a key role of hepatocyte apoptosis in the pathogenesis of viral hepatitis B. It was found in this study that stable expression of small hepatitis B surface antigen (SHBs) in HepG2 and Huh7 cells increased susceptibility to apoptosis. Proteomic analysis of SHBs expressing HepG2 cells revealed 43 down-regulated and 38 up-regulated proteins. Some have been implicated in apoptosis, including glucose-regulated protein 78 kDa (GRP78), heterogeneous nuclear ribonucleoprotein H3 (hnRNP H), Rho GDP dissociation inhibitor (GDI), cystatin B, far upstream element-binding protein (FUSEbp), and TNF receptor-associated protein 1 (TRAP1). Differential expression of GRP78 and several other proteins was confirmed by Western blot analysis. Replenishing GRP78 improved cellular resistance to apoptosis, whereas reduction of GRP78 by siRNA increased susceptibility even in the absence of SHBs. Taken together, these results suggest that HBsAg plays a pro-apoptotic role through down-regulation of GRP78.

  15. Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

    PubMed Central

    Agutter, P S; McCaldin, B; McArdle, H J

    1979-01-01

    The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process. PMID:229828

  16. Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

    PubMed

    Agutter, P S; McCaldin, B; McArdle, H J

    1979-09-15

    The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.

  17. Biological significance of the importin-β family-dependent nucleocytoplasmic transport pathways.

    PubMed

    Kimura, Makoto; Imamoto, Naoko

    2014-07-01

    Importin-β family proteins (Imp-βs) are nucleocytoplasmic transport receptors (NTRs) that import and export proteins and RNAs through the nuclear pores. The family consists of 14-20 members depending on the biological species, and each member transports a specific group of cargoes. Thus, the Imp-βs mediate multiple, parallel transport pathways that can be regulated separately. In fact, the spatiotemporally differential expressions and the functional regulations of Imp-βs have been reported. Additionally, the biological significance of each pathway has been characterized by linking the function of a member of Imp-βs to a cellular consequence. Connecting these concepts, the regulation of the transport pathways conceivably induces alterations in the cellular physiological states. However, few studies have linked the regulation of an importin-β family NTR to an induced cellular response and the corresponding cargoes, despite the significance of this linkage in comprehending the biological relevance of the transport pathways. This review of recent reports on the regulation and biological functions of the Imp-βs highlights the significance of the transport pathways in physiological contexts and points out the possibility that the identification of yet unknown specific cargoes will reinforce the importance of transport regulation.

  18. Nucleocytoplasmic Transport: A Paradigm for Molecular Logistics in Artificial Systems.

    PubMed

    Vujica, Suncica; Zelmer, Christina; Panatala, Radhakrishnan; Lim, Roderick Y H

    2016-01-01

    Artificial organelles, molecular factories and nanoreactors are membrane-bound systems envisaged to exhibit cell-like functionality. These constitute liposomes, polymersomes or hybrid lipo-polymersomes that display different membrane-spanning channels and/or enclose molecular modules. To achieve more complex functionality, an artificial organelle should ideally sustain a continuous influx of essential macromolecular modules (i.e. cargoes) and metabolites against an outflow of reaction products. This would benefit from the incorporation of selective nanopores as well as specific trafficking factors that facilitate cargo selectivity, translocation efficiency, and directionality. Towards this goal, we describe how proteinaceous cargoes are transported between the nucleus and cytoplasm by nuclear pore complexes and the biological trafficking machinery in living cells (i.e. nucleocytoplasmic transport). On this basis, we discuss how biomimetic control may be implemented to selectively import, compartmentalize and accumulate diverse macromolecular modules against concentration gradients in artificial organelles. PMID:27363369

  19. Nucleocytoplasmic Transport: A Paradigm for Molecular Logistics in Artificial Systems.

    PubMed

    Vujica, Suncica; Zelmer, Christina; Panatala, Radhakrishnan; Lim, Roderick Y H

    2016-01-01

    Artificial organelles, molecular factories and nanoreactors are membrane-bound systems envisaged to exhibit cell-like functionality. These constitute liposomes, polymersomes or hybrid lipo-polymersomes that display different membrane-spanning channels and/or enclose molecular modules. To achieve more complex functionality, an artificial organelle should ideally sustain a continuous influx of essential macromolecular modules (i.e. cargoes) and metabolites against an outflow of reaction products. This would benefit from the incorporation of selective nanopores as well as specific trafficking factors that facilitate cargo selectivity, translocation efficiency, and directionality. Towards this goal, we describe how proteinaceous cargoes are transported between the nucleus and cytoplasm by nuclear pore complexes and the biological trafficking machinery in living cells (i.e. nucleocytoplasmic transport). On this basis, we discuss how biomimetic control may be implemented to selectively import, compartmentalize and accumulate diverse macromolecular modules against concentration gradients in artificial organelles.

  20. Identification of a Novel Nucleocytoplasmic Shuttling RNA Helicase of Trypanosomes

    PubMed Central

    Inoue, Alexandre Haruo; Serpeloni, Mariana; Hiraiwa, Priscila Mazzocchi; Yamada-Ogatta, Sueli Fumie; Muniz, João Renato Carvalho; Motta, Maria Cristina Machado; Vidal, Newton Medeiros; Goldenberg, Samuel; Ávila, Andréa Rodrigues

    2014-01-01

    Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor. PMID:25313564

  1. The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

    PubMed Central

    Cavazza, Tommaso; Vernos, Isabelle

    2016-01-01

    The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

  2. Involvement of Nucleocytoplasmic Shuttling of Yeast Nap1 in Mitotic Progression

    PubMed Central

    Miyaji-Yamaguchi, Mary; Kato, Kohsuke; Nakano, Ryosuke; Akashi, Tomohiro; Kikuchi, Akihiko; Nagata, Kyosuke

    2003-01-01

    Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus. PMID:12944491

  3. BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress

    SciTech Connect

    Jin, Young-Hee; Ahn, Sang-Gun; Kim, Soo-A.

    2015-08-21

    Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1{sup −/−}) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress. - Highlights: • The expression level of BAG3 is induced by heat stress. • BAG3 translocates to the nucleus upon heat stress. • BAG3 interacts with HSF1 and co-localizes to the nucleus. • BAG3 is a key regulator for HSF1 nuclear shuttling.

  4. Respiratory virus modulation of host nucleocytoplasmic transport; target for therapeutic intervention?

    PubMed Central

    Caly, Leon; Ghildyal, Reena; Jans, David A.

    2015-01-01

    The respiratory diseases caused by rhinovirus, respiratory syncytial virus, and influenza virus represent a large social and financial burden on healthcare worldwide. Although all three viruses have distinctly unique properties in terms of infection and replication, they share the ability to exploit/manipulate the host-cell nucleocytoplasmic transport system in order to replicate effectively and efficiently. This review outlines the various ways in which infection by these viruses impacts on the host nucleocytoplasmic transport system, and examples where inhibition thereof in turn decreases viral replication. The highly conserved nature of the nucleocytoplasmic transport system and the viral proteins that interact with it make this virus–host interface a prime candidate for the development of specific antiviral therapeutics in the future. PMID:26322040

  5. Binding and endocytosis of 39 kDa protein by MDBK cells.

    PubMed

    Vettenranta, K; Bu, G; Schwartz, A L

    1995-08-01

    A 39 kDa protein copurifies with the low density lipoprotein receptor-related protein (LRP) and regulates ligand interactions with LRP. In our recent studies on the clearance of the 39 kDa protein in vivo, we demonstrated that once the liver LRP receptors were saturated, the kidney became the major organ responsible for the 39 kDa protein clearance (Warshawsky et al., 1993, J. Clin. Invest., 92:937-944). The current study was undertaken in order to investigate the potential binding and cellular processing of the 39 kDa protein by kidney-derived MDBK cells. Herein we demonstrate specific, high-affinity, saturable, and Ca(2+)-dependent binding of the 125I-39 kDa protein to MDBK cells (Kd approximately 10-15 nM, 50-70,000 binding sites per cell). Cellular uptake and degradation of the 125I-39 kDa protein by MDBK cells was also demonstrated with kinetics typical of receptor-mediated endocytosis. Using chemical crosslinking we show that LRP in part mediates the binding of 125I-39 kDa protein to the MDBK cell surface. In addition, the presence of functional LRP on the MDBK cell surface was confirmed by the specific binding of activated alpha 2-macroglobulin, another ligand of LRP. Our data thus demonstrate the ability of kidney-derived MDBK cells to specifically bind, endocytose, and degrade the 39 kDa protein.

  6. Characterization of molecular determinants for nucleocytoplasmic shuttling of PRV UL54

    SciTech Connect

    Li Meili; Wang Shuai; Cai Mingsheng; Guo Hong; Zheng Chunfu

    2011-09-01

    The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin {beta}1- and {alpha}5-dependent nuclear import mechanism.

  7. Vesicular Nucleo-Cytoplasmic Transport—Herpesviruses as Pioneers in Cell Biology

    PubMed Central

    Mettenleiter, Thomas C.

    2016-01-01

    Herpesviruses use a vesicle-mediated transfer of intranuclearly assembled nucleocapsids through the nuclear envelope (NE) for final maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex (NEC), conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane (INM) by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane (ONM). Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the NE has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport. PMID:27690080

  8. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

    PubMed

    Woerner, Andreas C; Frottin, Frédéric; Hornburg, Daniel; Feng, Li R; Meissner, Felix; Patra, Maria; Tatzelt, Jörg; Mann, Matthias; Winklhofer, Konstanze F; Hartl, F Ulrich; Hipp, Mark S

    2016-01-01

    Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. PMID:26634439

  9. Phosphorylation modulates rapid nucleocytoplasmic shuttling and cytoplasmic accumulation of Neurospora clock protein FRQ on a circadian time scale

    PubMed Central

    Diernfellner, Axel C.R.; Querfurth, Christina; Salazar, Carlos; Höfer, Thomas; Brunner, Michael

    2009-01-01

    The Neurospora clock protein FREQUENCY (FRQ) is an essential regulator of the circadian transcription factor WHITE COLLAR COMPLEX (WCC). In the course of a circadian period, the subcellular distribution of FRQ shifts from mainly nuclear to mainly cytosolic. This shift is crucial for coordinating the negative and positive limbs of the clock. We show that the subcellular redistribution of FRQ on a circadian time scale is governed by rapid, noncircadian cycles of nuclear import and export. The rate of nuclear import of newly synthesized FRQ is progressively reduced in a phosphorylation-dependent manner, leading to an increase in the steady-state level of cytoplasmic FRQ. The long-period frq7 mutant displays reduced kinetics of FRQ7 protein phosphorylation and a prolonged accumulation in the nucleus. We present a mathematical model that describes the cytoplasmic accumulation of wild-type and mutant FRQ on a circadian time scale on the basis of frequency-modulated rapid nucleocytoplasmic shuttling cycles. PMID:19759264

  10. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

    PubMed Central

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E.; Gehring, Niels H.; Mouland, Andrew J.

    2015-01-01

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. PMID:26492277

  11. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.

    PubMed

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E; Gehring, Niels H; Mouland, Andrew J

    2015-01-01

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. PMID:26492277

  12. 115 kDa serine protease confers sustained protection to visceral leishmaniasis caused by Leishmania donovani via IFN-γ induced down-regulation of TNF-α mediated MMP-9 activity.

    PubMed

    Choudhury, Rajdeep; Das, Partha; De, Tripti; Chakraborti, Tapati

    2013-01-01

    Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. The emergence of increasing number of L. donovani strains resistance to antimonial drugs recommended worldwide requires the intervention of effective vaccine strategy for treatment of VL. In the present study L. donovani culture derived, soluble, secretory serine protease (pSP) has been shown to be vaccine target of VL. Protection from VL could be achieved by the use of safer vaccine which generally requires an adjuvant for induction of strong Th1 response. To assess the safety, immunogenicity and efficacy of pSP as vaccine candidate in mouse model we used IL-12 as adjuvant. BALB/c mice immunized with pSP+IL-12 were protected significantly from challenged infection even after four months by reducing the parasite load in liver and spleen and suppressed the development of the disease along with an increase in IgG2a antibody level in serum, enhanced delayed type hypersensitivity and strong T-cell proliferation. Groups receiving pSP+IL-12 had an augmented pSP antigen specific Th1 cytokines like IFN-γ and TNF-α response with concomitant decrease of Th2 cytokines IL-4 and IL-10 after vaccination. In this study the vaccine efficacy of pSP was further assessed for its prophylactic potential by enumerating matrix metalloprotease-9 (MMP-9) profile which has been implicated in various diseases. MMP-9 associated with different microbial infections is controlled by their natural inhibitors (TIMPS) and by some cytokines. In this study pSP was found to regulate excessive inflammation by modulating the balance between MMP-9 and TIMP-1 expression. This modulatory effect has also been demonstrated by IFN-γ mediated down regulation of TNF-α induced MMP-9 expression in activated murine macrophages. This is the first report where a secretory L. donovani serine protease (pSP) adjuvanted with IL-12 could also act as protective imunogen by modifying

  13. ERK1 nucleocytoplasmic shuttling rate depends on specific N-terminal aminoacids

    SciTech Connect

    Marchi, Matilde; Pancrazi, Laura; Maffei, Margherita; Ratto, G. Michele; Costa, Mario

    2010-07-23

    Despite ERK1 and ERK2 were considered interchangeable isoforms for a long time, their roles are now emerging as only partially overlapping. We recently reported that the nucleocytoplasmic trafficking of GFP-tagged ERK1 is slower than that of ERK2, this difference being caused by a unique domain of ERK1 located at its N-terminus (ERK1-Nt). In the present report we further investigated this issue by asking which were the specific aminoacids involved in such process. By photobleaching strategy, we demonstrated that ERK1-Nt is a domain capable to slow down the nucleocytoplasmic shuttling rate even of a small cargo protein. ERK1-Nt was then dissected into three regions as follows: 1 (aa 1-9), 2 (aa 10-29) and 3, (aa 30-39) that were deleted or mutated at specific sites. Dynamic imaging assessment of the role played by each region in determining the shuttling rate revealed that: region 1 has no significant role, region 2 and specific aminoacids of region 3 (V{sub 31}, K{sub 33,} P{sub 36}) are critical, but singularly do not totally account for the difference in the shuttling rate between ERK1 and 2. Finally, we demonstrated that the nucleocytoplasmic shuttling rate of a passively diffusing protein (mRED) is inversely related to ERK1-Nt-GFP concentrations inside the cell, thus suggesting that ERK1-Nt-GFP occupies the nuclear pore perhaps because of an important affinity of ERK1-Nt for nucleoporins. In conclusion, ERK1-Nt is a domain able per se to confer a slower shuttling rate to a cargo protein. Specific regions within this domain were identified as responsible for this biophysical property.

  14. Effect of colchicine on mammalian liver nuclear envelope and on nucleo-cytoplasmic RNA transport.

    PubMed

    Agutter, P S; Suckling, K E

    1982-09-27

    The binding of colchicine to nuclear envelopes was studied in order to elucidate the mechanism whereby this compound inhibits nucleocytoplasmic RNA transport. The results suggest that a single class of colchicine-binding site (dissociation constant=approx. 0.7 mM, concentration=approx. 330 nmol colchicine/mg protein) is localised in the nuclear periphery (pore-lamina) and that binding to these sites effects a constriction of the pore-complexes with concomitant inhibition of RNA egress and disordering of the nuclear membrane phospholipid bilayers.

  15. Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies*

    PubMed Central

    Pankiv, Serhiy; Lamark, Trond; Bruun, Jack-Ansgar; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje

    2010-01-01

    p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84. PMID:20018885

  16. Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies.

    PubMed

    Pankiv, Serhiy; Lamark, Trond; Bruun, Jack-Ansgar; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje

    2010-02-19

    p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.

  17. Nucleocytoplasmic shuttling of hexokinase II in a cancer cell

    SciTech Connect

    Neary, Catherine L.; Pastorino, John G.

    2010-04-16

    In yeast, the hexokinase type II enzyme (HXKII) translocates to the nucleus in the presence of excess glucose, and participates in glucose repression. However, no evidence has suggested a nuclear function for HXKII in mammalian cells. Herein, we present data showing nuclear localization of HXKII in HeLa cells, both by immunocytochemistry and subcellular fractionation. HXKII is extruded from the nucleus, at least in part, by the activity of the exportin 1/CrmA system, as demonstrated by increased nuclear expression and decreased cytoplasmic expression after incubation with leptomycin B, a bacterially-derived exportin inhibitor. Furthermore, cytoplasmic localization of HXKII is dependent on its enzymatic activity, as inhibiting HXKII activity using 2-deoxy-D-glucose (2DG) increased nuclear localization. This effect was more significant in cells incubated in the absence of glucose for 24 h prior to addition of 2DG. Regulated translocation of HXKII to the nucleus of mammalian cells could represent a previously unknown glucose-sensing mechanism.

  18. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

    SciTech Connect

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2015-10-15

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases.

  19. The Effects of Modeled Microgravity on Nucleocytoplasmic Localization of Human Apurinic/Apyrimidinic

    NASA Technical Reports Server (NTRS)

    Gonda, Steve; Jackson, E.B.

    2004-01-01

    Exposure to space radiation and microgravity occurs to humans during space flight. In order to have accurate risk estimations, answering questions to whether increased DNA damage seen during space flight in modified by microgravity are important. Several studies have examined whether intercellular repair of radiation-induced DNA lesions are modified by microgravity. Results from these studies show no modification of the repair processes due to microgravity. However, it is known that in studies not involving radiation that microgravity interferes with normal development. Interestingly, there is no data that attempts to analyze the possible effects of microgravity on the trafficking of DNA repair proteins. In this study, we analyze the effects of modeled microgravity on nucleocytoplasmic shuttling of the human DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1/Ref1) which is involved in base excision repair. We examined nuclear translocation of APE1 using enhanced green fluorescent protein (EGFP) fused to APE1 as a reporter. While APE1 under normal gravity showed normal nuclear localization, APE1 nuclear localization under modeled microgravity was decreased. These results suggest that nucleocytoplasmic translocation of APE1 is modified under modeled microgravity.

  20. Import and export of nuclear proteins: focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor.

    PubMed

    Sebastian, Thomas; Sreeja, S; Thampan, Raghava Varman

    2004-05-01

    There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor alpha from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER alpha appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg2+ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex. PMID:15228090

  1. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways.

    PubMed

    Ciomperlik, Jessica J; Basta, Holly A; Palmenberg, Ann C

    2015-10-01

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler׳s murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions.

  2. Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

    PubMed Central

    Lucena, Rafael; Dephoure, Noah; Gygi, Steve P.; Kellogg, Douglas R.; Tallada, Victor A.

    2015-01-01

    During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast. PMID:25963819

  3. An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

    PubMed

    Kendirgi, Frederic; Barry, Dianne M; Griffis, Eric R; Powers, Maureen A; Wente, Susan R

    2003-03-31

    Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

  4. Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head.

    PubMed

    Muhlrad, A; Kasprzak, A A; Ue, K; Ajtai, K; Burghardt, T P

    1986-01-30

    We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.

  5. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    SciTech Connect

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania; Bertini, Enrico; Zanni, Ginevra

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.

  6. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.

    PubMed

    Ciomperlik, Jessica J; Basta, Holly A; Palmenberg, Ann C

    2016-01-01

    Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

  7. A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning

    PubMed Central

    Kırlı, Koray; Karaca, Samir; Dehne, Heinz Jürgen; Samwer, Matthias; Pan, Kuan Ting; Lenz, Christof; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction. DOI: http://dx.doi.org/10.7554/eLife.11466.001 PMID:26673895

  8. Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes.

    PubMed

    Hingamp, Pascal; Grimsley, Nigel; Acinas, Silvia G; Clerissi, Camille; Subirana, Lucie; Poulain, Julie; Ferrera, Isabel; Sarmento, Hugo; Villar, Emilie; Lima-Mendez, Gipsi; Faust, Karoline; Sunagawa, Shinichi; Claverie, Jean-Michel; Moreau, Hervé; Desdevises, Yves; Bork, Peer; Raes, Jeroen; de Vargas, Colomban; Karsenti, Eric; Kandels-Lewis, Stefanie; Jaillon, Olivier; Not, Fabrice; Pesant, Stéphane; Wincker, Patrick; Ogata, Hiroyuki

    2013-09-01

    Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2-1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10(4)-10(5) genomes ml(-1) for the samples from the photic zone and 10(2)-10(3) genomes ml(-1) for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.

  9. Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium

    PubMed Central

    Dundon, Samantha E. R.; Chang, Shyr-Shea; Kumar, Abhishek; Occhipinti, Patricia; Shroff, Hari; Roper, Marcus; Gladfelter, Amy S.

    2016-01-01

    Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus’ transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale. PMID:27193301

  10. Abnormal expression of TIP30 and arrested nucleocytoplasmic transport within oligodendrocyte precursor cells in multiple sclerosis

    PubMed Central

    Nakahara, Jin; Kanekura, Kohsuke; Nawa, Mikiro; Aiso, Sadakazu; Suzuki, Norihiro

    2008-01-01

    Oligodendrocyte precursor cells (OPCs) persist near the demyelinated axons arising in MS but inefficiently differentiate into oligodendrocytes and remyelinate these axons. The pathogenesis of differentiation failure remains elusive. We initially hypothesized that injured axons fail to present Contactin, a positive ligand for the oligodendroglial Notch1 receptor to induce myelination, and thus tracked axoglial Contactin/Notch1 signaling in situ, using immunohistochemistry in brain tissue from MS patients containing chronic demyelinated lesions. Instead, we found that Contactin was saturated on demyelinated axons, Notch1-positive OPCs accumulated in Contactin-positive lesions, and the receptor was engaged, as demonstrated by cleavage to Notch1-intracellular domain (NICD). However, nuclear translocalization of NICD, required for myelinogenesis, was virtually absent in these cells. NICD and related proteins carrying nuclear localization signals were associated with the nuclear transporter Importin but were trapped in the cytoplasm. Abnormal expression of TIP30, a direct inhibitor of Importin, was observed in these OPCs. Overexpression of TIP30 in a rat OPC cell line resulted in cytoplasmic entrapment of NICD and arrest of differentiation upon stimulation with Contactin-Fc. Our results suggest that extracellular inhibitory factors as well as an intrinsic nucleocytoplasmic transport blockade within OPCs may be involved in the pathogenesis of remyelination failure in MS. PMID:19104151

  11. Repair of base damage and genome maintenance in the nucleo-cytoplasmic large DNA viruses.

    PubMed

    Redrejo-Rodríguez, Modesto; Salas, María L

    2014-01-22

    Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins.

  12. Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes

    PubMed Central

    Hingamp, Pascal; Grimsley, Nigel; Acinas, Silvia G; Clerissi, Camille; Subirana, Lucie; Poulain, Julie; Ferrera, Isabel; Sarmento, Hugo; Villar, Emilie; Lima-Mendez, Gipsi; Faust, Karoline; Sunagawa, Shinichi; Claverie, Jean-Michel; Moreau, Hervé; Desdevises, Yves; Bork, Peer; Raes, Jeroen; de Vargas, Colomban; Karsenti, Eric; Kandels-Lewis, Stefanie; Jaillon, Olivier; Not, Fabrice; Pesant, Stéphane; Wincker, Patrick; Ogata, Hiroyuki

    2013-01-01

    Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2–1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 104–105 genomes ml−1 for the samples from the photic zone and 102–103 genomes ml−1 for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts. PMID:23575371

  13. Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium.

    PubMed

    Dundon, Samantha E R; Chang, Shyr-Shea; Kumar, Abhishek; Occhipinti, Patricia; Shroff, Hari; Roper, Marcus; Gladfelter, Amy S

    2016-07-01

    Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus' transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale. PMID:27193301

  14. Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65.

    PubMed

    Frankenberg, Nadine; Lischka, Peter; Pepperl-Klindworth, Sandra; Stamminger, Thomas; Plachter, Bodo

    2012-11-01

    The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.

  15. GGGGCC repeat expansion in C9ORF72 compromises nucleocytoplasmic transport

    PubMed Central

    Freibaum, Brian D.; Lu, Yubing; Lopez-Gonzalez, Rodrigo; Kim, Nam Chul; Almeida, Sandra; Lee, Kyung-Ha; Badders, Nisha; Valentine, Marc; Miller, Bruce L.; Wong, Philip C.; Petrucelli, Leonard; Kim, Hong Joo; Gao, Fen-Biao; Taylor, J. Paul

    2015-01-01

    GGGGCC (G4C2) repeat expansion in a noncoding region of C9ORF72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1,2. The basis for pathogenesis is unknown. To capture the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2 repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein (GFP) to detect repeat-associated non-AUG (RAN) translation. These transgenic animals show dosage-dependent, repeat length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins as observed in patients. This model was used in a large-scale, unbiased genetic screen ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC) as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged iPSC-derived neurons from C9ORF72 patients. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration. PMID:26308899

  16. The carp-goldfish nucleocytoplasmic hybrid has mitochondria from the carp as the nuclear donor species.

    PubMed

    Hu, Guangfu; Zou, Guiwei; Liu, Xiangjiang; Liang, Hongwei; Li, Zhong; Hu, Shaona

    2014-02-25

    It is widely accepted that mitochondria and its DNA (mtDNA) exhibit strict maternal inheritance, with sperm contributing no or non-detectable mitochondria to the next generation. In fish, nuclear transfer (NT) through the combination of a donor nucleus and an enucleated oocyte can produce fertile nucleocytoplasmic hybrids (NCHs) even between different genera and subfamilies. One of the best studied fish NCHs is CyCa produced by transplanting the nuclei plus cytoplasm from the common carp (Cyprinus carpio var. wuyuanensis) into the oocytes of the wild goldfish (Carassius auratus), which has been propagated by self-mating for three generations. These NCH fish thus provide a unique model to study the origin of mitochondria. Here we report the complete mtDNA sequence of the CyCa hybrid and its parental species carp and goldfish as nuclear donor and cytoplasm host, respectively. Interestingly, the mtDNA of NCH fish CyCa is 99.69% identical to the nuclear donor species carp, and 89.25% identical to the oocyte host species goldfish. Furthermore, an amino acid sequence comparison of 13 mitochondrial proteins reveals that CyCa is 99.68% identical to the carp and 87.68% identical to the goldfish. On an mtDNA-based phylogenetic tree, CyCa is clustered with the carp but separated from the goldfish. A real-time PCR analysis revealed the presence of carp mtDNA but the absence of goldfish mtDNA. These results demonstrate--for the first time to our knowledge--that the mtDNA of a NCH such as CyCa fish may originate from its nuclear donor rather than its oocyte host.

  17. Sturgeon nucleo-cytoplasmic large DNA virus phylogeny and PCR tests.

    PubMed

    Clouthier, Sharon C; VanWalleghem, Elissa; Anderson, Eric D

    2015-12-01

    Sturgeon epitheliotropic nucleo-cytoplasmic large DNA viruses (NCLDVs) can cause a lethal disease of the integumentary system. These viruses have not been assigned to any currently recognized family or genus. In this study, phylogenetic analyses using the major capsid protein (MCP) showed that the sturgeon NCLDVs formed a cohesive taxonomic group, could be identified to the species or possibly sub-species level and formed a distinct evolutionary lineage within the Megavirales. The genetic relatedness of the sturgeon virus MCP allowed design of 3 PCR diagnostic tests with analytical specificity (ASp) inclusive of this group of viruses. The conventional PCR test, C1, had broader ASp than the 2 quantitative PCR tests, Q1 and Q2, and was inclusive of the sturgeon viruses as well as some viruses belonging to the families Mimi-, Phycodna-, or Iridoviridae. Q2 had broader specificity than Q1 but both tests recognized the sturgeon NCLDVs and did not cross-react with co-localizing sturgeon herpesviruses. Analytical test performance characteristics evaluated for Q1 and Q2 revealed sensitive assays with observed 50% limits of detection between 3 and 6.25 plasmid copies and high intra- and inter-assay repeatability. Q1 was used to test for sturgeon viruses in endangered populations of lake sturgeon Acipenser fulvescens within the Winnipeg River or Nelson River drainage systems of Manitoba, Canada. Test results indicated that namao virus is endemic in the Nelson River water basin. These tests meet the analytical requirements for diagnostic testing in Canada and are useful tools for disease management in sturgeon conservation stocking programs in North America. PMID:26648102

  18. Sturgeon nucleo-cytoplasmic large DNA virus phylogeny and PCR tests.

    PubMed

    Clouthier, Sharon C; VanWalleghem, Elissa; Anderson, Eric D

    2015-12-01

    Sturgeon epitheliotropic nucleo-cytoplasmic large DNA viruses (NCLDVs) can cause a lethal disease of the integumentary system. These viruses have not been assigned to any currently recognized family or genus. In this study, phylogenetic analyses using the major capsid protein (MCP) showed that the sturgeon NCLDVs formed a cohesive taxonomic group, could be identified to the species or possibly sub-species level and formed a distinct evolutionary lineage within the Megavirales. The genetic relatedness of the sturgeon virus MCP allowed design of 3 PCR diagnostic tests with analytical specificity (ASp) inclusive of this group of viruses. The conventional PCR test, C1, had broader ASp than the 2 quantitative PCR tests, Q1 and Q2, and was inclusive of the sturgeon viruses as well as some viruses belonging to the families Mimi-, Phycodna-, or Iridoviridae. Q2 had broader specificity than Q1 but both tests recognized the sturgeon NCLDVs and did not cross-react with co-localizing sturgeon herpesviruses. Analytical test performance characteristics evaluated for Q1 and Q2 revealed sensitive assays with observed 50% limits of detection between 3 and 6.25 plasmid copies and high intra- and inter-assay repeatability. Q1 was used to test for sturgeon viruses in endangered populations of lake sturgeon Acipenser fulvescens within the Winnipeg River or Nelson River drainage systems of Manitoba, Canada. Test results indicated that namao virus is endemic in the Nelson River water basin. These tests meet the analytical requirements for diagnostic testing in Canada and are useful tools for disease management in sturgeon conservation stocking programs in North America.

  19. Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme.

    PubMed

    Gal, S W; Choi, J Y; Kim, C Y; Cheong, Y H; Choi, Y J; Lee, S Y; Bahk, J D; Cho, M J

    1998-03-01

    A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45 degrees C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 mumol min-1 mg-1 and 60 mumol min-1 mg-1, respectively.

  20. Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport.

    PubMed

    Bollmann, Franziska; Fechir, Katrin; Nowag, Sebastian; Koch, Kathrin; Art, Julia; Kleinert, Hartmut; Pautz, Andrea

    2013-04-01

    Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.

  1. The high risk HPV16 L2 minor capsid protein has multiple transport signals that mediate its nucleocytoplasmic traffic

    SciTech Connect

    Mamoor, Shahan; Onder, Zeynep; Karanam, Balasubramanyam; Kwak, Kihyuck; Bordeaux, Jennifer; Crosby, Lauren; Roden, Richard B.S.; Moroianu, Junona

    2012-01-20

    In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ({sub 462}LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.

  2. Subcellular Localization of Class II HDAs in Arabidopsis thaliana: Nucleocytoplasmic Shuttling of HDA15 Is Driven by Light

    PubMed Central

    Alinsug, Malona V.; Chen, Fang Fang; Luo, Ming; Tai, Ready; Jiang, Liwen; Wu, Keqiang

    2012-01-01

    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. PMID:22363501

  3. The Drosophila 110-kDa transcription factor TFIID subunit directly interacts with the N-terminal region of the 230-kDa subunit.

    PubMed Central

    Kokubo, T; Gong, D W; Roeder, R G; Horikoshi, M; Nakatani, Y

    1993-01-01

    Transcription initiation factor TFIID is a multimeric protein complex that plays a central role in transcriptional regulation by facilitating promoter responses to various activators. cDNAs encoding the 110-kDa subunit of Drosophila TFIID (p110) were isolated with a degenerate oligodeoxynucleotide probe based on an amino acid sequence of the purified protein. The entire cDNA sequence contains an open reading frame encoding a 921-amino acid polypeptide with a calculated molecular mass of 99,337 Da. The recombinant protein expressed in Sf9 cells via a baculovirus vector interacts directly with the 230-kDa subunit of TFIID (p230). Together with the previous observation that the TATA box-binding subunit of TFIID (TFIID tau or TBP) interacts directly with only p230 among the TFIID subunits, this result suggests that p110 forms a complex with TFIID tau via p230. A binding study using various p230 mutants indicated that both p110 and TFIID tau interact with the N-terminal 352-amino acid portion of p230, suggesting a functional communication between p110 and TFIID tau via p230 interactions. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8327460

  4. Enhanced efficacy and immunogenicity of 78kDa antigen formulated in various adjuvants against murine visceral leishmaniasis.

    PubMed

    Nagill, Rajeev; Kaur, Sukhbir

    2010-05-21

    Leishmania infection causes localized cutaneous to severe visceral disease in humans and animals. Current control measures, based on antimonial compounds, are not effective because of resistance in Leishmania. Vaccination would be a feasible alternative, but as yet no vaccine to protect humans against infection has been commercialized. Parasite antigens that preferentially stimulate the induction of significant protection through Th1 response presents a rational approach for a vaccine against leishmaniasis. With this view in mind, we investigated the potential of 78kDa antigen of Leishmania donovani alone and along with different adjuvants against murine visceral leishmaniasis. Various adjuvants used along with 78kDa antigen include monophosphoryl lipid A (MPL-A), liposomal encapsulation, recombinant IL-12, autoclaved Leishmania antigen (ALD) and Freund's adjuvant (FCA). BALB/c mice were immunized subcutaneously thrice with respective vaccine formulation. Challenge infection was given intracardially after 2 weeks of second booster. A significant decrease in parasite burden was seen in vaccinees over the infected controls on all post challenge days and was found that maximum protection was provided by 78kDa+rIL-12 vaccine and it was highly immunogenic as depicted by the reduction in parasite load (71-94.8%), reduction in infection rate of peritoneal macrophages (92.9-98%), enhanced DTH response (6.5-10.5 fold), increase in IgG2a anti-leishmanial antibody production (3-3.7 fold) and up-regulation of IFN-gamma (3.7-6.5 fold) and IL-2 levels (7.7-12.3 fold), which demonstrate the generation of protective Th1 type of immune response. Comparable results were also observed in 78kDa+MPL-A and liposome-encapsulated 78kDa vaccines with 56.5-92% and 62.9-93.4% reduction in parasite load respectively. Significant results have also been obtained with 78kDa antigen+ALD, 78kDa antigen+FCA and 78kDa antigen alone group but the protective efficacy was reduced as compared to the

  5. Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

    PubMed Central

    Mineo, I; Holmes, E W

    1991-01-01

    Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene. Images PMID:1922051

  6. Components and regulation of nuclear transport processes.

    PubMed

    Cautain, Bastien; Hill, Richard; de Pedro, Nuria; Link, Wolfgang

    2015-02-01

    The spatial separation of DNA replication and gene transcription in the nucleus and protein translation in the cytoplasm is a uniform principle of eukaryotic cells. This compartmentalization imposes a requirement for a transport network of macromolecules to shuttle these components in and out of the nucleus. This nucleo-cytoplasmic transport of macromolecules is critical for both cell physiology and pathology. Consequently, investigating its regulation and disease-associated alterations can reveal novel therapeutic approaches to fight human diseases, such as cancer or viral infection. The characterization of the nuclear pore complex, the identification of transport signals and transport receptors, as well as the characterization of the Ran system (providing the energy source for efficient cargo transport) has greatly facilitated our understanding of the components, mechanisms and regulation of the nucleo-cytoplasmic transport of proteins in our cells. Here we review this knowledge with a specific emphasis on the selection of disease-relevant molecular targets for potential therapeutic intervention.

  7. A novel 29-kDa chicken heat shock protein.

    PubMed

    Einat, M F; Haberfeld, A; Shamay, A; Horev, G; Hurwitz, S; Yahav, S

    1996-12-01

    The family of small heat shock proteins is the more variable among the highly conserved superfamily of heat shock proteins (HSP). Using a metabolic labeling procedure with tissue explants, we have detected in chickens a new member of the small HSP family with an apparent molecular weight of 29-kDa. This protein was induced in broiler chickens' heart muscle and lungs following an in vivo heat stress. The 29-kDa band appears after 3 h of heat stress, much later than the induction of HSP 90, HSP 70, and HSP 27. The late onset of induction suggests that HSP 29 plays a more specific role of a "second stage defense protein". PMID:9000279

  8. 16-kDa prolactin and bromocriptine in postpartum cardiomyopathy.

    PubMed

    Hilfiker-Kleiner, Denise; Struman, Ingrid; Hoch, Melanie; Podewski, Edith; Sliwa, Karen

    2012-09-01

    Peripartum cardiomyopathy (PPCM) is a potentially life-threatening heart disease emerging toward the end of pregnancy or in the first postpartal months in previously healthy women. Recent data suggest a central role of unbalanced peri-/postpartum oxidative stress that triggers the proteolytic cleavage of the nursing hormone prolactin (PRL) into a potent antiangiogenic, proapoptotic, and proinflammatory 16-kDa PRL fragment. This notion is supported by the observation that inhibition of PRL secretion by bromocriptine, a dopamine D2-receptor agonist, prevented the onset of disease in an animal model of PPCM and by first clinical experiences where bromocriptine seem to exert positive effects with respect to prevention or treatment of PPCM patients. Here, we highlight the current state of knowledge on diagnosis of PPCM, provide insights into the biology and pathophysiology of 16-kDa PRL and bromocriptine, and outline potential consequences for the clinical management and treatment options for PPCM patients.

  9. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

    SciTech Connect

    Kang, Won Kyung . E-mail: wkkang@riken.jp; Kurihara, Masaaki . E-mail: mkuri@riken.jp; Matsumoto, Shogo . E-mail: smatsu@riken.jp

    2006-06-20

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

  10. The fluidity of the nuclear envelope lipid does not affect the rate of nucleocytoplasmic RNA transport in mammalian liver.

    PubMed

    Agutter, P S; Suckling, K E

    1982-03-29

    The effects of in vitro and in vivo modifications of nuclear envelope lipid on DNa leakage and on ATP-stimulated RNA release from isolated rat liver nuclei were investigated. The modifications included corn-oil feeding of the animals to alter the fatty acid composition of the lipids, phospholipase treatment of the isolated nuclei, and extraction of the total lipid with Triton X-100. Significant changes in lipid composition and approximate order parameter values of the spin-label 5-doxylstearate resulted, but there was no significant effect on RNA transport rate. It was concluded that the nuclear envelope lipid does not play any important part in nucleocytoplasmic RNA transport in mammalian liver.

  11. Analysis of nucleo-cytoplasmic shuttling of the proto-oncogene SET/I2PP2A.

    PubMed

    Lam, B Daniel; Anthony, Eloise C; Hordijk, Peter L

    2012-01-01

    SET/I2PP2A is a nuclear protein that was initially identified as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. In addition, SET is a potent inhibitior of the phosphatase PP2A. Previously, we proposed a model in which the small GTPase Rac1 recruits SET from the nucleus to the plasma membrane to promote cell migration. This event represents an entirely novel concept in the field of cell migration. Now, fluorescent versions of the SET protein are generated to analyze its nucleo-cytoplasmic shuttling in live cells. Our studies showed that under steady-state conditions a fraction of the SET protein, which is primarily localized in the nucleus, translocates to the cytosol in an apparently random fashion. SET exiting the nucleus was also seen in spreading as well as dividing cells. We designed an image analysis method to quantify the frequency of nuclear exit of the SET proteins, based on 4D confocal imaging. This straightforward method was validated by analysis of SET wild-type and mutant proteins. This showed that the frequency of nuclear exit of a Ser-9 phosphomimetic mutant (S9E) is enhanced compared to wild-type SET or a S9A mutant. Thus, we have developed a novel method to analyze the nucleo-cytoplasmic shuttling of the proto-oncogene SET dynamics in live cells. This method will also be applicable to monitor dynamic localization of other nuclear and/or cytoplasmic signaling proteins.

  12. Structural requirements to obtain highly potent and selective 18 kDa Translocator Protein (TSPO) Ligands.

    PubMed

    Taliani, Sabrina; Pugliesi, Isabella; Da Settimo, Federico

    2011-01-01

    The (18 kDa) Translocator Protein (TSPO), was initially identified in 1977 as peripheral binding site for the benzodiazepine diazepam and named "Peripheral-type benzodiazepine receptor (PBR)". It is an evolutionarily well-conserved protein particularly located at the outer/inner mitochondrial membrane contact sites, in closely association with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT), thus forming the mitochondrial permeability transition pore (MPTP). TSPO is ubiquitary expressed in peripheral tissues (steroid producing tissues, liver, heart, kidney, lung, immune system) and in lower levels in the central nervous system, where it is mainly located in glial cells, and in neurons. TSPO is involved in a variety of biological processes such as cholesterol transport, steroidogenesis, calcium homeostasis, lipid metabolism, mitochondrial oxidation, cell growth and differentiation, apoptosis induction, and regulation of immune functions. In the last decade, many studies have reported that TSPO basal expression is altered in a number of human pathologies, such as cancer and neurodegenerative disorders (Huntington's and Alzheimer's diseases), as well as in various forms of brain injury and inflammation and anxiety. Consequently, TSPO has not only been suggested as a promising drug target for a number of therapeutic applications (anticonvulsant, anxiolytic, immunomodulating, etc.), but also as valid diagnostic marker for related-disease state and progression, prompting the development of specific labelled ligands as powerful tools for imaging techniques. A number of structurally different classes of ligands have been reported, showing high affinity and selectivity towards TSPO. Indeed, most of these ligands have been designed starting from selective CBR ligands which were structurally modified in order to shift their affinity towards TSPO. Extensive structure-activity relationship studies were performed allowing to

  13. Calbindin 28 kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells.

    PubMed

    Buffa, R; Mare, P; Salvadore, M; Solcia, E; Furness, J B; Lawson, D E

    1989-01-01

    The distribution of calbindin in some endocrine glands (thyroid, parathyroid, ultimobranchial body, pituitary and adrenals) and in the diffuse endocrine cells of the gut and pancreas has been investigated immunohistochemically using an antiserum raised against the 28 kDa calbindin from chicken duodenum. The identity of calbindin-immunoreactive cells in a number of avian and mammalian species was ascertained by comparison with hormone-reactive cells in consecutive sections or by double immunostaining of the same section with both calbindin and hormone antibodies. Calcitonin-producing C cells of the mammalian and avian thyroid, parathyroid or ultimobranchial body, PP, glucagon and insulin cells of the mammalian and avian pancreas, enteroglucagon cells of the avian intestine, secretin cells of the mammalian duodenum, histamine-producing ECL cells of the mammalian stomach, as well as noradrenaline-producing cells of the adrenal medulla and some (TSH?) cells of the adenohypophysis were among the calbindin-immunoreactive cells. Although some species variability has been observed in the intensity and distribution of the immunoreactivity, especially in the pancreas and the gut, a role for calbindin in the mechanisms of calcium-mediated endocrine cell stimulation or of intracellular and extracellular calcium homeostasis is suggested. PMID:2737922

  14. In vivo breakdown products of the 32 kDa thylakoid herbicide binding protein. [Spirodela oligorrhiza

    SciTech Connect

    Greenberg, B.M.; Mattoo, A.K.; Gaba, V.; Edelman, M.

    1986-04-01

    The 32 kDa herbicide binding protein of PSII is degraded rapidly in the light. This phenomenon was investigated in Spirodela oligorrhiza. When fronds were radiolabeled in the presence of cycloheximide only the chloroplast-encoded proteins were labeled. Under these conditions, a breakdown product of 23.5 kDa was observed. This polypeptide was further degraded with kinetics similar to that of the 32 kDa protein. The 23.5 kDa polypeptide cross-reacted with an antibody specific to the 32 kDa protein. By protease digestion it was determined that the 23.5 kDa polypeptide is the intact N-terminal piece of the 32 kDa protein. Thus, this product corresponds to the membrane anchor of the 32 kDa protein. Using the same antibody, breakdown products of 16 kDa, 14 kDa and 12 kDa were observed. These products have also been observed in Zea mays and Solanum nigrum. Using DCMU during pulse-chase experiments at different light intensities, evidence was obtained that the 23.5 kDa breakdown product is generated in vivo.

  15. Evolving understanding of translocator protein 18 kDa (TSPO).

    PubMed

    Li, Fei; Liu, Jian; Garavito, R Michael; Ferguson-Miller, Shelagh

    2015-09-01

    The translocator protein 18 kDa (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive. A recent breakthrough in determining the high-resolution crystal structures of bacterial homologs of mitochondrial TSPO provides new insight into the structural and functional properties at a molecular level and new opportunities for investigating the significance of this ancient and highly conserved protein family. The availability of atomic level structural information from different species also provides a platform for structure-based drug development. Here we briefly review current knowledge regarding TSPO and the implications of the new structures with respect to hypotheses and controversies in the field.

  16. A 92-kDa human immunostimulatory protein.

    PubMed Central

    Fontan, E; Briend, E; Saklani-Jusforgues, H; d'Alayer, J; Vandekerckhove, J; Fauve, R M

    1994-01-01

    We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety. Images PMID:8078887

  17. Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects.

    PubMed Central

    Künzler, M; Trueheart, J; Sette, C; Hurt, E; Thorner, J

    2001-01-01

    We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response. PMID:11238397

  18. Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation

    SciTech Connect

    Guinea, Barbara; Gonzalez de la Pena, Manuel . E-mail: abernad@cnb.uam.es

    2006-01-15

    PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator.

  19. Identification of sequence similarity between 60 kDa and 70 kDa molecular chaperones: evidence for a common evolutionary background?

    PubMed Central

    Flores, A I; Cuezva, J M

    1997-01-01

    Recent findings support the premise that chaperonins (60 kDa stress-proteins) and alpha-subunits of F-type ATPases (alpha-ATPase) are evolutionary related protein families. Two-dimensional gel patterns of synthesized proteins in unstressed and heat-shocked embryonic Drosophila melanogaster SL2 cells revealed that antibodies raised against the alpha-subunit of the F1-ATPase complex from rat liver recognize an inducible p71 member of the 70 kDa stress-responsive protein family. Molecular recognition of this stress-responsive 70 kDa protein by antibodies raised against the F1-ATPase alpha-subunit suggests the possibility of partial sequence similarity within these ATP-binding protein families. A multiple sequence alignment between alpha-ATPases and 60 kDa and 70 kDa molecular chaperones is presented. Statistical evaluation of sequence similarity reveals a significant degree of sequence conservation within the three protein families. The finding suggests a common evolutionary origin for the ATPases and molecular chaperone protein families of 60 kDa and 70 kDa, despite the lack of obvious structural resemblance between them. PMID:9065788

  20. The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal alga Scenedesmus obliquus.

    PubMed

    Cepák, V; Pribyl, P; Vítová, M

    2006-01-01

    The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed. PMID:17007440

  1. Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins

    PubMed Central

    Trapannone, Riccardo; Mariappa, Daniel; Ferenbach, Andrew T.; vanAalten, Daan M.F.

    2016-01-01

    O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome. PMID:27048592

  2. Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins.

    PubMed

    Trapannone, Riccardo; Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

    2016-06-15

    O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome.

  3. Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae

    PubMed Central

    Jacquet, Michel; Renault, Georges; Lallet, Sylvie; De Mey, Jan; Goldbeter, Albert

    2003-01-01

    Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes. In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus. Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration. Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes. A large heterogeneity in the oscillatory response to stress is observed between individual cells. This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed. The cAMP–PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling. In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus. We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors. PMID:12732613

  4. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo

    PubMed Central

    1988-01-01

    A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport. PMID:2459127

  5. The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal alga Scenedesmus obliquus.

    PubMed

    Cepák, V; Pribyl, P; Vítová, M

    2006-01-01

    The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.

  6. Nucleocytoplasmic shuttling of tRNAs and implication of the cytosolic Hsp70 system in tRNA import.

    PubMed

    Yoshihisa, Tohru

    2015-01-01

    tRNAs, a class of non-coding RNAs essential for translation, are unique among cytosolic RNA species in that they shuttle between the nucleus and cytoplasm during their life. Although their export from the nucleus has been studied in detail, limited information on import machinery was available. Our group recently reported that Ssa2p, one of major cytosolic Hsp70s in Saccharomyces cerevisiae, acts as a crucial factor for tRNA import upon nutrient starvation. Ssa2p can bind tRNAs and a nucleoporin directly in an ATP-sensitive manner, suggesting that it acts as a nuclear import carrier for tRNAs, like importin-β proteins. In vitro assays revealed that Ssa2p binds tRNA specifically but has preference for loosely folded tRNAs. In this Extra View, these features of Ssa2p as a new import factor is discussed with other recent findings related to nucleocytoplasmic transport of tRNAs reported from other groups. PMID:26280499

  7. Reconstitution of surfactant activity by using the 6 kDa apoprotein associated with pulmonary surfactant.

    PubMed Central

    Yu, S H; Possmayer, F

    1986-01-01

    Lipid extracts of bovine pulmonary surfactant containing the 6 kDa apoprotein, but lacking the 35 kDa apoprotein, can mimic the essential characteristics of pulmonary surfactant on a pulsating-bubble surfactometer. Reconstituted surfactant can be produced by combining silicic acid fractions containing 6 kDa apoprotein and phosphatidylglycerol with phosphatidylcholine. Treatment of the protein-containing fraction with proteolytic enzymes abolishes its efficacy. These results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant. Immunodiffusion studies indicate that, contrary to earlier suggestions, the 6 kDa apoprotein is not structurally related to the major surfactant apoprotein that has a molecular mass of 35 kDa. Images Fig. 2. Fig. 3. Fig. 4. PMID:3098235

  8. Accumulation of 23 kDa lipocalin during brain development and injury in Hyphantria cunea.

    PubMed

    Kim, Hong Ja; Je, Hyun Jeong; Cheon, Hyang Mi; Kong, Sun Young; Han, JikHyun; Yun, Chi Young; Han, Yeon Su; Lee, In Hee; Kang, Young Jin; Seo, Sook Jae

    2005-10-01

    The cDNA corresponding to a novel lipocalin was identified from the fall webworm, Hyphantria cunea. This lipocalin cDNA encodes a 194 residue protein with a calculated molecular mass of 23 kDa. Sequence analyses revealed that the 23 kDa lipocalin cDNA is most similar to Drosophila lazarillo, human apolipoprotein D, and Bombyrin. Northern blot analyses showed that 23 kDa lipocalin transcript is expressed in the whole body only in 4- and 6-day-old pupae. By Western blot analysis it was confirmed that 23 kDa lipocalin is mainly accumulated in brain and subesophageal ganglion, though it is detected in a small amount in fat body and epidermis of Hyphantria cunea. The accumulation of 23 kDa lipocalin in brain tissue was upregulated in response to injury. The putative function of 23 kDa lipocalin in brain is discussed.

  9. Accumulation of 52 kDa glycine rich protein in auxin-deprived strawberry fruits and its role in fruit growth. [Fragaria ananassa

    SciTech Connect

    Reddy, A.S.N.; Poovaiah, B.W.

    1987-04-01

    Growth of strawberry (Fragaria ananassa Duch) receptacles can be stopped at any stage by deachening the fruits and can be resumed by exogenous application of auxin. In their earlier studies they demonstrated auxin regulated polypeptide changes at different stages of strawberry fruit development. Removal of achenes from fruits to deprive auxin resulted in the accumulation of 52 KDa polypeptide. This polypeptide is associated with cell wall and its concentration is increased in a time-dependent manner in auxin deprived receptacles. Incorporation studies with (/sup 35/S) methionine showed the promotion of labelling of 52 kDa polypeptide in the auxin-deprived receptacles within 12 h after removal of the achenes. Amino acid analysis revealed that the 52 KDa polypeptide is rich in glycine. Their studies, with normal and mutant strawberry receptacles, indicate that the synthesis and accumulation of this glycine rich protein correlates with cessation of receptacle growth. These results suggest a role for the glycine rich protein in growth.

  10. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

  11. Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors.

    PubMed

    Klose, Cornelia; Viczián, András; Kircher, Stefan; Schäfer, Eberhard; Nagy, Ferenc

    2015-05-01

    The photoreceptors phytochromes monitor the red/far-red part of the spectrum, exist in the biologically active Pfr (far-red absorbing) or inactive Pr (red absorbing) forms, and function as red/far-red light-regulated molecular switches to modulate plant development and growth. Phytochromes are synthesized in the cytoplasm, and light induces translocation of the Pfr conformer into the nucleus. Nuclear import of phytochromes is a highly regulated process and is fine-tuned by the quality and quantity of light. It appears that phytochrome A (phyA) and phytochrome B (phyB) do not possess active endogenous nuclear import signals (NLSs), thus light-induced translocation of these photoreceptors into the nucleus requires direct protein–protein interactions with their NLS-containing signaling partners. Sub-cellular partitioning of the various phytochrome species is mediated by different molecular machineries. Translocation of phyA into the nucleus is promoted by FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL), but the identity of nuclear transport facilitators mediating the import of phyB-E into the nucleus remains elusive. Phytochromes localized in the nucleus are associated with specific protein complexes, termed photobodies. The size and distribution of these structures are regulated by the intensity and duration of irradiation, and circumstantial evidence indicates that they are involved in fine-tuning phytochrome signaling.

  12. Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors

    PubMed Central

    Klose, Cornelia; Viczián, András; Kircher, Stefan; Schäfer, Eberhard; Nagy, Ferenc

    2015-01-01

    The photoreceptors phytochromes monitor the red/far-red part of the spectrum, exist in the biologically active Pfr (far-red absorbing) or inactive Pr (red absorbing) forms, and function as red/far-red light-regulated molecular switches to modulate plant development and growth. Phytochromes are synthesized in the cytoplasm, and light induces translocation of the Pfr conformer into the nucleus. Nuclear import of phytochromes is a highly regulated process and is fine-tuned by the quality and quantity of light. It appears that phytochrome A (phyA) and phytochrome B (phyB) do not possess active endogenous nuclear import signals (NLSs), thus light-induced translocation of these photoreceptors into the nucleus requires direct protein–protein interactions with their NLS-containing signaling partners. Sub-cellular partitioning of the various phytochrome species is mediated by different molecular machineries. Translocation of phyA into the nucleus is promoted by FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL), but the identity of nuclear transport facilitators mediating the import of phyB-E into the nucleus remains elusive. Phytochromes localized in the nucleus are associated with specific protein complexes, termed photobodies. The size and distribution of these structures are regulated by the intensity and duration of irradiation, and circumstantial evidence indicates that they are involved in fine-tuning phytochrome signaling. PMID:26042244

  13. 28 kDa adenosine-binding proteins of brain and other tissues.

    PubMed Central

    Ravid, K; Rosenthal, R A; Doctrow, S R; Lowenstein, J M

    1989-01-01

    Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the

  14. Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands.

    PubMed

    Falchi, Angela Maria; Battetta, Barbara; Sanna, Francesca; Piludu, Marco; Sogos, Valeria; Serra, Mariangela; Melis, Marta; Putzolu, Martina; Diaz, Giacomo

    2007-08-01

    One of the main functions of the translocator protein (18 kDa) or TSPO, previously known as peripheral-type benzodiazepine receptor, is the regulation of cholesterol import into mitochondria for steroid biosynthesis. In this paper we show that TSPO ligands induce changes in the distribution of intracellular cholesterol in astrocytes and fibroblasts. NBD-cholesterol, a fluorescent analog of cholesterol, was rapidly removed from membranes and accumulated into lipid droplets. This change was followed by a block of cholesterol esterification, but not by modification of intracellular cholesterol synthesis. NBD-cholesterol droplets were in part released in the medium, and increased cholesterol efflux was observed in [(3)H]cholesterol-prelabeled cells. TSPO ligands also induced a prominent shrinkage and depolarization of mitochondria and depletion of acidic vesicles with cytoplasmic acidification. Consistent with NBD-cholesterol changes, MTT assay showed enhanced accumulation of formazan into lipid droplets and inhibition of formazan exocytosis after treatment with TSPO ligands. The effects of specific TSPO ligands PK 11195 and Ro5-4864 were reproduced by diazepam, which binds with high affinity both TSPO and central benzodiazepine receptors, but not by clonazepam, which binds exclusively to GABA receptor, and other amphiphilic substances such as DIDS and propranolol. All these effects and the parallel immunocytochemical detection of TSPO in potentially steroidogenic cells (astrocytes) and non-steroidogenic cells (fibroblasts) suggest that TSPO is involved in the regulation and trafficking of intracellular cholesterol by means of mechanisms not necessarily related to steroid biosynthesis.

  15. TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria.

    PubMed

    Gatliff, Jemma; Campanella, Michelangelo

    2016-01-15

    The 18-kDa translocator protein (TSPO) localizes in the outer mitochondrial membrane (OMM) of cells and is readily up-regulated under various pathological conditions such as cancer, inflammation, mechanical lesions and neurological diseases. Able to bind with high affinity synthetic and endogenous ligands, its core biochemical function resides in the translocation of cholesterol into the mitochondria influencing the subsequent steps of (neuro-)steroid synthesis and systemic endocrine regulation. Over the years, however, TSPO has also been linked to core cellular processes such as apoptosis and autophagy. It interacts and forms complexes with other mitochondrial proteins such as the voltage-dependent anion channel (VDAC) via which signalling and regulatory transduction of these core cellular events may be influenced. Despite nearly 40 years of study, the precise functional role of TSPO beyond cholesterol trafficking remains elusive even though the recent breakthroughs on its high-resolution crystal structure and contribution to quality-control signalling of mitochondria. All this along with a captivating pharmacological profile provides novel opportunities to investigate and understand the significance of this highly conserved protein as well as contribute the development of specific therapeutics as presented and discussed in the present review.

  16. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

    PubMed

    Fortes, P; Beloso, A; Ortín, J

    1994-02-01

    The influenza virus RNA segment 8 encodes two proteins, NS1 and NS2, by differential splicing. The collinear transcript acts as mRNA for NS1 protein, while the spliced mRNA encodes NS2 protein. The splicing of NS1 mRNA was studied in cells transfected with a recombinant plasmid that has the cDNA of RNA segment 8 cloned under the SV40 late promoter and polyadenylation signals. As described for influenza virus-infected cells, NS1 mRNA was poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene, but not the NS2 gene, led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs. This effect was not specific for NS1 mRNA, since the splicing of the endogenous SV40 early transcript was altered in such a way that t-Ag mRNA was almost eliminated. These changes in the splicing pattern coincided with a strong inhibition of the mRNA nucleocytoplasmic transport. Both NS1 and NS2 mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed. Furthermore, other mRNAs tested, such as T-Ag mRNA and the non-spliceable nucleoprotein transcript, were also retained in the nucleus upon expression of NS1 protein, suggesting that it induced a generalized block of mRNA export from the nucleus.

  17. Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

    PubMed

    Amazit, Larbi; Alj, Youssef; Tyagi, Rakesh Kumar; Chauchereau, Anne; Loosfelt, Hugues; Pichon, Christophe; Pantel, Jacques; Foulon-Guinchard, Emmanuelle; Leclerc, Philippe; Milgrom, Edwin; Guiochon-Mantel, Anne

    2003-08-22

    Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation. PMID:12791702

  18. A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation.

    PubMed Central

    Bischoff, F R; Maier, G; Tilz, G; Ponstingl, H

    1990-01-01

    Several autoimmune sera from patients with Raynaud phenomenon decorated mammalian kinetochores and bound to a 47-kDa protein on immunoblots of nuclear lysates. Antibody affinity-purified from immunoblots of the 47-kDa band recognized kinetochores, but due to crossreaction with an 18-kDa protein, localization remains elusive. We used one of these sera to purify the antigen from HeLa cells synchronized in mitosis as a noncovalent complex with a 25-kDa protein. The antigen was released from DNA by intercalation with 25 mM chloroquine. Ion-exchange chromatography yielded the pure complex with an apparent molecular size of 68 kDa, which was separated into its components by gel filtration in 6 M guanidinium chloride. Upon two-dimensional gel electrophoresis the 47-kDa protein gave two main spots of pI 6.6 and 6.7, respectively. Posttranslational modification is indicated by additional antigenic spots, by lack of a free alpha-amino group, and by chromatographic behavior of peptides on reversed-phase chromatography. The amino acid sequence for 205 residues of the 47-kDa protein has been established. This sequence is highly homologous with the translated reading frame of RCC1, a gene reportedly involved in regulating onset of mammalian chromosome condensation. Images PMID:2236072

  19. Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system.

    PubMed

    Finn, Michael W; Clayberger, Carol; Krensky, Alan M

    2011-01-01

    Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.

  20. Prostaglandin E2 requirement for transforming growth factor beta 1 inhibition of elicited macrophage 14 kDa phospholipase A2 release.

    PubMed Central

    McCord, M.; Bolognese, B.; Marshall, L. A.

    1995-01-01

    1. Cultured elicited-peritoneal macrophages release a soluble type II 14 kDa phospholipase A2 (PLA2) over time, reaching a plateau by 20-24 h of incubation and maintaining these levels over 72 h. Prostaglandin E2 (PGE2) is also produced but does not plateau until 48-72 h. 2. Transforming growth factor beta 1 (TGF beta 1) reduces cellular 14 kDa PLA2 and its subsequent release by approximately half, but does not alter PGE2 production. Co-incubation of TGF beta 1 with indomethacin interfered, in a concentration-dependent manner, with the ability of TGF beta 1 to reduce cellular 14 kDa PLA2 and its subsequent release over 24 h. The regulation of TGF beta 1 was not specific to indomethacin since other non-steroidal anti-inflammatory drugs had the same effect. This suggested that cyclooxygenase activity was essential for TGF beta 1 to exert its effect and indeed, the addition of exogenous PGE2 restored the TGF beta 1 action. 3. PGE2 alone exerted a concentration-dependent negative feedback action on elicited-macrophage 14 kDa PLA2 release. The inhibitory concentration (IC50 = approximately 180 ng PGE2 ml-1) approximated the PGE2 levels measured in the 24 h macrophage conditioned media (85-140 ng PGE2 ml-1) where PLA2 release began to plateau. Further, incubation of cells with indomethacin over 48 h resulted in the enhancement of 14 kDa PLA2 activity compared to that released from untreated cells. Forskolin failed to inhibit 14 kDa PLA2 release, suggesting PGE2 was not acting through an increase in adenylate cyclase. 4. Taken together, the data are consistent with the immunosuppressive aspects reported for both mediators during inflammation and demonstrates the requirement of PGE2 for TGF beta 1 action on the elicited macrophage. Images Figure 3 PMID:8590973

  1. Human MHC class I antigens are associated with a 90-kDa cell surface protein.

    PubMed

    Ferm, M T; Grönberg, A

    1991-08-01

    Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

  2. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.

    PubMed

    Harauz, George; Boggs, Joan M

    2013-05-01

    The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP's protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.

  3. Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

    PubMed Central

    Delgado, Miguel; Garrido, Francisco; Pérez-Miguelsanz, Juliana; Pacheco, María; Partearroyo, Teresa; Pérez-Sala, Dolores

    2014-01-01

    Abstract Aims: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results: Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. Innovation: Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion: Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of

  4. Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine

    PubMed Central

    Holy, Jon; Lamont, Genelle; Perkins, Edward

    2006-01-01

    Background The quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells. Results 5 – 10 μM sanguinarine effectively inhibits MCF-7 proliferation after a single application of drug. This growth inhibition is accompanied by a striking relocalization of cyclin D1 and topoisomerase II from the nucleus to the cytoplasm, and this effect persists for at least three days after drug addition. DNA synthesis is transiently inhibited by sanguinarine, but cells recover their ability to synthesize DNA within 24 hours. Taking advantage of the fluorescence characteristics of sanguinarine to follow its uptake and distribution suggests that these effects arise from a window of activity of a few hours immediately after drug addition, when sanguinarine is concentrated in the nucleus. These effects occur in morphologically healthy-looking cells, and thus do not simply represent part of an apoptotic response. Conclusion It appears that sub-apoptotic concentrations of sanguinarine can suppress breast cancer cell proliferation for extended lengths of time, and that this effect results from a relatively brief period of activity when the drug is concentrated in the nucleus. Sanguinarine transiently inhibits DNA synthesis, but a novel mechanism of action appears to involve disrupting the trafficking of a number of molecules involved in cell cycle regulation and progression. The ability of sub-apoptotic concentrations of sanguinarine to inhibit cell growth may be a useful

  5. A 25 kDa polypeptide is the ligand for p185neu and is secreted by activated macrophages.

    PubMed

    Tarakhovsky, A; Zaichuk, T; Prassolov, V; Butenko, Z A

    1991-12-01

    Medium conditioned by mouse peritoneal macrophages, activated by muramyl dipeptide (MDP), was used as a possible source of p185neu-specific ligand. MDP-activated macrophage-conditioned medium (MDP-CM) was shown to induce p185neu down-regulation in NEU-expressing NIH3T3 cells in a dose-dependent and temperature-sensitive manner. To exclude the possibility of an indirect action of proteins/metabolites present in MDP-CM on p185neu turnover, a ligand-trapping approach was used. Secreted NEU protein possessing only the extracellular domain but lacking transmembrane and protein kinase domains was expressed in HeLa cells and then purified from conditioned medium, using affinity chromatography on WGA-Sepharose. Co-incubation of the truncated, soluble NEU protein preparation with MDP-CM abolished MDP-CM-induced p185neu down-regulation and reduced self-phosphorylation. It is concluded that a putative p185neu-specific ligand is produced by macrophages activated by MDP. Using MDP-CM, the presence of a 25 kDa polypeptide distinct from EGF, PDGF, FGF, IGF, TGF-alpha and TGF-beta and TNF-alpha, could be demonstrated by decorating a Western blot with soluble NEU and anti-NEU antibodies. Thus, a 25 kDa (non-reduced) p185neu ligand has been described.

  6. Antimicrobial Properties of an Immunomodulator - 15 kDa Human Granulysin

    PubMed Central

    Wei, Hung-Mu; Lin, Li-Chih; Wang, Chiu-Feng; Lee, Yi-Jang; Chen, Yuan-Tsong; Liao, You-Di

    2016-01-01

    Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against Pseudomonas aeruginosa within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by P. aeruginosa. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases. PMID:27276051

  7. Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

    PubMed

    Li, Fei; Liu, Jian; Liu, Nan; Kuhn, Leslie A; Garavito, R Michael; Ferguson-Miller, Shelagh

    2016-05-24

    Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

  8. Nucleocytoplasmic Translocation of UBXN2A Is Required for Apoptosis during DNA Damage Stresses in Colon Cancer Cells

    PubMed Central

    Abdullah, Ammara; Sane, Sanam; Freeling, Jessica L; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-01-01

    The subcellular localization, expression level, and activity of anti-cancer proteins alter in response to intrinsic and extrinsic cellular stresses to reverse tumor progression. The purpose of this study is to determine whether UBXN2A, an activator of the p53 tumor suppressor protein, has different subcellular compartmentalization in response to the stress of DNA damage. We measured trafficking of the UBXN2A protein in response to two different DNA damage stresses, UVB irradiation and the genotoxic agent Etoposide, in colon cancer cell lines. Using a cytosol-nuclear fractionation technique followed by western blot and immunofluorescence staining, we monitored and quantitated UBXN2A and p53 proteins as well as p53's downstream apoptotic pathway. We showed that the anti-cancer protein UBXN2A acts in the early phase of cell response to two different DNA damage stresses, being induced to translocate into the cytoplasm in a dose- and time-dependent manner. UVB-induced cytoplasmic UBXN2A binds to mortalin-2 (mot-2), a known oncoprotein in colon tumors. UVB-dependent upregulation of UBXN2A in the cytoplasm decreases p53 binding to mot-2 and activates apoptotic events in colon cancer cells. In contrast, the shRNA-mediated depletion of UBXN2A leads to significant reduction in apoptosis in colon cancer cells exposed to UVB and Etoposide. Leptomycin B (LMB), which was able to block UBXN2A nuclear export following Etoposide treatment, sustained p53-mot-2 interaction and had partially antagonistic effects with Etoposide on cell apoptosis. The present study shows that nucleocytoplasmic translocation of UBXN2A in response to stresses is necessary for its anti-cancer function in the cytoplasm. In addition, LMB-dependent suppression of UBXN2A's translocation to the cytoplasm upon stress allows the presence of an active mot-2 oncoprotein in the cytoplasm, resulting in p53 sequestration as well as activation of other mot-2-dependent growth promoting pathways. PMID:26516353

  9. Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype.

    PubMed

    Taylor, Janice M W; Allen, Anne-Marie; Graham, Annette

    2014-11-01

    The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors.

  10. The 32 kDa Enamelin Undergoes Conformational Transitions upon Calcium Binding

    PubMed Central

    Fan, Daming; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet

    2008-01-01

    The 32 kDa hydrophilic and acidic enamelin, the most stable cleavage fragment of the enamel specific glycoprotein, is believed to play vital roles in controlling crystal nucleation or growth during enamel biomineralization. Circular dichroism and Fourier transform infrared spectra demonstrate that the secondary structure of the 32 kDa enamelin has a high content of α-helix (81.5%). Quantitative analysis on the circular dichroism data revealed that the 32 kDa enamelin undergoes conformational changes with a structural preference to β-sheet as a function of calcium ions. We suggest that the increase of β-sheet conformation upon presence of Ca2+ may allow preferable interaction of the 32 kDa enamelin with apatite crystal surfaces during enamel biomineralization. The calcium association constant of the 32 kDa enamelin calculated from the fitting curve of ellipticity at 222 nm is Ka = 1.55 (±0.13) × 103 M−1, indicating a relatively low affinity. Our current biophysical studies on the 32 kDa enamelin structure provide novel insights towards understanding the enamelin-mineral interaction and subsequently the functions of enamelin during enamel formation. PMID:18508280

  11. Investigation of hydrophobic organic carbon (HOC) partitioning to 1 kDa fractionated municipal wastewater colloids.

    PubMed

    McPhedran, Kerry N; Seth, Rajesh; Drouillard, Ken G

    2013-03-19

    Natural organic matter from the aquatic environment passing a 1 kDa filter has been hypothesized to not contribute appreciably to hydrophobic organic compound (HOC) partitioning; however, to our knowledge this limit has not been verified experimentally for any sorbate/sorbent system. Presently, colloidal organic carbon (COC) < 1 kDa approached 70% of the total COC (<1.5 μm) mass in primary effluent (PE) from a municipal wastewater treatment plant. Partitioning of HOCs 1,2,4,5-tetrachlorobenzene, pentachlorobenzene, and hexachlorobenzene to COC for both 1.5 μm and 1 kDa filtrates of PE was investigated using the gas-stripping technique. Contrary to the hypothesis, significant HOC-COC partitioning to the 1 kDa filtrate was observed with organic carbon-normalized partitioning coefficients (logKCOC) of 4.30, 4.36, and 3.74 for 1,2,4,5-TeCB, PeCB, and HCB, respectively. Further, partitioning to COC < 1 kDa dominated the overall partitioning of the three chlorobenzenes in the 1.5 μm filtrate, and the partitioning behavior did not follow the trend based on hydrophobicity (KOW). The results show that significant partitioning of HOC may occur to OC < 1 kDa and highlights the need for further experiments with other HOCs and COC characterization to better understand and explain the observed partitioning.

  12. In vitro degradation of the 32kDa PS II reaction centre protein

    SciTech Connect

    Eckenswiller, L.C.; Greenberg, B.M. )

    1989-04-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components.

  13. Integrated and Functional Genomics Analysis Validates the Relevance of the Nuclear Variant ErbB380kDa in Prostate Cancer Progression

    PubMed Central

    El Maassarani, Mahmoud; Barbarin, Alice; Fromont, Gaëlle; Kaissi, Ouafae; Lebbe, Margot; Vannier, Brigitte; Moussa, Ahmed; Séité, Paule

    2016-01-01

    The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB380kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB380kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB380kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB380kDa. Detecting ErbB380kDa could thus constitute a useful marker of prognosis and response to therapy. PMID:27191720

  14. Homology cloning of rat 72 kDa type IV collagenase: cytokine and second-messenger inducibility in glomerular mesangial cells.

    PubMed Central

    Marti, H P; McNeil, L; Davies, M; Martin, J; Lovett, D H

    1993-01-01

    Glomerular mesangial cells (MC) play a central role in the synthesis and turnover of the glomerular extracellular matrix. Prior studies [Davies, Thomas, Martin and Lovett (1988) Biochem. J. 251, 419-425; Martin, Davies, Thomas and Lovett (1989) Kidney Int. 36, 790-801] have characterized at the protein level a 72 kDa type IV collagenase that is secreted by cultured human and rat MC. While exposure of most cell types to interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) or phorbol ester has little or even an inhibitory effect on 72 kDa type IV collagenase secretion, these factors significantly increased the synthesis of this enzyme by rat MC. Given this divergent pattern of expression, a homology-based PCR cloning strategy using rat MC cDNA templates was employed to define at the molecular level the structure of the mesangial 72 kDa type IV collagenase. The nucleotide sequence within the open reading frame of the rat mesangial 72 kDa type IV collagenase cDNA diverges from the sequence of the human homologue by approx. 9%. The divergence in the 3' untranslated region was much more extensive. Steady-state levels of the 3.1 kb transcript of the 72 kDa type IV collagenase were low or undetectable in resting MC, but were greatly stimulated following incubation with IL-beta, TNF-alpha or phorbol ester. None of these factors induced synthesis by MC of the closely related 92 kDa type IV collagenase. Synthesis by MC of the 72 kDa type IV collagenase was also induced by second-messenger analogues, including 8-bromo-cyclic AMP and forskolin. It is concluded that MC regulate the expression of this enzyme in an unusual, tissue-specific fashion. Cytokine and second-messenger inducibility may contribute to the enhanced expression of the enzyme during glomerular inflammatory disorders. Images Figure 1 Figure 2 PMID:7916617

  15. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  16. Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro*

    PubMed Central

    Kwak, Seo-Young; Wiedemann-Bidlack, Felicitas B.; Beniash, Elia; Yamakoshi, Yasuo; Simmer, James P.; Litman, Amy; Margolis, Henry C.

    2009-01-01

    The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin. PMID:19443653

  17. Tandem Phosphorylation of Serines 221 and 318 by Protein Kinase Cδ Coordinates mRNA Binding and Nucleocytoplasmic Shuttling of HuR▿

    PubMed Central

    Doller, Anke; Schlepckow, Kai; Schwalbe, Harald; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2010-01-01

    Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cδ (PKCδ)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCδ mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D1, and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCδ coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions. PMID:20086103

  18. Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

    PubMed Central

    Park, Young-Jin; Park, Jeong-Hyun; Chai, Jong-Yil

    2014-01-01

    This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species. PMID:24850966

  19. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

    PubMed Central

    Song, Hyun-Ouk

    2016-01-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis. PMID:26951982

  20. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk

    2016-02-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis. PMID:26951982

  1. In vitro study on the interaction between the 32 kDa enamelin and amelogenin

    PubMed Central

    Fan, Daming; Du, Chang; Sun, Zhi; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet

    2015-01-01

    Enamel extracelluar matrix components play vital roles in controlling crystal nucleation and growth during enamel formation. We investigated the interaction between the 32 kDa enamelin fragment and amelogenin using immunochemical and biophysical methods. Immunoprecipitation studies revealed that the 32 kDa enamelin and amelogenin eluted together from a Protein A column. Dynamic light scattering results showed that the 32 kDa enamelin had a profound effect on amelogenin assembly at pH 8.0, causing partial dissociation of the nanospheres, in a dose-dependent manner. The appearance of an isodichroic point and the shifting and intensity decrease of the ellipticity minima in the circular dichroism spectra of amelogenin following the addition of the 32 kDa enamelin were indicative of conformational changes in amelogenin and of a direct interaction between the two macromolecules. Our results collectively demonstrate that the 32 kDa enamelin has a direct interaction with amelogenin in vitro. Our current studies provide novel insights into understanding possible cooperation between enamelin and amelogenin in macromolecular self-assembly and in controlling enamel mineral formation. PMID:19263522

  2. Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase

    SciTech Connect

    Shoseyov, O.; Doi, R.H. )

    1990-03-01

    The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. The authors propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits.

  3. Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase.

    PubMed Central

    Shoseyov, O; Doi, R H

    1990-01-01

    The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. We propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits. Images PMID:2107547

  4. Characterization of the 70 kDa polypeptide of the Na/Ca exchanger.

    PubMed Central

    Saba, R I; Bollen, A; Herchuelz, A

    1999-01-01

    The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1-21; NCX1, residues 393-406; and Exon F, residues 622-644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger. PMID:9931309

  5. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  6. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions.

    PubMed

    Winick-Ng, Warren; Caetano, Fabiana A; Winick-Ng, Jennifer; Morey, Trevor M; Heit, Bryan; Rylett, R Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1-42 (Aβ1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1-42-induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ-exposure. PMID:27052102

  7. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions

    PubMed Central

    Winick-Ng, Warren; Caetano, Fabiana A.; Winick-Ng, Jennifer; Morey, Trevor M.; Heit, Bryan; Rylett, R. Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure. PMID:27052102

  8. Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

    SciTech Connect

    Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz; Burger, Gertraud; Hori, Manabu; Fujishima, Masahiro . E-mail: fujishim@yamaguchi-u.ac.jp

    2005-12-02

    The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.

  9. Specific binding of victorin to a 100-kDa protein from oats

    SciTech Connect

    Wolpert, T.J.; Macko, V. )

    1989-06-01

    Susceptibility of oats to victoria blight, caused by the fungus Cochliobolus victoriae, and sensitivity to the host-specific toxin victorin, produced by the fungus, are controlled by the dominant allele at the Vb locus. It has been postulated that the Vb locus encodes a toxin receptor, although direct evidence for such a receptor is not available. Recent studies on structure-activity relationships of the toxin established a methodology for producing {sup 125}I-labeled victorin. Electrophoretic analysis of proteins from isogenic susceptible and resistant oat genotypes following treatment of leaves with radiolabeled victorin showed that victorin binds in a covalent and a genotype-specific manner to a 100-kDa protein only in susceptible oat leaf slices. This in vivo binding was competitively displaced by reduced victorin, a nontoxic protective compound, and appeared to be correlated with biological activity. In vitro binding to the 100-kDa protein in leaf extracts showed several differences from in vivo binding. Binding was not genotype specific and required a reducing agent that was not required for in vivo binding. Differential centrifugation showed that the 100-kDa victorin binding protein was not a cytosolic protein but was enriched in a high-speed particulate fraction. The data support the hypothesis that the 100-kDa protein is the victorin receptor.

  10. Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum.

    PubMed

    Delplace, P; Fortier, B; Tronchin, G; Dubremetz, J F; Vernes, A

    1987-04-01

    Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.

  11. Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

    PubMed

    Naidoo, Kerrilyn; Malindisa, Sibusiso T; Otgaar, Tyrone C; Bernert, Martin; Da Costa Dias, Bianca; Ferreira, Eloise; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F T; Letsolo, Boitelo T

    2015-01-01

    Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity. PMID:26545108

  12. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase.

    PubMed

    Krishnan, Hari B; Chen, Ming-Hsuan

    2013-06-01

    Rice, the staple food of south and east Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16, 26, 33, and 56 kDa have been identified as allergens. Recently, it was documented that the 56 kDa rice allergen was responsible for rice-induced anaphylaxis. The 14-16 kDa allergens have been identified as α-amylase inhibitors; the 26 kDa protein has been identified as α-globulin; and the 33 kDa protein has been identified as glyoxalase I. However, the identity of the 56 kDa rice allergen has not yet been determined. In this study, we demonstrate that serum from patients allergic to maize shows IgE binding to a 56 kDa protein that was present in both maize and rice but not in the oil seeds soybean and peanut. The 56 kDa IgE-binding protein was abundant in the rice endosperm. We have purified this protein from rice endosperm and demonstrated its reactivity to IgE antibodies from the serum of maize-allergic patients. The purified protein was subjected to matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry analysis, resulting in identification of this rice allergen as granule-bound starch synthase, a product of the Waxy gene. Immunoblot analysis using protein extracts from a waxy mutant of rice revealed the absence of the 56 kDa IgE-binding protein. Our results demonstrate that the 56 kDa rice allergen is granule-bound starch synthase and raise the possibility of using waxy mutants of rice as a potential source of the hypoallergenic diet for patients sensitized to the 56 kDa rice allergen.

  13. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 Is Required for Circadian Periodicity through the Promotion of Nucleo-Cytoplasmic mRNA Export in Arabidopsis[W][OPEN

    PubMed Central

    MacGregor, Dana R.; Gould, Peter; Foreman, Julia; Griffiths, Jayne; Bird, Susannah; Page, Rhiannon; Stewart, Kelly; Steel, Gavin; Young, Jack; Paszkiewicz, Konrad; Millar, Andrew J.; Halliday, Karen J.; Hall, Anthony J.; Penfield, Steven

    2013-01-01

    Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock. PMID:24254125

  14. The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms.

    PubMed

    Haas, Muriel; Geldreich, Angèle; Bureau, Marina; Dupuis, Laurence; Leh, Véronique; Vetter, Guillaume; Kobayashi, Kappei; Hohn, Thomas; Ryabova, Lyubov; Yot, Pierre; Keller, Mario

    2005-03-01

    The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

  15. The defective nuclear lamina in Hutchinson-gilford progeria syndrome disrupts the nucleocytoplasmic Ran gradient and inhibits nuclear localization of Ubc9.

    PubMed

    Kelley, Joshua B; Datta, Sutirtha; Snow, Chelsi J; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J; Paschal, Bryce M

    2011-08-01

    The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.

  16. The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms.

    PubMed

    Haas, Muriel; Geldreich, Angèle; Bureau, Marina; Dupuis, Laurence; Leh, Véronique; Vetter, Guillaume; Kobayashi, Kappei; Hohn, Thomas; Ryabova, Lyubov; Yot, Pierre; Keller, Mario

    2005-03-01

    The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle. PMID:15746075

  17. A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

    PubMed

    Hawes, T C; Marshall, C J; Wharton, D A

    2014-08-01

    A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component. PMID:25025820

  18. Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein

    SciTech Connect

    Lin, Jihjing; Thach, R.E. ); Patino, M.M.; Gaffield, L.; Walden. W.E. ); Smith, A. )

    1991-07-15

    Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. ({sup 14}C)Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H{sub 2}O{sub 2} is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.

  19. Attachment of group B streptococci to macrophages is mediated by a 21-kDa protein.

    PubMed

    Smith, L M; Laganas, V; Pistole, T G

    1998-02-01

    Group B Streptococcus (GBS) is able to bind to human macrophages in vitro in the absence of exogenous opsonins. The exact mechanisms that mediate this attachment are unclear. This study was undertaken to determine what protein adhesins are present on the surface of GBS that mediate attachment to macrophages. We have identified a 21-kDa protein from the envelope of GBS type III that directly binds to macrophages as determined by Western blot analysis. Antiserum against this protein was able to inhibit binding of GBS to macrophages by greater than 80% as measured by flow cytometry. Antiserum against the 21-kDa protein cross-reacted with 21-kDa proteins from GBS type Ib, type II, type III (COH31 and MR732) and type IV, as well as Staphyloccus epidermidis, but not GBS type Ia, Listeria monocytogenes or Enterococcus faecalis. This protein may be important in mediating the attachment of GBS to macrophages in an opsonin-poor environment.

  20. The alpha- and beta-subunits of the jacalins are cleavage products from a 17-kDa precursor.

    PubMed

    Ngoc, L D; Brillard, M; Hoebeke, J

    1993-02-13

    The jacalins of three Artocarpus species were purified by affinity chromatography on a desialylated mucin-CNBr-Sepharose 4B column. The beta-chains and the 14 kDa alpha-chains were separated by high pressure liquid chromatography and the 17 kDa chains by preparative electrophoresis. The 17 kDa and 14 kDa chains had a similar highly conserved N-terminal sequence. The beta-chains were different for the three species and Artocarpus champeden contained two different beta-chains. CNBr cleavage of the 17 kDa polypeptide of Artocarpus tonkinensis yielded one peptide more than the 14 kDa. The N-terminal sequence of this fragment was similar to that of the beta-chain proving that this chain results from a proteolytic cleavage at the C-terminus of the 17 kDa peptide. The large heterogeneity of the beta-chains of jacalins from different species could be used as a marker for evolutionary studies on the Artocarpus family.

  1. A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase.

    PubMed Central

    Treptau, T; Kissmehl, R; Wissmann, J D; Plattner, H

    1995-01-01

    We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that

  2. Identification of a major PAF acetylhydrolase in human serum/plasma as a 43 kDa glycoprotein containing about 9 kDa asparagine-conjugated sugar chain(s).

    PubMed

    Akiyama, M; Sugatani, J; Suzuki, T; Suzuki, Y; Miwa, M

    1998-05-01

    Platelet-activating factor (PAF) acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma. PMID:9562606

  3. Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

    PubMed

    Kimura, Tominori; Hashimoto, Iwao; Nishikawa, Masao; Yamada, Hisao

    2009-06-01

    Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

  4. Identification and characterization of a novel 34 kDa merozoite protein in Babesia orientalis.

    PubMed

    He, Lan; Fan, Lizhe; Liu, Qin; Hu, Jinfang; Miao, Xiaoyan; Huang, Yuan; He, Pei; He, Junwei; Yu, Long; Khan, Muhammad Kasib; Zhou, Yanqin; Shen, Bang; Zhao, Junlong

    2015-09-15

    A novel Babesia orientalis 34 kDa protein (designated BoP34) was obtained by immunoscreening of a cDNA expression library using B. orientalis infected water buffalo serum. The complete nucleotide sequence of the BoP34 was 1088 bp, which contained one open reading frame (ORF), two untranslated regions (UTRs) and a poly (A) tail. The length of ORF was 933 bp, encoding a polypeptide of 310 aa with a predicted size of 34 kDa. BLAST analysis showed that the nucleotide sequence of BoP34 had 71% similarity with that of the Babesia bovis gene XM_001611335, which encodes a nuclear movement family protein. This suggested that BoP34 is a homologous of the movement family protein. Structural analysis of the BoP34 protein indicated a CS domain which may interact with the ATPase domain of the heat shock protein 90. A truncated version of BoP34 was cloned into the expression vector pET-32a and subsequently expressed and purified from the Escherichia coli Rosetta™ (DE3) pLysS stain as a Trx-fusion (designated rBoP34-T). Antibodies in the serum of a B. orientalis-infected water buffalo were able to recognize this protein in immune-bloting analysis. Rabbit antibodies raised against rBoP34-T could detecte native BoP34 (34 kDa) in B. orientalis-infected water buffalo erythrocytes. These results suggested that BoP34 might be a good diagnostic antigen for the specific detection of anti-B. orientalis antibody in water buffalo. Further research is required to explore the biological function and diagnostic potential of this molecule.

  5. Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

    PubMed Central

    2010-01-01

    Background The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia). Results The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph. Conclusions In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O2 binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological

  6. Complexes of 3.6 kDa maltodextrin with some metals.

    PubMed

    Tomasik, Piotr; Anderegg, James W; Schilling, Christopher H

    2004-06-30

    Preparation of magnesium, lanthanum, and bismuth(III) complexes of 3.6 kDa maltodextrin and some properties of the resulting materials are presented. The metal derivatives contain metals bound to the oxygen atoms of the hydroxyl groups of maltodextrin. Additionally, the metal atoms are coordinated to the hydroxyl groups of the D-glucose units of the macroligand. Such coordination stabilized the metal - oxygen bond against hydrolysis, even in boiling water. The presence of magnesium and lanthanum atoms increased the thermal stability of maltodextrin, whereas bismuth atoms decreased it.

  7. A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii).

    PubMed

    Wade, M A; Lin, M

    1999-09-01

    This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility. PMID:10645248

  8. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.

    PubMed Central

    Takagaki, Y; MacDonald, C C; Shenk, T; Manley, J L

    1992-01-01

    Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs. Images PMID:1741396

  9. Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

    SciTech Connect

    Coseno,M.; Martin, G.; Berger, C.; Gilmartin, G.; Keller, W.; Doublie, S.

    2008-01-01

    Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.

  10. Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle.

    PubMed

    Henderson, Rory C; Gao, Feng; Jayanthi, Srinivas; Kight, Alicia; Sharma, Priyanka; Goforth, Robyn L; Heyes, Colin D; Henry, Ralph L; Suresh Kumar, Thallapuranam Krishnaswamy

    2016-09-20

    Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes. PMID:27653474

  11. Identification of a 41 kDa protein embedded in the biosilica of scales and bristles isolated from Mallomonas splendens (Synurophyceae, Ochrophyta).

    PubMed

    Schultz, T F; Egerton-Warburton, L; Crawford, S A; Wetherbee, R

    2001-12-01

    Cells of the photosynthetic protist Mallomonas splendens (Synurophyceae, Ochrophyta) are encased within a highly patterned wall or scale case that consists of silicified scales and bristles. In an effort to understand the mechanisms that unicellular protists utilize to produce elaborate, mineralized structures of great complexity and hierarchical structure, we identified and characterized a 41 kDa protein from purified scales/bristles isolated from M. splendens (SP41 for Scale Protein of 41 kDa). A cDNA encoding this protein was isolated and sequence analysis indicated that it is a novel protein. Polyclonal antibodies were generated against bacterially expressed SP41 and used to localize the protein throughout scale and bristle morphogenesis. Immunoelectron microscopy confirmed the biochemical data that SP41 is a component of mature scales and bristles, the protein localizing to silicified components of the purified extracellular matrix. During scale and bristle biogenesis within the cell, SP41 is deposited into a specialized Silica Deposition Vesicle (SDV) concomitant with silica deposition, a highly regulated event during scale and bristle formation. These results argue for SP41 playing a role in morphogenesis and/or silicification within the SDV during scale and bristle biogenesis.

  12. Identification of a 115kDa MAP-kinase activated by freezing and anoxic stresses in the marine periwinkle, Littorina littorea.

    PubMed

    MacDonald, Justin A; Storey, Kenneth B

    2006-06-15

    The mitogen-activated protein kinase (MAPK) cascade regulates changes in gene transcription by transmitting extracellular stimuli from the plasma membrane to the cell nucleus and has an important role to play in organismal responses to environmental stresses. The activities of MAPKs were investigated in the marine gastropod mollusk, Littorina littorea, a species that tolerates both extracellular freezing and long term oxygen deprivation. In-gel kinase assays revealed the presence of two MAPKs in foot muscle and hepatopancreas, a 42 and a 115kDa protein. Immunoblot analysis showed that both were MAPK proteins and that one was the periwinkle homologue of p42(ERK2). Size exclusion chromatography confirmed the 115kDa size of the novel snail MAPK and its role as the dominant MAPK activity in foot muscle. In-gel kinase assays, immunoblotting with phospho-specific ERK antibody, as well as kinase activity profiles from hydroxyapatite chromatography demonstrated that p115 MAPK kinase activity was increased in foot muscle in response to in vivo freezing or anoxia exposures. The results suggest a role for this novel kinase in environmental stress response. PMID:16620767

  13. Tissue Localization of Lymphocystis Disease Virus (LCDV) Receptor-27.8 kDa and Its Expression Kinetics Induced by the Viral Infection in Turbot (Scophthalmus maximus)

    PubMed Central

    Sheng, Xiuzhen; Wu, Ronghua; Tang, Xiaoqian; Xing, Jing; Zhan, Wenbin

    2015-01-01

    The 27.8 kDa membrane protein expressed in flounder (Paralichthys olivaceus) gill cells was proved to be a receptor mediating lymphocystis disease virus (LCDV) infection. In this study, SDS-PAGE and Western blotting demonstrated that 27.8 kDa receptor (27.8R) was shared by flounder and turbot (Scophthalmus maximus). Indirect immunofluorescence assay (IIFA) and immunohistochemistry showed that 27.8R was widely expressed in tested tissues of healthy turbot. The indirect enzyme-linked immunosorbent assay indicated that 27.8R expression was relatively higher in stomach, gill, heart, and intestine, followed by skin, head kidney, spleen, blood cells, kidney and liver, and lower in ovary and brain in healthy turbot, and it was significantly up-regulated after LCDV infection. Meanwhile, real-time quantitative PCR demonstrated that LCDV was detected in heart, peripheral blood cells, and head kidney at 3 h post infection (p.i.), and then in other tested tissues at 12 h p.i. LCDV copies increased in a time-dependent manner, and were generally higher in the tissues with higher 27.8R expression. Additionally, IIFA showed that 27.8R and LCDV were detected at 3 h p.i. in some leukocytes. These results suggested that 27.8R also served as a receptor in turbot, and LCDV can infect some leukocytes which might result in LCDV spreading to different tissues in turbot. PMID:26556346

  14. Tissue Localization of Lymphocystis Disease Virus (LCDV) Receptor-27.8 kDa and Its Expression Kinetics Induced by the Viral Infection in Turbot (Scophthalmus maximus).

    PubMed

    Sheng, Xiuzhen; Wu, Ronghua; Tang, Xiaoqian; Xing, Jing; Zhan, Wenbin

    2015-01-01

    The 27.8 kDa membrane protein expressed in flounder (Paralichthys olivaceus) gill cells was proved to be a receptor mediating lymphocystis disease virus (LCDV) infection. In this study, SDS-PAGE and Western blotting demonstrated that 27.8 kDa receptor (27.8R) was shared by flounder and turbot (Scophthalmus maximus). Indirect immunofluorescence assay (IIFA) and immunohistochemistry showed that 27.8R was widely expressed in tested tissues of healthy turbot. The indirect enzyme-linked immunosorbent assay indicated that 27.8R expression was relatively higher in stomach, gill, heart, and intestine, followed by skin, head kidney, spleen, blood cells, kidney and liver, and lower in ovary and brain in healthy turbot, and it was significantly up-regulated after LCDV infection. Meanwhile, real-time quantitative PCR demonstrated that LCDV was detected in heart, peripheral blood cells, and head kidney at 3 h post infection (p.i.), and then in other tested tissues at 12 h p.i. LCDV copies increased in a time-dependent manner, and were generally higher in the tissues with higher 27.8R expression. Additionally, IIFA showed that 27.8R and LCDV were detected at 3 h p.i. in some leukocytes. These results suggested that 27.8R also served as a receptor in turbot, and LCDV can infect some leukocytes which might result in LCDV spreading to different tissues in turbot.

  15. Tissue Localization of Lymphocystis Disease Virus (LCDV) Receptor-27.8 kDa and Its Expression Kinetics Induced by the Viral Infection in Turbot (Scophthalmus maximus).

    PubMed

    Sheng, Xiuzhen; Wu, Ronghua; Tang, Xiaoqian; Xing, Jing; Zhan, Wenbin

    2015-01-01

    The 27.8 kDa membrane protein expressed in flounder (Paralichthys olivaceus) gill cells was proved to be a receptor mediating lymphocystis disease virus (LCDV) infection. In this study, SDS-PAGE and Western blotting demonstrated that 27.8 kDa receptor (27.8R) was shared by flounder and turbot (Scophthalmus maximus). Indirect immunofluorescence assay (IIFA) and immunohistochemistry showed that 27.8R was widely expressed in tested tissues of healthy turbot. The indirect enzyme-linked immunosorbent assay indicated that 27.8R expression was relatively higher in stomach, gill, heart, and intestine, followed by skin, head kidney, spleen, blood cells, kidney and liver, and lower in ovary and brain in healthy turbot, and it was significantly up-regulated after LCDV infection. Meanwhile, real-time quantitative PCR demonstrated that LCDV was detected in heart, peripheral blood cells, and head kidney at 3 h post infection (p.i.), and then in other tested tissues at 12 h p.i. LCDV copies increased in a time-dependent manner, and were generally higher in the tissues with higher 27.8R expression. Additionally, IIFA showed that 27.8R and LCDV were detected at 3 h p.i. in some leukocytes. These results suggested that 27.8R also served as a receptor in turbot, and LCDV can infect some leukocytes which might result in LCDV spreading to different tissues in turbot. PMID:26556346

  16. The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

    SciTech Connect

    Kitayama, Kumiko; Kamo, Mariko; Oma, Yukako; Matsuda, Ryo; Uchida, Takafumi; Ikura, Tsuyoshi; Tashiro, Satoshi; Ohyama, Takashi; Winsor, Barbara; Harata, Masahiko

    2009-01-15

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5{delta} yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX ({gamma}-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced {gamma}-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

  17. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

    PubMed Central

    Rousseau, D; Kaspar, R; Rosenwald, I; Gehrke, L; Sonenberg, N

    1996-01-01

    The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8577715

  18. The prolactin gene is expressed in the hypothalamic-neurohypophyseal system and the protein is processed into a 14-kDa fragment with activity like 16-kDa prolactin.

    PubMed Central

    Clapp, C; Torner, L; Gutiérrez-Ospina, G; Alcántara, E; López-Gómez, F J; Nagano, M; Kelly, P A; Mejía, S; Morales, M A; Martínez de la Escalera, G

    1994-01-01

    The 23-kDa form of prolactin (PRL) has been proposed to function as both a mature hormone and a prohormone precursor for different uniquely bioactive forms of the molecule. We have shown that the 16-kDa N-terminal fragment of PRL (16K PRL) inhibits angiogenesis via a specific receptor. In addition, 16K PRL stimulates natriuresis and diuresis in the rat, and kidney membranes contain high-affinity specific binding sites for this PRL fragment. 16K PRL can be derived from an enzymatically cleaved form of PRL (cleaved PRL). With the use of a specific 16K PRL antiserum, we have localized a 14-kDa immunoreactive protein in the paraventricular and supraoptic nuclei of the hypothalamus and in the neurohypophysis. Reverse transcription-polymerase chain reaction of RNA from isolated paraventricular nuclei showed the expression of the full-length PRL mRNA. The neurohypophysis was found to contain the enzymes that produce cleaved PRL, small amounts of PRL, and cleaved PRL. Medium conditioned by neurohypophyseal cultures, enriched with the 14-kDa immunoreactive protein, has antiangiogenic effects that are blocked by the 16K PRL antiserum. These results are consistent with the expression of PRL in the hypothalamic-neurohypophyseal system, and the preferential processing of the protein into a 14-kDa fragment with biological and immunological properties of 16K PRL. Images PMID:7937959

  19. [Alterations in heat shock protein 70 kDa levels in human neutrophils under the heat shock conditions].

    PubMed

    Boĭko, A A; Vetchinin, S S; Sapozhnikov, A M; Kovalenko, E I

    2014-01-01

    Intracellular content of heat shock proteins of 70 kDa family (HSP70) possessing chaperone and cytoprotective functions depends on specialization and functional activity of the cells. The aim of this study was to analyze the dynamics of constitutive and inducible HSP70 levels evoked by heat shock in human neutrophils, the short-lived fraction of white blood cells providing non-specific defense against bacterial pathogens. Biphasic dynamics of the intracellular HSP70 level with an increase and following decrease in 15-30 min after the heat shock was revealed by flow cytometry. This dynamics was similar for constitutive and inducible forms of HSP70. Pre-incubation of neutrophils with cycloheximide, the inhibitor of protein synthesis, did not change the intracellular HSP70 dynamics registered by flow cytometry indicating that the increased HSP70 level detected immediately after the heat shock was not mediated by de novo protein synthesis. It was confirmed by Western blotting analysis of HSP70 intracellular content. It was suggested that the elevated HSP70 level was related to conformational HSP70 molecule changes and to increased availability of HSP70 epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or C-terminal substrate-binding domains of HSP70 it has been demonstrated by cell immunofluorescence and flow cytometry methods that the heat shock-associated increase of the intracellular HSP70 level was mediated by HSP70 interaction with antibodies recognizing HSP70 substrate-binding domain. It was demonstrated that the decrease of intracellular HSP70 level after heat treatment could be connected with a release of both inducible and constitutive HSP70 into extracellular space. Our data suggest that stress-induced release of HSP70 from neutrophils is regulated by ABC-transporters.

  20. A 43 kDa recombinant plasmepsin elicits immune response in mice against Plasmodium berghei malaria.

    PubMed

    Pirta, Chhaya; Sharma, Nitya Nand; Banyal, H S

    2016-01-01

    Intraerythrocytic parasites degrade haemoglobin to make available nutrients for their growth and maturation. Plasmepsins, the aspartic proteases of Plasmodium play a significant role in haemoglobin degradation and are proposed as attractive drug targets. In the present study the gene which encodes plasmepsin in rodent malaria parasite, Plasmodium berghei, was cloned and expressed. The gene was sequenced and expressed in Escherichia coli BL21DE3 and a recombinant plasmepsin of molecular weight 43 kDa was obtained. The sequence obtained was analysed and compared with plasmepsins of other Plasmodium spp. Mice immunized with the recombinant plasmepsin induced a strong humoral immune response. ELISA and IFA performed on the serum of immunized mice showed high antibody titres. Along with this, in vivo study exhibited partial protection against P. berghei infection suggesting role of plasmepsin in malaria control.

  1. TSPO 18 kDa (PBR) Targeted Photosensitizers for Cancer Imaging (PET) and PDT.

    PubMed

    Chen, Yihui; Sajjad, Munawwar; Wang, Yanfang; Batt, Carrie; Nabi, Hani A; Pandey, Ravindra K

    2011-02-10

    Translocator protein (TSPO) 18 kDa overexpression has been observed in a large variety of human cancers, especially breast cancers. PK 11195, an isoquinoline analogue, is one of the ligands of highest TSPO binding affinity. Due to the long biological half life of our photosensitizers, there is a need to label them with a long lived radioisotope, for example I-124. Our objectives are to find translocator protein targeted photosensitizers for both tumor imaging (PET) and photodynamic therapy (PDT). I-PK 11195 is conjugated with the tumor avid photosensitizer HPPH. We find that those two tumor avid components complement each other and make the conjugate molecule even more tumor avid; compared to the photosensitizer itself, the conjugate is found to show improved PDT efficacy. It is concluded that I-PK 11195 can be a good vehicle to deliver radionuclide and photosensitizer to TSPO overexpressed tumor regions. Such conjugates could be useful for both tumor imaging (PET) and PDT.

  2. Chromosomal assignment of the gene encoding the human 58-kDa inhibitor (PRKRI) of the interferon-induced dsRNA-activated protein kinase to chromosome 13q32

    SciTech Connect

    Korth, M.J.; Katze, M.G.; Edelhoff, S.; Disteche, C.M.

    1996-01-15

    The 58-kDa inhibitor (p58) of the interferon-induced dsRNA-activated protein kinase (PKR) is a cellular protein recruited by the influenza virus to down-regulate the activity of PKR during virus infection. The inhibitor also appears to play a role in the regulation of cellular gene expression in the absence of viral infection and has oncogenic properties when overexpressed. Using fluorescence in situ hybridization, we have mapped the p58 gene (PRKRI) to human chromosome 13 band q32. Aberrations in the structure or number of chromosome 13 have been identified in a variety of human cancers, particularly in acute leukemia. 15 refs., 1 fig.

  3. Diffusion into human islets is limited to molecules below 10 kDa.

    PubMed

    Williams, S J; Schwasinger-Schmidt, T; Zamierowski, D; Stehno-Bittel, L

    2012-10-01

    Isolated islets are important tools in diabetes research and are used for islet transplantation as a treatment for type 1 diabetes. Yet these cell clusters have a dramatic diffusion barrier that leads to core cell death. Computer modeling has provided theoretical size limitations, but little has been done to measure the actual rate of diffusion in islets. The purpose of this study was to directly measure the diffusion barrier in intact human islets and determine its role in restricting insulin secretion. Impeded diffusion into islets was monitored with fluorescent dextran beads. Dextran beads of 10-70 kDa failed to diffuse into the core of the intact islets, while 0.9 kDa probe was observed within the core of smaller islets. Diffusion of the fluorescent form of glucose, 2-NBDG, had similar diffusion limitations as the beads, with an average intra-islet diffusion rate of 1.5 ± 0.2 μm/min. The poor diffusion properties were associated with core cell death from necrosis, not apoptosis. Short-term exposure to a mild papain/0 Ca(2+) cocktail, dramatically reduced the diffusion barrier so that all cells within islets were exposed to media components. Lowering the diffusion barrier increased the immediate and long-term viability of islet cells, and tended to increase the amount of insulin released, especially in low glucose conditions. However, it failed to improve the large islet's glucose-stimulated insulin secretion. Thus, the islet diffusion barrier leads to low viability and poor survival of large islets, but is not solely responsible for the reduced insulin secretion of large isolated islets.

  4. Immunogenicity and immunolocalization of the 22.6 kDa antigen of Schistosoma japonicum.

    PubMed

    Li, Y; Auliff, A; Jones, M K; Yi, X; McManus, D P

    2000-08-01

    The 22.6 kDa tegumental-associated antigens of Schistosoma mansoni (Sm22.6) and Schistosoma japonicum (Sj22.6) are of recognized interest in schistosomiasis vaccine development, although no direct vaccination/challenge experiments using either Sm22.6 or Sj22.6 had been previously described. We report that Escherichia coli-expressed reSj22.6 failed to protect mice or water buffaloes against subsequent challenge with S. japonicum cercariae. This was despite the fact that specific IgG (buffaloes) and IgG and IgE (CBA mice) antibodies were generated against the 22.6 kDa molecule, observations consistent with some of our earlier findings. We could find no evidence from immunolocalization studies that Sj22.6 is expressed or exposed on the surface of the adult parasite since it appears to be restricted to the apical cytoplasm of the tegument and is not associated with the apical or basal membrane or any membrane-bound structures in the apical cytoplasm. Nevertheless, Sj22.6 must be released to the immune system during the course of infection because specific anti-Sj22.6 IgG antibodies were present in the sera of nonvaccinated but challenged mice. We conclude that it may be necessary to produce reSj22.6 in a more relevant expression system, such as baculovirus, to further establish its vaccine potential and that detailed immunochemical and immunolocalization studies of early developmental stages may be necessary to determine how Sj22.6 is released or shed in S. japonicum infections.

  5. Circulating non-22 kDa growth hormone isoforms after a repeated GHRH stimulus in normal subjects.

    PubMed

    Coya, R; Algorta, J; Boguszewski, C L; Vela, A; Carlsson, L M S; Aniel-Quiroga, A; Busturia, M A; Martul, P

    2005-04-01

    The aim of this study was to evaluate the proportion of non-22 kDa GH isoforms in relation to total GH concentration after a repeated GHRH stimulus in healthy subjects. We studied 25 normal volunteers (12 males and 13 females, mean age 13.1 years, range 6-35), who received two GHRH bolus (1.5 mug/kg body weight, i.v.) administered separately by an interval of 120 minutes. The proportion of non-22 kDa GH was determined by the 22 kDa GH exclusion assay (GHEA), which is based on immunomagnetic extraction of monomeric and dimeric 22 kDa GH from serum, and quantitation of non-22 kDa GH isoforms using a polyclonal GH assay. Samples were collected at baseline and at 15-30 min intervals up to 240 min for total GH concentration. Non-22 kDa GH isoforms were measured in samples where peak GH after GHRH was observed. Total GH peaked after the first GHRH bolus in all subjects (median 37.2 ng/ml; range: 10.4-94.6). According to GH response to the second GHRH stimulus, the study group was divided in "non-responders" (n=7; 28%), with GH peak levels lower than 10 ng/ml (median GH: 8.7 ng/ml; range 7.3-9.6) and "responders" (n=18; 72%), who showed a GH response greater than 10 ng/ml (median 17 ng/ml; range 10.1-47.0). The median proportion of non-22 kDa GH on the peak of GH secretion after the first GHRH administration was similar in both groups ("responders" median: 8.6%, range 7-10.9%; "non-responders" median: 8.7%, range 6.7-10.3%), independently of the type of response after the second GHRH. In contrast, the median proportion of non-22 kDa GH was greater at time of GH peak after the second GHRH bolus in the "non-responders" (median 11.4%; range 9.1-14.3%) in comparison with the "responders" (median 9.1%; range 6.7-11.9%; p=0.003). A significant negative correlation between the total GH secreted and the percentage of non-22 kDa isoforms was seen in the "non-responders" (p=0.003). These differences in GH response to repeated GHRH stimulation and in the pattern of GH isoforms at GH

  6. Systematic Analysis of Translocator Protein 18 kDa (TSPO) Ligands on Toll-like Receptors-mediated Pro-inflammatory Responses in Microglia and Astrocytes

    PubMed Central

    Lee, Ji-Won; Nam, Hyeri

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a mitochondrial protein highly expressed on reactive microglia and astrocytes, and is considered as a biomarker for neurodegeneration and brain damage, especially neuroinflammation. Toll-like receptors (TLRs) are closely related with inflammatory responses of microglia and astrocytes and these signaling pathways regulate neuroinflammation. Previous reports have identified the anti-inflammatory effects of TSPO ligands, however study of their effects in relation to the TLR signaling was limited. Here, we investigated the effects of five representative TSPO ligands on microglia and astrocytes following activation by various TLR ligands. Our results show that TSPO ligands reduce the pro-inflammatory response elicited by the TLR ligands with more profound effects on microglia than astrocytes. PMID:27790060

  7. Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize.

    PubMed

    Liu, Hongjun; Shi, Junpeng; Sun, Chuanlong; Gong, Hao; Fan, Xingming; Qiu, Fazhan; Huang, Xuehui; Feng, Qi; Zheng, Xixi; Yuan, Ningning; Li, Changsheng; Zhang, Zhiyong; Deng, Yiting; Wang, Jiechen; Pan, Guangtang; Han, Bin; Lai, Jinsheng; Wu, Yongrui

    2016-05-01

    The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

  8. The HtrA-Like Serine Protease PepD Interacts with and Modulates the Mycobacterium tuberculosis 35-kDa Antigen Outer Envelope Protein

    PubMed Central

    White, Mark J.; Savaryn, John P.; Bretl, Daniel J.; He, Hongjun; Penoske, Renee M.; Terhune, Scott S.; Zahrt, Thomas C.

    2011-01-01

    Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress. PMID:21445360

  9. The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis.

    PubMed

    Ren, Chunhua; Chen, Ting; Sun, Hongyan; Jiang, Xiao; Hu, Chaoqun; Qian, Jing; Wang, Yanhong

    2015-11-01

    Poly-U-binding factor 60 kDa (PUF60), also known as Ro RNA binding protein (RoBPI) and FBP interacting repressor (FIR), is a multifunctional protein that is involved in a variety of nuclear processes including pre-mRNA splicing, apoptosis and transcription regulation. In this study, the first echinoderm PUF60 named StmPUF60 was identified from sea cucumber (Stichopus monotuberculatus). The StmPUF60 cDNA is 4503 bp in length, containing a 5'-untranslated region (UTR) of 34 bp, a 3'-UTR of 2963 bp and an open reading frame (ORF) of 1506 bp that encoding a protein of 501 amino acids with a deduced molecular weight of 54.15 kDa and a predicted isoelectric point of 5.15. The putative StmPUF60 protein possesses all the main characteristics of known PUF60 proteins, including two RNA recognition motifs (RRM1 and RRM2), a C-terminal PUMP domain and two conserved nucleic acid-binding ribonucleoprotein sequences (RNP1 and RNP2). For the gene structure, StmPUF60 contains nine exons separated by eight introns. In addition, the highest level of StmPUF60 mRNA expression was noticed in the gonad, followed by coelomocytes, intestine, respiratory tree and body wall. In in vivo experiments, the expression of StmPUF60 mRNA in coelomocytes and intestine was significantly up-regulated by lipopolysaccharides (LPS) challenge, suggesting that the sea cucumber PUF60 might play critical roles in the innate immune defense against bacterial infections. Moreover, we further confirmed that overexpressed StmPUF60 could induce apoptosis, and this function of StmPUF60 may be one of the innate immune defense mechanisms for sea cucumber against pathogen infections.

  10. Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

    PubMed

    Chaudhuri, Pallab; Kumar, S Vinoth; Prasad, Rajeev; Srivastava, S K; Yadav, M P

    2005-09-01

    Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

  11. Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.

    PubMed

    Verma, Alok Kumar; Sharma, Akanksha; Kumar, Sandeep; Gupta, Rinkesh Kumar; Kumar, Dinesh; Gupta, Kriti; Giridhar, B H; Das, Mukul; Dwivedi, Premendra D

    2016-06-01

    Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to

  12. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    SciTech Connect

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-09-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  13. Purification and characterization of a novel ~18 kDa antioxidant protein from Ginkgo biloba seeds.

    PubMed

    Zhou, Hao; Chen, Xijuan; Wang, Chengzhang; Ye, Jianzhong; Chen, Hongxia

    2012-12-11

    Ginkgo biloba seeds are widely used as a food and traditional medicine in China. In the present study, a novel antioxidant protein named GBSP was purified from Ginkgo biloba seeds. The protein (GBSP) was purified by homogenization of Ginkgo biloba seed powder in saline solution, 70% ammonium sulphate precipitation, filtration on a DEAE-Cellulose52 anion exchange column, gel filtration on a Sephadex G-50 column, and preparative chromatography on a C(18) column using RP-HPLC. GBSP showed an apparent molecular weight of 18 kDa by SDS-PAGE and MALDI-TOF/MS analyses. The amino acid sequence obtained by MALDI-TOF/TOF MS analysis showed GBSP was a novel protein, as no matching protein in was found the database. The protein exhibited significant antioxidant activities against free radicals such as DPPH, ABTS and superoxide anion and showed higher activity than α-tocopherol in a linoleic acid emulsion assay system. Furthermore, GBSP exhibited notable reducing power and a strong chelating effect on Cu(2+) and Fe(2+). Therefore, the present study demonstrates, for the first time, that this novel protein from Ginkgo biloba seeds is an excellent antioxidant.

  14. Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1990-05-16

    Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.

  15. A 32 kDa surface antigen of Theileria parva: characterization and immunization studies.

    PubMed

    Skilton, R A; Musoke, A J; Wells, C W; Yagi, Y; Nene, V; Spooner, P R; Gachanja, J; Osaso, J; Bishop, R P; Morzaria, S P

    2000-06-01

    Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 degrees C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed. PMID:10874718

  16. Detrimental Effect of Fungal 60-kDa Heat Shock Protein on Experimental Paracoccidioides brasiliensis Infection

    PubMed Central

    Fernandes, Fabrício Freitas; de Oliveira, Leandro Licursi; Landgraf, Taise Natali; Peron, Gabriela; Costa, Marcelo Vieira; Coelho-Castelo, Arlete A. M.; Bonato, Vânia L. D.; Roque-Barreira, Maria-Cristina; Panunto-Castelo, Ademilson

    2016-01-01

    The genus Paracoccidioides comprises species of dimorphic fungi that cause paracoccidioidomycosis (PCM), a systemic disease prevalent in Latin America. Here, we investigated whether administration of native 60-kDa heat shock protein of P. brasiliensis (nPbHsp60) or its recombinant counterpart (rPbHsp60) affected the course of experimental PCM. Mice were subcutaneously injected with nPbHsp60 or rPbHsp60 emulsified in complete’s Freund Adjuvant (CFA) at three weeks after intravenous injection of P. brasiliensis yeasts. Infected control mice were injected with CFA or isotonic saline solution alone. Thirty days after the nPbHsp60 or rPbHsp60 administration, mice showed remarkably increased fungal load, tissue inflammation, and granulomas in the lungs, liver, and spleen compared with control mice. Further, rPbHsp60 treatment (i) decreased the known protective effect of CFA against PCM and (ii) increased the concentrations of IL-17, TNF-α, IL-12, IFN-γ, IL-4, IL-10, and TGF-β in the lungs. Together, our results indicated that PbHsp60 induced a harmful immune response, exacerbated inflammation, and promoted fungal dissemination. Therefore, we propose that PbHsp60 contributes to the fungal pathogenesis. PMID:27598463

  17. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

    PubMed

    Hernández-Gutiérrez, Rodolfo; Avila-González, Leticia; Ortega-López, Jaime; Cruz-Talonia, Fernando; Gómez-Gutierrez, Guillermo; Arroyo, Rossana

    2004-01-01

    Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection.

  18. Detrimental Effect of Fungal 60-kDa Heat Shock Protein on Experimental Paracoccidioides brasiliensis Infection.

    PubMed

    Fernandes, Fabrício Freitas; Oliveira, Leandro Licursi de; Landgraf, Taise Natali; Peron, Gabriela; Costa, Marcelo Vieira; Coelho-Castelo, Arlete A M; Bonato, Vânia L D; Roque-Barreira, Maria-Cristina; Panunto-Castelo, Ademilson

    2016-01-01

    The genus Paracoccidioides comprises species of dimorphic fungi that cause paracoccidioidomycosis (PCM), a systemic disease prevalent in Latin America. Here, we investigated whether administration of native 60-kDa heat shock protein of P. brasiliensis (nPbHsp60) or its recombinant counterpart (rPbHsp60) affected the course of experimental PCM. Mice were subcutaneously injected with nPbHsp60 or rPbHsp60 emulsified in complete's Freund Adjuvant (CFA) at three weeks after intravenous injection of P. brasiliensis yeasts. Infected control mice were injected with CFA or isotonic saline solution alone. Thirty days after the nPbHsp60 or rPbHsp60 administration, mice showed remarkably increased fungal load, tissue inflammation, and granulomas in the lungs, liver, and spleen compared with control mice. Further, rPbHsp60 treatment (i) decreased the known protective effect of CFA against PCM and (ii) increased the concentrations of IL-17, TNF-α, IL-12, IFN-γ, IL-4, IL-10, and TGF-β in the lungs. Together, our results indicated that PbHsp60 induced a harmful immune response, exacerbated inflammation, and promoted fungal dissemination. Therefore, we propose that PbHsp60 contributes to the fungal pathogenesis. PMID:27598463

  19. The calmodulin-binding domain of the mouse 90-kDa heat shock protein.

    PubMed

    Minami, Y; Kawasaki, H; Suzuki, K; Yahara, I

    1993-05-01

    The mouse 90-kDa heat shock protein (HSP90) and Ca(2+)-calmodulin were cross-linked at an equimolar ratio using a carbodiimide zero-length cross-linker. To identify the calmodulin-binding domain(s) of HSP90, CNBr-cleaved peptide fragments of HSP90 were mixed with Ca(2+)-calmodulin and cross-linked. Amino acid sequence determination revealed that an HSP90 alpha-derived peptide starting at the 486th amino acid residue was contained in the cross-linked products, which contains a calmodulin-binding motif (from Lys500 to Ile520). A similar motif is present also in HSP90 beta (from Lys491 to Val511). The synthetic peptides corresponding to these putative calmodulin-binding sequences were found to be cross-linked with Ca(2+)-calmodulin and to prevent the cross-linking of HSP90 and Ca(2+)-calmodulin. Both HSP90 alpha and HSP90 beta bind Ca2+. The HSP90 peptides bind HSP90 and thereby inhibit the binding of Ca2+. In addition, the HSP90 peptides augment the self-oligomerization of HSP90 induced at elevated temperatures. These results suggest that the calmodulin-binding domain of HSP90 might interact with another part of the same molecule and that Ca(2+)-calmodulin might modulate the structure and function of HSP90 through abolishing the intramolecular interaction. PMID:8486648

  20. Chimeric Avidin--NMR structure and dynamics of a 56 kDa homotetrameric thermostable protein.

    PubMed

    Tossavainen, Helena; Kukkurainen, Sampo; Määttä, Juha A E; Kähkönen, Niklas; Pihlajamaa, Tero; Hytönen, Vesa P; Kulomaa, Markku S; Permi, Perttu

    2014-01-01

    Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin-bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin-free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability. Conventional triple resonance experiments for fully protonated proteins combined with methyl-detection optimized experiments acquired at 58°C were adequate for the structure determination of this 56 kDa protein. The model-free parameters derived from the 15N relaxation data reveal a remarkably rigid protein at 58°C in both the biotin-bound and the free forms. The H/D exchange experiments indicate a notable increase in hydrogen protection upon biotin binding.

  1. Purification and properties of a 90-kDa nuclear actin-binding protein.

    PubMed

    Yeoman, L C; Bremer, J W

    1986-04-01

    A 90 kDa actin-binding nuclear protein (ABNP) with a pI of 5.2 has been purified from the 0.7 M NaCl extracted residue fraction of chromatin prepared from Novikoff hepatoma cell nuclei. This residue fraction was previously shown to contain nuclear actin. Although twice the size, similar in pI, and similar in amino acid composition to actin, the tryptic peptide map for ABNP is distinct and contains the appropriate number of tyrosine-containing tryptic peptides for a protein of 90,000 molecular weight. A comparison of the amino acid composition of ABNP with those reported in the literature for gelsolin and villin, using a calculation of S delta Q as an indication of relatedness, results in values of 30 and 27, respectively. Actin-binding activity, however, was demonstrated for both crude and gel purified ABNP using a gel-overlay technique that employs 125I-G-actin to detect specific actin-binding proteins.

  2. Emerging roles for the Ro 60 kDa autoantigen in noncoding RNA metabolism

    PubMed Central

    Sim, Soyeong; Wolin, Sandra L.

    2011-01-01

    All cells contain an enormous variety of ribonucleoprotein complexes that function in diverse processes. Although the mechanisms by which many of these RNPs contribute to cell metabolism are well understood, the roles of others are only now beginning to be revealed. A member of this latter category, the Ro 60 kDa protein and its associated noncoding Y RNAs, was discovered because the protein component is a frequent target of the autoimmune response in patients with the rheumatic diseases systemic lupus erythematosus and Sjögren’s syndrome. Recent studies have shown that Ro is ring-shaped, binds the single-stranded ends of misfolded noncoding RNAs in its central cavity, and may function in noncoding RNA quality control. Although Ro is not present in yeast, many bacterial genomes contain potential Ro orthologs. In the radiation-resistant eubacterium Deinococcus radiodurans, the Ro ortholog functions with exoribonucleases during stress-induced changes in RNA metabolism. Moreover, in both D. radiodurans and animal cells, Ro is involved in the response to multiple types of environmental stress. Finally, Y RNAs can influence the subcellular location of Ro, inhibit access of the central cavity to other RNAs and may also act as binding sites for proteins that influence Ro function. PMID:21823229

  3. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

    PubMed Central

    Son, Mina; Lee, June Yong

    2016-01-01

    Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

  4. Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein.

    PubMed Central

    Walter, G; Ferre, F; Espiritu, O; Carbone-Wiley, A

    1989-01-01

    Polyoma virus medium tumor antigen forms specific complexes with several cellular proteins; among these is a protein of approximately 61 kDa. With antibodies directed against medium tumor antigen, the 61-kDa protein was purified from human 293 cells that were infected with a hybrid adenovirus and overexpressed medium tumor antigen. The purified 61-kDa protein was partially digested with protease V8, and one of the protease V8 fragments was isolated and partially sequenced. The amino acid sequence information was used to design mixed oligonucleotide probes for screening a cDNA library from human placenta. A clone was isolated that hybridized with two separate probes; the clone contained an insert with an open reading frame for 589 amino acids. By in vitro translation of the transcript from this insert, a protein was generated that had the same size and yielded the same pattern of protease V8 fragments as the original 61-kDa protein. Its amino acid sequence reveals 15 repeats, the majority of which are 39 amino acids long. This protein bears no resemblance to proteins in the data bank that was searched. Images PMID:2554323

  5. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  6. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

    PubMed

    Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

    2016-01-01

    Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

  7. Isolation of a 19-kDa mycobacterium, bovis-specific antigen, different from MPB70/80, by chromatofocusing.

    PubMed

    Varela, Elvira; Massó, Felipe; Páez, Araceli; Zenteno, Roberto; Zenteno, Edgar; Montaño, Luis F

    2002-11-01

    Two antigens, 19-kDa each, were purified from Mycobacterium bovis culture filtrate protein extract by chromatofocusing. Antigen I had a 4.5 pI, and its amino terminal (DPVDAVINTTCNYGQVVAALNATDP) showed a 100% homology with the hypothetical protein Rv1174c. Antigen II had a pI of 6.0 pI and its amino terminal (GDLVGPGCAEYAAANPTGPASVQGM) showed a 100% homology with M. bovis MPB70/80. Antigen I is a hetero-dimer formed by a glycosylated, 10.5-kDa, monomer and a non-glycosylated 8-kDa monomer with identical amino terminal sequences. Both antigens were recognized by the sera of PPD+ animals, but antigen I did not crossreact with sera of human PPD+ individuals. Antigen I was a weak inducer of lymphocyte proliferation and IFN-gamma production. Our results show that M. bovis expresses a 19 kDa glycoprotein, homologue to the product of M. tuberculosis gen Rv1174c, which may prove useful for bovine TB diagnostic assays.

  8. Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci.

    PubMed

    Peralta, Regina H; Espíndola, Noeli M; Pardini, Alessandra X; Iha, Alberto H; Moura, Hercules; Barr, John R; Vaz, Adelaide J; Peralta, José M

    2010-03-01

    Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.

  9. Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin.

    PubMed

    Kabir, S; Aebersold, R; Daar, A S

    1993-02-13

    Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphylococcus aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.

  10. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

    PubMed

    Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

    2016-01-01

    Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

  11. Topography of the 27- and 31-kDa electron transport proteins in the onion root plasma membrane.

    PubMed

    Córdoba, M C; Serrano, A; Córdoba, F; González-Reyes, J A; Navas, P; Villalba, J M

    1995-11-22

    Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane. PMID:7488179

  12. Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component.

    PubMed

    Goldkorn, I; Gleeson, P A; Toh, B H

    1989-11-01

    Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.

  13. Purification and characterization of a novel cold-regulated protein from an ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1.

    PubMed

    Obata, H; Ishigaki, H; Kawahara, H; Yamade, K

    1998-11-01

    The psychrotrophic ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs). We found the function of a 26-kDa protein of the CSPs in the strain KUIN-1. In strain KUIN-1, a cold shock from 18 to 4 degrees C induced the synthesis of the 26-kDa protein. By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4 degrees C. The 26-kDa protein was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies (QA52, phenyl Superose, Superose 12, and Mono Q). The purified 26-kDa protein is composed of 6 subunits of 26.5-kDa with a molecular mass of approximately 159-kDa according to gel filtration and SDS-PAGE. The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-Gln- Gln-Val-Gln-Gln-His-Trp-Gly-His-His-. Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing. The characteristics of 26-kDa protein are analogous to the cold-regulated protein of the plants. PMID:9972230

  14. Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

    PubMed Central

    Róna, Gergely; Borsos, Máté; Ellis, Jonathan J; Mehdi, Ahmed M; Christie, Mary; Környei, Zsuzsanna; Neubrandt, Máté; Tóth, Judit; Bozóky, Zoltán; Buday, László; Madarász, Emília; Bodén, Mikael; Kobe, Bostjan; Vértessy, Beáta G

    2014-01-01

    Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle. PMID:25483092

  15. Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize

    PubMed Central

    Liu, Hongjun; Shi, Junpeng; Sun, Chuanlong; Gong, Hao; Fan, Xingming; Qiu, Fazhan; Huang, Xuehui; Feng, Qi; Zheng, Xixi; Yuan, Ningning; Li, Changsheng; Zhang, Zhiyong; Deng, Yiting; Wang, Jiechen; Pan, Guangtang; Han, Bin; Lai, Jinsheng; Wu, Yongrui

    2016-01-01

    The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding. PMID:27092004

  16. A novel 70-kDa Triton X-114-soluble antigen of Plasmodium falciparum that contains interspecies-conserved epitopes.

    PubMed

    Ma, H W; Ray, P; Dhanda, V; Das, P K; Paliwal, S; Sahoo, N; Patra, K P; Das, L K; Singh, B; Kironde, F A

    1996-08-01

    In order to identify novel conserved integral membrane and other membrane-associated proteins of Plasmodium falciparum, lambda gt11-P. falciparum DNA library phages were immunoscreened with convalescent-phase mouse sera and rabbit antiserum against Triton X-114-soluble proteins of P. falciparum. One recombinant phage clone, L857, reacted with both of the antibody probes. Insert DNA (857 bp long) in L857 was 69% dA+dT rich and hybridized to a fragment of 1800 bp from mung bean nuclease-digested P. falciparum genomic DNA. The cloned parasite DNA did not show notable sequence homology with any known protein gene. The L857-encoded polypeptide, p34 (M(r) 34 kDa) was expressed in bacteria, fused to glutathione S-transferase (GST). The fusion peptide, GST-p34 (M(r) 62 kDa), was recognized by immune serum against Triton X-114-soluble antigens of P. falciparum and was reactive with anti-P. falciparum, anti-Plasmodium yoelii, and anti-GST sera. Rabbit antiserum raised against the fusion peptide recognized a 70-kDa protein from lysates of P. falciparum cells and a putative homologous 100-kDa protein from lysates of P. yoelii. The rabbit serum anti-fusion peptide antibodies bound to acetone-fixed P. falciparum-infected erythrocytes and, in immunofluorescent antibody tests, produced a punctate pattern of fluorescence suggesting that the 70-kDa native protein is associated with an apical organelle of the parasite.

  17. Chondroitin sulfate and hyaluronic acid (500-730 kda) inhibit stromelysin-1 synthesis in human osteoarthritic chondrocytes.

    PubMed

    Monfort, J; Nacher, M; Montell, E; Vila, J; Verges, J; Benito, P

    2005-01-01

    Chondroitin sulfate (CS) and 500-730 kDa hyaluronic acid (HA) are symptomatic slow-acting drugs for the treatment of osteoarthritis (OA). In addition, a growing body of evidence suggests a role for CS and this specific HA as modifiers of the course of OA. The therapeutic efficacy of CS and HA lies in their different mechanisms of action. Stromelysin-1 (metalloprotease-3 [MMP-3]) is a cartilage proteolytic enzyme, which induces cartilage destruction and acts as a mediator of the inflammatory response. However, there are few studies evaluating the in vitro effect of CS and HA on MMP-3 synthesis in human chondrocyte cultures from OA patients. Thus, the aim of the present study was to analyze the effect of CS and HA (500-730 kDa) on MMP-3 synthesis induced by interleukin-1beta (IL-1beta) in chondrocytes from patients with hip OA. Chondrocyte cultures were incubated for 48 h with IL-1beta (2.5 ng/ml) in the absence or presence of different HA 500-730 kDa (Hyalgan, Bioibérica Farma, Barcelona, Spain) concentrations, or alternatively, CS (Condro.san, Bioibérica Farma) at concentrations of 10, 50, 100, 150, 200 and 1,000 microg/ml. The results revealed that both CS and HA (500-730 kDa) inhibited MMP-3 synthesis induced by IL-1beta in human OA chondrocytes. Specifically, CS and HA (500-730 kDa) reduced MMP-3 expression levels at all tested concentrations. Therefore, our study provides new data on the mechanism of action of these drugs, which could help to explain their clinical efficacy in OA patients.

  18. An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

    PubMed Central

    Zobiak, Melanie; Büttner, Henning; Franke, Gefion; Christner, Martin; Saß, Katharina; Zobiak, Bernd; Henke, Hanae A.; Horswill, Alexander R.; Bischoff, Markus; Bur, Stephanie; Hartmann, Torsten; Schaeffer, Carolyn R.; Fey, Paul D.; Rohde, Holger

    2015-01-01

    Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces. PMID:25799153

  19. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

    PubMed

    Hernández-Gutiérrez, Rodolfo; Avila-González, Leticia; Ortega-López, Jaime; Cruz-Talonia, Fernando; Gómez-Gutierrez, Guillermo; Arroyo, Rossana

    2004-01-01

    Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection. PMID:15363938

  20. An 18 kDa scaffold protein is critical for Staphylococcus epidermidis biofilm formation.

    PubMed

    Decker, Rahel; Burdelski, Christoph; Zobiak, Melanie; Büttner, Henning; Franke, Gefion; Christner, Martin; Saß, Katharina; Zobiak, Bernd; Henke, Hanae A; Horswill, Alexander R; Bischoff, Markus; Bur, Stephanie; Hartmann, Torsten; Schaeffer, Carolyn R; Fey, Paul D; Rohde, Holger

    2015-03-01

    Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm-substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.

  1. Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

    PubMed

    Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

    2015-05-01

    Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. PMID:25792295

  2. Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes

    PubMed Central

    Wang, Lei; Gu, Yuan; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis. Methods and Findings The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response. Conclusion The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis. PMID:26288365

  3. Stabilization of mutant 46-kDa mannose 6-phosphate receptors by proteasomal inhibitor lactacystin.

    PubMed

    Breuer, P; Braulke, T

    1998-12-11

    Palmitoylation of cysteine residue 34 within the 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR 46), which may be anchored to the lipid bilayer, prevents the receptor from entering lysosomes (Schweizer, A., Kornfeld, S., and Rohrer, J. (1996) J. Cell Biol. 132, 577-584). In the present study, we examined the importance of the spacing between the transmembrane domain and the palmitoylation anchor site in the cytoplasmic domain for stability and trafficking of MPR 46. MPR 46 mutants with deletions of residues 20-23 and 24-29 expressed in baby hamster kidney cells were rapidly degraded with half-lives of less than 10 h. The replacement of residues 24-29 by alanine resulted in prolongation of receptor stability (t(1)/(2) approximately 20 h). Whereas mutant MPR 46 could not be detected in lysosomal fractions and inhibitors of lysosomal proteases failed to prevent degradation, treatment with the proteasome inhibitor lactacystin resulted in increased stability of mutant MPR 46. Pulse-chase experiments at low temperature and the acquirement of endoglucosaminidase H-resistant oligosaccharides indicate that the majority of mutant MPR 46 is degraded after leaving the Golgi compartment. Altered trafficking of mutant MPR 46 may be the result of decreased palmitoylation reaching 40% of wild type receptors. The data suggest that the spacing between the transmembrane domain and the proposed palmitoylation anchor site in the cytoplasmic domain of MPR 46 is important for a post Golgi sorting step preventing receptor degradation by multiple proteolytic systems including the proteasome. PMID:9837896

  4. Extraribosomal Functions Associated with the C Terminus of the 37/67 kDa Laminin Receptor are Required for Maintaining Cell Viability

    SciTech Connect

    J Scheiman; K Jamieson; J Ziello; J Tseng; D Meruelo

    2011-12-31

    The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G{sub 1} phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

  5. Sumoylation of SAE2 C terminus regulates SAE nuclear localization.

    PubMed

    Truong, Khue; Lee, Terry D; Li, Baozong; Chen, Yuan

    2012-12-14

    SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

  6. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    SciTech Connect

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  7. A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity.

    PubMed

    Bernstein, J A; Richardson, C C

    1988-01-01

    Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of 56- and 63-kDa species that provides helicase and primase activities required for T7 DNA replication. The 56-kDa species has been purified independently of the colinear 63-kDa species. Like a mixture of the two proteins, the 56-kDa protein binds single-stranded DNA in the presence of dTTP, catalyzes DNA-dependent hydrolysis of dTTP, and has helicase activity. In contrast to the mixture, the 56-kDa protein cannot catalyze template-dependent RNA primer synthesis. In the absence of a DNA template, both the 56-kDa protein and the mixture of the two species synthesize low levels of diribonucleotide. A putative "zinc finger" present near the amino terminus of the 63-kDa protein but absent from the 56-kDa protein may play a major role in the recognition of primase sites in the template.

  8. A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity.

    PubMed Central

    Bernstein, J A; Richardson, C C

    1988-01-01

    Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of 56- and 63-kDa species that provides helicase and primase activities required for T7 DNA replication. The 56-kDa species has been purified independently of the colinear 63-kDa species. Like a mixture of the two proteins, the 56-kDa protein binds single-stranded DNA in the presence of dTTP, catalyzes DNA-dependent hydrolysis of dTTP, and has helicase activity. In contrast to the mixture, the 56-kDa protein cannot catalyze template-dependent RNA primer synthesis. In the absence of a DNA template, both the 56-kDa protein and the mixture of the two species synthesize low levels of diribonucleotide. A putative "zinc finger" present near the amino terminus of the 63-kDa protein but absent from the 56-kDa protein may play a major role in the recognition of primase sites in the template. Images PMID:2829184

  9. Tyrosine phosphorylation of a 58 kDa protein induced by morphine in SK-N-SH cells.

    PubMed

    Nakano, K; Osugi, T; Kuo, C H; Higuchi, H; Miki, N

    1994-04-29

    A 58 kDa protein which was phosphorylated on tyrosine residues with morphine was found in human neuroblastoma cells (SK-N-SH cells) by immunoblot with monoclonal anti-phosphotyrosine antibody. The tyrosine phosphorylation was induced by morphine in 5 min in a dose-dependent manner and the increment was completely inhibited by naloxone. A Delta (d) agonist, [D-Pen2,Pen5]-enkephalin (DPDPE), but not a m agonist, [D-Ala2,N-Me-Phe,Gly5-ol]-enkephalin (DAGO), stimulated the phosphorylation and treatment of the cells with pertussis toxin inhibited the phosphorylation by morphine. These data suggest that d receptor-stimulation increases tyrosine phosphorylation of the 58 kDa protein through Gi protein in SK-N-SH cells.

  10. Immunological evaluation of a 26-kDa antigen from Taenia solium larvae for specific immunodiagnosis of human neurocysticercosis.

    PubMed

    Ev, L V; Maia, A A; Pianetti, G; Nascimento, E

    1999-02-01

    Human neurocysticercosis, due to infection of the central nervous system by cysticerci of Taenia solium, is a severe form of neurologic disease occurring in Central and South America. Specific proteins from scolex antigen from cysticerci were purified by polyacrylamide gel electrophoresis and electroelution and recognized in Western blots by antibodies present in sera from patients with neurocysticercosis. The proteins appeared as 13-, 17-, and 26-kDa bands on Coomassie blue-stained gels and proved to be specific to cysticerci of T. solium. No cross-reactivity with sera from patients with taeniasis or hydatidosis was observed. Enzyme-linked immunosorbent assay using the purified proteins of 13, 17, and 26 kDa demonstrated rates of 53%, 88%, and 100% specificity, respectively, at the cutoff serum dilution of 1:32 for the specific immunodiagnosis of human neurocysticercosis.

  11. A 21-kDa chloroplast heat shock protein assembles into high molecular weight complexes in vivo and in Organelle.

    PubMed

    Chen, Q; Osteryoung, K; Vierling, E

    1994-05-01

    The conservation of the carboxyl-terminal "heat shock" domain among small (sm) cytoplasmic and chloroplast heat shock proteins (HSPs) suggests that these smHSPs perform similar functions. Previous studies have established that cytoplasmic smHSPs are found in higher order structures in vivo (approximately 500 kDa). To determine if the chloroplast smHSP is found in similar complexes, we examined the size of the 21-kDa chloroplast smHSP from Pisum sativum, PsHSP21, under non-denaturing conditions. Following sedimentation of chloroplast stromal extracts on sucrose gradients PsHSP21 is detected in fractions corresponding to 10-11 S. Upon non-denaturing gel electrophoresis, PsHSP21 was detected in two high molecular mass complexes of approximately 230 and 200 kDa, in good agreement with the sucrose gradient data. These PsHSP21-containing particles were stable under different salt and Mg2+ conditions, and their integrity was not affected by 1.0% Triton X-100 or 10 mM ATP. To study assembly of the high molecular weight complexes containing PsHSP21, in vitro translated PsHSP21 was imported into chloroplasts and its size was examined. Following import into chloroplasts isolated from heat-stressed plants, greater than 50% of PsHSP21 was recovered in the higher molecular weight forms. In contrast, following import into chloroplasts isolated from control plants the protein was recovered exclusively in a 5 S (approximately 42-kDa) form. These data suggest that preexisting PsHSP21 or other heat-induced factors may be required for assembly of the higher molecular weight particles. We propose that the 10-11 S particles are the functional form of PsHSP21.

  12. Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide

    SciTech Connect

    Price, D.J.; Gunsalus, J.R.; Avruch, J. )

    1990-10-01

    The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated {sup 32}P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa {sup 32}P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified {sup 32}P-labeled H4 hepatoma insulin-stimulated S6 kinase. Immune complexes prepared from the cytosol of {sup 32}P-labeled H4 cells contain several {sup 32}P-labeled polypeptides. Insulin treatment increases the {sup 32}P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from {sup 32}P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.

  13. A 43-kDa TDP-43 Species Is Present in Aggregates Associated with Frontotemporal Lobar Degeneration

    PubMed Central

    Bosque, Patrick J.; Boyer, Philip J.; Mishra, Priya

    2013-01-01

    The transactive response DNA-binding protein (TDP-43) is a major component of the abnormal intracellular inclusions that occur in two common neurodegenerative diseases of humans: (1) a subtype of frontotemporal lobar degeneration and (2) amyotrophic lateral sclerosis. Genetics, experiments in cultured cells and animals, and analogy with other neurodegenerative diseases indicate that the process of TDP-43 aggregation is fundamental to the pathogenesis of these 2 diseases, but the process by which this aggregation occurs is not understood. Biochemical fractionation has revealed truncated, phosphorylated and ubiquitinated forms of TDP-43 in a detergent-insoluble fraction from diseased CNS tissue, while these forms are absent from controls. However, a large amount of the normally predominant 43-kDa form of TDP-43 is present in the detergent-insoluble fraction even from control brains, so it has not been possible to determine if this form of TDP-43 is part of pathological aggregates in frontotemporal lobe degeneration. We used semi-denaturing detergent-agarose gel electrophoresis to isolate high molecular weight aggregates containing TDP-43 that are present in the cerebral cortex of individuals with frontotemporal lobar degeneration but not that of controls. These aggregates include the same covalently modified forms of TDP-43 seen in detergent-insoluble extracts. In addition, aggregates include a 43-kDa TDP-43 species. This aggregated 43-kDa form of TDP-43 is absent or present only at low levels in controls. The presence of 43-kDa TDP-43 in aggregates raises the possibility that covalent modification is not a primary step in the pathogenic aggregation of TDP-43 associated with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. PMID:23704877

  14. A 43-kDa TDP-43 species is present in aggregates associated with frontotemporal lobar degeneration.

    PubMed

    Bosque, Patrick J; Boyer, Philip J; Mishra, Priya

    2013-01-01

    The transactive response DNA-binding protein (TDP-43) is a major component of the abnormal intracellular inclusions that occur in two common neurodegenerative diseases of humans: (1) a subtype of frontotemporal lobar degeneration and (2) amyotrophic lateral sclerosis. Genetics, experiments in cultured cells and animals, and analogy with other neurodegenerative diseases indicate that the process of TDP-43 aggregation is fundamental to the pathogenesis of these 2 diseases, but the process by which this aggregation occurs is not understood. Biochemical fractionation has revealed truncated, phosphorylated and ubiquitinated forms of TDP-43 in a detergent-insoluble fraction from diseased CNS tissue, while these forms are absent from controls. However, a large amount of the normally predominant 43-kDa form of TDP-43 is present in the detergent-insoluble fraction even from control brains, so it has not been possible to determine if this form of TDP-43 is part of pathological aggregates in frontotemporal lobe degeneration. We used semi-denaturing detergent-agarose gel electrophoresis to isolate high molecular weight aggregates containing TDP-43 that are present in the cerebral cortex of individuals with frontotemporal lobar degeneration but not that of controls. These aggregates include the same covalently modified forms of TDP-43 seen in detergent-insoluble extracts. In addition, aggregates include a 43-kDa TDP-43 species. This aggregated 43-kDa form of TDP-43 is absent or present only at low levels in controls. The presence of 43-kDa TDP-43 in aggregates raises the possibility that covalent modification is not a primary step in the pathogenic aggregation of TDP-43 associated with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. PMID:23704877

  15. The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

    PubMed

    Lesot, H; Fausser, J L; Akiyama, S K; Staub, A; Black, D; Kubler, M D; Ruch, J V

    1992-03-01

    Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation. PMID:1597256

  16. Detection of tissue origin of a 43 kDa diabetogenic protein from alloxan-induced diabetic rats

    PubMed Central

    Chauhan, Shivkumar D.; Nath, Nirmalendu M.; Tule, Vinay K.

    2009-01-01

    BACKGROUND: Earlier, we had found high levels of circulating immune complexes (CICs) in the serum of type 2 diabetes mellitus patients along with a novel 43 kDa protein. METHODS: Different tissues of alloxan-induced, diabetic, male albino rats (200–250 g in body weight) were collected for the present study. Tissue proteins were isolated and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A primary cell culture of polymorphonuclear neutrophils (PMNs) was used to evaluate the effects of the diabetogenic protein. Cell proliferative index, oxidant/antioxidant status, and ion-transporting ability were chosen as study parameters. RESULTS: SDS-PAGE of different tissues shows that the diabetic liver alone was the only tissue that contained the 43 kDa protein band compared to the normal liver. In vitro effects of the new liver protein on PMNs include significantly decreased cell proliferative activity, increased free radical levels, and decreased levels of antioxidant enzymes as well as ionic transporters. The new liver protein also exhibited protease activity when compared with standard trypsin. CONCLUSIONS: This study concluded that a novel 43 kDa protein obtained from the livers of alloxan-induced diabetic rats shows protease activity as well as antiproliferative activity. Also, this protein may act as a diabetogenic factor as it elicited a significantly gross elevation in the oxidant status level as well as in the levels of lysosomal enzymes and a decrease in the levels of antioxidative enzymes and ionic transporters of PMNs. PMID:20062560

  17. Calmyonemin: a 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate).

    PubMed

    David, C; Viguès, B

    1994-01-01

    Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calcium-binding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein cross-reacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myoneme-mediated retraction of the ciliature.

  18. The Transition of the 37-Kda Laminin Receptor (Rpsa) to Higher Molecular Weight Species: Sumoylation or Artifact?

    PubMed

    Digiacomo, Vincent; Gando, Ivan A; Venticinque, Lisa; Hurtado, Alicia; Meruelo, Daniel

    2015-12-01

    The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

  19. Myotubularin-related proteins 3 and 4 interact with polo-like kinase 1 and centrosomal protein of 55 kDa to ensure proper abscission.

    PubMed

    St-Denis, Nicole; Gupta, Gagan D; Lin, Zhen Yuan; Gonzalez-Badillo, Beatriz; Pelletier, Laurence; Gingras, Anne-Claude

    2015-04-01

    The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to

  20. Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals.

    PubMed

    Teixeira, A M S; Plavnik, F L; Fernandes, F B; Marson, O; Christofalo, D M J; Ajzen, S A; Sesso, R; Franco, M C; Casarini, D E

    2008-05-01

    We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 +/- 5.0 vs 16.1 +/- 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes. PMID:18516470

  1. Cell Proliferation and Motility Are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells

    PubMed Central

    Bang, Jeyoung; Huh, Jang Hoe; Na, Ji-Woon; Lu, Qiao; Carlson, Bradley A.; Tobe, Ryuta; Tsuji, Petra A.; Gladyshev, Vadim N.; Hatfield, Dolph L.; Lee, Byeong Jae

    2015-01-01

    The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility. PMID:25728752

  2. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  3. Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network.

    PubMed Central

    Ramos-Morales, F; Vime, C; Bornens, M; Fedriani, C; Rios, R M

    2001-01-01

    GMAP-210 (Golgi-microtubule-associated protein of 210 kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83--98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105--196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed. PMID:11463340

  4. Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa from orange-spotted grouper, Epinephelus coioides.

    PubMed

    Wei, Jingguang; Ji, Huasong; Guo, Minglan; Yan, Yang; Qin, Qiwei

    2013-11-01

    Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, a thioredoxin-related protein of 14 kDa (named as Ec-TRP14) was identified from the marine fish grouper, Epinephelus coioides by RACE PCR. The full-length cDNA of Ec-TRP14 was comprised of 1066 bp with a 372 bp open reading frame that encodes a putative protein of 123 amino acids. Similar to most TRP14s, Ec-TRP14 contained the conserved motif C-P-D-C. Ec-TRP14 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-TRP14 was up-regulated in the liver of grouper challenged with SGIV. Ec-TRP14 was recombined and expressed in Escherichia coli BL21 (DE3), and the rEc-Ec-TRP14 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-TRP14 (8.3 μg/ml), which significantly enhanced cells viability under damage caused by viral infection. These results together indicated that Ec-TRP14 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.

  5. Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

    PubMed

    Poustis-Delpont, C; Thaon, S; Auberger, P; Gerardi-Laffin, C; Sudaka, P; Rossi, B

    1994-05-20

    Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex

  6. Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many gram-negative bacteria.

    PubMed

    Menozzi, F D; Menozzi-Dejaiffe, C; Nano, F E

    1989-03-01

    In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.

  7. The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex.

    PubMed

    Capell, A; Grünberg, J; Pesold, B; Diehlmann, A; Citron, M; Nixon, R; Beyreuther, K; Selkoe, D J; Haass, C

    1998-02-01

    Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and

  8. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    SciTech Connect

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. ); Flores, B.M. ); Hagen, F.S. )

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  9. Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

    PubMed

    Sheikh, Inayat Hussain; Kaushal, Deep C; Chandra, Deepak; Kaushal, Nuzhat A

    2016-10-01

    Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses. PMID:27311385

  10. Soluble recombinant merozoite surface antigen-142kDa of Plasmodium vivax: An improved diagnostic antigen for vivax malaria.

    PubMed

    Mirahmadi, Hadi; Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad

    2016-04-01

    Enzyme Linked Immunosorbent Assay (ELISA), as a serological test, can be a beneficial tool for epidemiological studies by screening blood donors and diagnosis of specific antibodies from Plasmodium vivax (P. vivax) infected cases. Since P. vivax cannot easily be acquired in vitro, ELISA assays using total or semi-purified antigens are seldom used. On the basis of this restriction, we examined whether recombinant protein 42 kDa related to C-terminal region of the merozoite surface antigen-1 of P. vivax (MSA-1(42)) could be suitable for serological detection of vivax malaria infection. Purified recombinant protein produced in Escherichia coli (E. coli) (GST-MSA-1(42)) was examined for its ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 262 serum samples collected from individuals living in the south and southeastern regions of Iran where malaria is endemic. Samples exposed to Plasmodium falciparum (P. falciparum) infection and patients with other infectious disease (toxoplasmosis, Leishmania infantum infection, echinococcosis and FUO (fever with unknown origin)) except for P. falciparum were residing in non- malaria endemic areas in Iran. Generally, the sensitivity of ELISA evaluated with sera from naturally infected individuals was 86.9%. The specificity value of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases was 94.05%. The positive predictive value (PPV), negative predictive value (NPV) provided, and the diagnostic efficiency of anti-rPvMSA-1(42) antibody using indirect ELISA were determined 93.58, 87.77 and 91.06% respectively. Our study demonstrated that, because MSA-1(42) kDa contains both the 33 and 19 kDa fragments in its structure, it can serve as the basis for the development of a sensitive serological test which can be used for epidemiological studies, screening blood donors and diagnosis of P. vivax malaria. PMID:26851675

  11. [Autoantibodies against U1-n-RNP (ENA)--detection using a recombinant 70-kDa protein].

    PubMed

    Seelig, H P; Wieland, C; Heim, C; Renz, M

    1990-02-01

    An ELISA for the demonstration of antibodies to RNP antigens (ENA) in sera of patients with systemic rheumatic diseases was developed using the recombinant 70-kDa protein, a marker antigen of U1-n-RNP. The specificity and sensitivity of the method was evaluated with 3588 patients' sera. The results were compared with those obtained by natural antigens isolated from calf thymus and Western blot analysis using HeLa-cell nuclear extracts. The test was found to be specific and sensitive and to be superior in routine laboratory screening than the other tests commonly used.

  12. High Resolution Imaging Study of Interactions between the 37 kDa/67 kDa Laminin Receptor and APP, Beta-Secretase and Gamma-Secretase in Alzheimer's Disease

    PubMed Central

    Jovanovic, Katarina; Loos, Ben; Da Costa Dias, Bianca; Penny, Clement; Weiss, Stefan F. T.

    2014-01-01

    Alzheimer's disease (AD) is the most prevalent form of dementia affecting the elderly. Neurodegeneration is caused by the amyloid beta (Aβ) peptide which is generated from the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by the β– and γ- secretases. Previous reports revealed that the 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in APP processing, however, the exact mechanism by which this occurs remains largely unclear. This study sought to assess whether LRP/LR interacted with APP, β- or γ-secretase. Detailed confocal microscopy revealed that LRP/LR showed a strong co-localisation with APP, β- and γ-secretase, respectively, at various sub-cellular locations. Superresolution Structured Illumination Microscopy (SR-SIM) showed that interactions were unlikely between LRP/LR and APP and β-secretase, respectively, while there was strong co-localisation between LRP/LR and γ-secretase at this 80 nm resolution. FRET was further employed to assess the possibility of protein-protein interactions and only an interaction between LRP/LR and γ-secretase was found. FLAG co-immunoprecipitation confirmed these findings as LRP/LR co-immunoprecipitated with γ-secretase, but failed to do so with APP. These findings indicate that LRP/LR exerts its influence on Aβ shedding via a direct interaction with the γ-secretase and possibly an indirect interaction with the β-secretase. PMID:24972054

  13. Prevalence of bands other than 160 and 130 kDa in pemphigus sera (a multicenter immunoblotting study). Gruppo Italiano Studi Epidemiologici in Dermatologia (GISED).

    PubMed

    Cozzani, E; Parodi, A; Rebora, A

    1998-05-01

    Patients with pemphigus may produce antibodies against molecules other than the classical transmembranal ones. Recently, for example antibodies to 230 kDa antigens have been found in association with antibodies to intercellular substance. To better understand their prevalence, clinical correlates and prognostic significance of bands other than 130 and 160 kDa, we studied 67 pemphigus sera. About one-fourth of patients revealed multiple heterogeneous bands and 13% the 230 kDa band. When challenged with the recombinant protein rBP55, the carbossiterminal portion of bullous pemphigoid major antigen, all 230 kDa-positive-sera proved negative. Caution is to be recommended in interpreting pemphigus sera with a band migrating at the 230 kDa level.

  14. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells.

    PubMed Central

    Häring, H U; White, M F; Machicao, F; Ermel, B; Schleicher, E; Obermaier, B

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission. Images PMID:3540953

  15. A phospholipid is the membrane-anchoring domain of a protein growth factor of molecular mass 34 kDa in placental trophoblasts.

    PubMed Central

    Roy-Choudhury, S; Mishra, V S; Low, M G; Das, M

    1988-01-01

    Recently we isolated a protein growth factor of 34 kDa from trophoblastic membranes of human placenta. A fraction (approximately equal to 50%) of the membrane-associated 34-kDa protein is peripherally associated--i.e., it can be released by high salt treatment. The remainder shows the characteristics of an integral membrane protein--i.e., its release requires detergent treatment. Here we report studies on the structural basis for membrane anchorage of the protein. Phospholipase C was found to release an immunoreactive 34-kDa polypeptide from intact isolated cytotrophoblasts. Studies with isolated trophoblastic membranes showed that phospholipase C specifically released the salt-resistant fraction of the 34-kDa polypeptide. The polypeptide released by phospholipase C showed the same electrophoretic mobility in NaDodSO4/PAGE as the polypeptide prior to phospholipase C treatment. The identity of the released protein with the 34-kDa growth factor has been established by both immunologic and receptor-binding assays. Other studies show that there is biosynthetic incorporation of [3H]myristate into the 34-kDa protein. The myristate label is labile to phospholipase C treatment. These results suggest that some of the 34-kDa protein is anchored to the plasma membrane via a posttranslationally added phospholipid. This mode of anchorage has been observed for some other membrane proteins and raises interesting questions regarding the role of this novel linkage in the mitogenic function of the 34-kDa polypeptide. Images PMID:3162324

  16. Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

    SciTech Connect

    Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

    1988-09-01

    Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing /sup 125/I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.

  17. Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts

    SciTech Connect

    Keyse, S.M.; Tyrrell, R.M.

    1987-10-25

    We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H/sub 2/O/sub 2/, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H/sub 2/O/sub 2/, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.

  18. The biological function of DMP-1 in osteocyte maturation is mediated by its 57-kDa C-terminal fragment.

    PubMed

    Lu, Yongbo; Yuan, Baozhi; Qin, Chunlin; Cao, Zhengguo; Xie, Yixia; Dallas, Sarah L; McKee, Marc D; Drezner, Marc K; Bonewald, Lynda F; Feng, Jian Q

    2011-02-01

    Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism.

  19. 78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

    SciTech Connect

    Gonzalez-Noriega, Alfonso . E-mail: gonor@biomedicas.unam.mx; Ortega Cuellar, Daniel D.; Michalak, Colette

    2006-04-15

    Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH{sub 4}Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.

  20. Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

    SciTech Connect

    Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

    1986-04-01

    The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-(/sup 14/C)-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed.

  1. 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A

    SciTech Connect

    Samelson, L.E.; Harford, J.; Schwartz, R.H.; Klausner, R.D.

    1985-04-01

    Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell receptor.

  2. The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

    PubMed Central

    Wulfhorst, Hannes; Franken, Linda E.; Wessinghage, Thomas; Boekema, Egbert J.; Nowaczyk, Marc M.

    2014-01-01

    The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant. PMID:25119998

  3. Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp. bovine group 7.

    PubMed

    Frey, J; Cheng, X; Monnerat, M P; Abdo, E M; Krawinkler, M; Bölske, G; Nicolet, J

    1998-01-01

    The gene encoding a lipoprotein of 67 kDa, named P67, was cloned from Mycoplasma sp. bovine group 7 strain PG50 and expressed in Escherichia coli K12. Analysis of the amino acid sequence derived from the DNA sequence of the P67 gene revealed a typical prokaryotic signal peptidase II membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. Protein P67 showed 91% identical amino acid residues to the lipoprotein P72 of Mycoplasma mycoides subsp. mycoides small colony type (SC) and 53% identical amino acid residues to a peptide of an unassigned gene on the genome of Mycoplasma capricolum subsp. capricolum. Antibodies made against recombinant P67 reacted with a 67-kDa protein in all Mycoplasma sp. bovine group 7 strains tested and also, to some extent, with P72 of Mycoplasma mycoides subsp. mycoides SC. The gene encoding P67 was present in all strains of Mycoplasma sp. bovine group 7 analysed, but not in other Mycoplasma sp. of the "mycoides cluster" and not in the phylogenetically related Mycoplasma putrefaciens. PCR and restriction fragment analysis revealed that the gene of P67 is conserved in all strains of Mycoplasma sp. bovine group 7. A specific PCR reaction based on the P67 gene sequence enabled rapid identification of strains belonging to Mycoplasma sp. bovine group 7.

  4. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus.

    PubMed

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-05-01

    We developed a rapid dot-enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available.

  5. Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia.

    PubMed

    Ognibene, Marzia; Della Giovampaola, Cinzia; Trielli, Francesca; Focarelli, Riccardo; Rosati, Floriana; Umberta Delmonte Corrado, Maria

    2008-05-01

    In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays. PMID:17870426

  6. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

    PubMed Central

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-01-01

    We developed a rapid dot–enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available. PMID:25802430

  7. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme.

    PubMed Central

    Wing, S S; Bedard, N

    1996-01-01

    Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E2(14K)), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am.J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1-3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E2(14K) mRNA levels in L6 myotubes. Insulin suppressed levels of E2(14K) mRNA with an IC50 of 4 x 10(-9) M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E2(14K), also increase in skeletal muscle upon fasting. Reduction of E2(14K) mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5 x 10(-10) M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E2(14K) mRNA levels with an IC50 of 3 x 10(-11) M. DES-IGF-I did not alter rates of transcription of the E2(14K) gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3' non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E2(14K) expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates. PMID

  8. Extractable low mass proteins <30kDa from peanut display elevated antigenicity (IgG-binding) and allergenicity (IgE-binding) in vitro and are attenuated by thermal reactivity with non-peanut food ingredients.

    PubMed

    Bennett, Louise; Lee, Alvin

    2016-03-01

    Human allergic reactions to peanut proteins and the associated risk of life-threatening anaphylaxis requires vigilant management of peanuts in food processing. Processed forms of peanuts with attenuated antigenicity and less severe immunogenic responses may lower the risk. Molecular subfractions of raw (UP), blanched (BP) and roasted (RP) peanuts were prepared including water-insoluble (P1), water-soluble high mass (>30kDa, P2) and water-soluble low mass (<30kDa, P3) fractions. Products were screened by measuring binding to IgG (polyclonal antibody against peanut allergen) and IgE (sera from peanut-allergic donors, RAST>3). The results showed that IgE titres were highest for total extracts of RP, particularly for P3 fractions of UP and RP, and were affected by further heating. Antigenicity was also modulated by heating in the presence of either peanut oil or non-peanut food ingredients (lactose, coconut oil). Results support several alternative methods for regulating peanut antigenicity using food processing approaches but require further substantiation in larger numbers of allergic and control donor sera.

  9. Extractable low mass proteins <30kDa from peanut display elevated antigenicity (IgG-binding) and allergenicity (IgE-binding) in vitro and are attenuated by thermal reactivity with non-peanut food ingredients.

    PubMed

    Bennett, Louise; Lee, Alvin

    2016-03-01

    Human allergic reactions to peanut proteins and the associated risk of life-threatening anaphylaxis requires vigilant management of peanuts in food processing. Processed forms of peanuts with attenuated antigenicity and less severe immunogenic responses may lower the risk. Molecular subfractions of raw (UP), blanched (BP) and roasted (RP) peanuts were prepared including water-insoluble (P1), water-soluble high mass (>30kDa, P2) and water-soluble low mass (<30kDa, P3) fractions. Products were screened by measuring binding to IgG (polyclonal antibody against peanut allergen) and IgE (sera from peanut-allergic donors, RAST>3). The results showed that IgE titres were highest for total extracts of RP, particularly for P3 fractions of UP and RP, and were affected by further heating. Antigenicity was also modulated by heating in the presence of either peanut oil or non-peanut food ingredients (lactose, coconut oil). Results support several alternative methods for regulating peanut antigenicity using food processing approaches but require further substantiation in larger numbers of allergic and control donor sera. PMID:26471622

  10. Identification of the Interactors of Human Nibrin (NBN) and of Its 26 kDa and 70 kDa Fragments Arising from the NBN 657del5 Founder Mutation

    PubMed Central

    Pennisi, Rosa; Pallotta, Valeria; D'Alessandro, Angelo; Antoccia, Antonio; Zolla, Lello; Ascenzi, Paolo; di Masi, Alessandra

    2014-01-01

    Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS. PMID:25485873

  11. Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex

    NASA Astrophysics Data System (ADS)

    Wu, Yi-Min; Wang, Chun-Hsiung; Chang, Jen-wei; Chen, Yi-yun; Miyazaki, Naoyuki; Murata, Kazuyoshi; Nagayama, Kuniaki; Chang, Wei-Hau

    2013-12-01

    Cryo-electron microscopy (cryo-EM) has become a powerful technique for obtaining near atomic structures for large protein assemblies or large virus particles, but the application to protein particles smaller than 200-300 kDa has been hampered by the feeble phase contrast obtained for such small samples and the limited number of electrons tolerated by them without incurring excessive radiation damage. By implementing a thin-film quarter-wave phase plate to a cryo-EM, Nagayama, one of the present authors, has recently restored the long-lost very low spatial frequencies, generating in-focus phase contrast superior to that of conventional defocusing phase contrast, and successfully applied the so-called Zernike phase-plate cryo-EM to target various biological samples in native state. Nevertheless, the sought-after goal of using enhanced phase contrast to reveal a native protein as small as 100 kDa waits to be realized. Here, we report a study in which 200 kV Zernike phase-plate cryo-EM with a plate cut-on periodicity of 36 nm was applied to visualize 100 kDa components of various protein complexes, including the small domains on the surface of an icosahedral particle of ˜38 nm derived from the dragon grouper nervous necrosis virus (DGNNV) and the labile sub-complex dissociated from yeast RNA polymerase III of 17 nm. In the former case, we observed a phase contrast reversal phenomenon at the centre of the icosahedral particle and traced its root cause to the near matching of the cut-on size and the particle size. In summary, our work has demonstrated that Zernike phase-plate implementation can indeed expand the size range of proteins that can be successfully investigated by cryo-EM, opening the door for countless proteins. Finally, we briefly discuss the possibility of using a transfer lens system to enlarge the cut-on periodicity without further miniaturizing the plate pinhole.

  12. Protective effect of recombinant Trichinella 53-kDa protein in sepsis and the effect on macrophages

    PubMed Central

    Yang, Chuwei; Liu, Hui; Li, Xiangdong; Sui, Shaoguang; Liu, Yufei

    2016-01-01

    The survival rate of the recombinant Trichinella 53-kDa protein infected by the cecal ligation and puncture (CLP) model of sepsis in rats and the expression difference of macrophages were analyzed. Eighteen clean SD rats were selected for the present study. The rats were divided into the sham operation (n=5), control (n=5) and experimental (n=8) groups. The rats in the sham operation group underwent cecum division and suture with routine therapy for cure. The rats in the control and experimental groups were placed in the CLP model of sepsis in rats. The experimental group was administered recombinant Trichinella 53-kDa protein in advance, and the control group was administered the same dose of placebo. The survival rate of the rats within 6 and 12 h, the macrophage expression ratio, and the differences of the expression levels Th1-type cytokines IFN-γ and tumor necrosis factor (TNF)-α, and the Th2 type cytokines interleukin-4 (IL-4) and IL-13 were analyzed. The survival rate of rats in the experimental group was higher than that of the control group with a statistically significant difference (P<0.05). The expression ratio of the macrophages received from the different handling methods of the rats in the experimental group was higher than that of the control group. The difference was statistically significant (P<0.05). The expression levels of the Th1-type cytokines IFN-γ and TNF-α of the rats in the experimental group was higher than that of the control group, while the expression level of the Th2-type cytokines IL-4 and IL-13 was higher than that of the control group. The difference was statistically significant (P<0.05). In conclusion, recombinant Trichinella 53-kDa protein can increase the survival rate following infection with CLP sepsis, which may be associated with the improvement of the macrophages and the adjustment of the expression of Th2 cytokines. PMID:27446304

  13. A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus.

    PubMed

    Geng, D; Baker, D P; Foley, S F; Zhou, C; Stieglitz, K; Roberts, M F

    1999-03-19

    Two phospholipase D (PLD) enzymes with both hydrolase and transferase activities were isolated from Streptomyces chromofuscus. There were substantial differences in the kinetic properties of the two PLD enzymes towards monomeric, micellar, and vesicle substrates. The most striking difference was that the higher molecular weight enzyme (PLD57 approximately 57 kDa) could be activated allosterically with a low mole fraction of phosphatidic acid (PA) incorporated into a PC bilayer (Geng et al., J. Biol. Chem. 273 (1998) 12195-12202). PLD42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4-6-fold higher Vmax toward PC substrates than PLD57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD42/20 were cleavage products of PLD57. The larger component included the N-terminal segment of PLD57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD57; this peptide had no PLD activity by itself. Increasing the pH of PLD42/20 to 8.9, followed by chromatography of PLD42/20 on a HiTrap Q column at pH 8.5 separated the 42- and 20-kDa proteins. The 42-kDa complex had about the same specific activity with or without the 20-kDa fragment. The lack of PA activation for the 42-kDa protein and for PLD42/20 indicates that an intact C-terminal region of PLD57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD57.

  14. Expression of a low-molecular-weight (10 kDa) calcium binding protein in glial cells of the brain of the trout (Teleostei).

    PubMed

    Manso, M J; Becerra, M; Becerra, M; Anadón, R

    1997-11-01

    Calcium-binding proteins of the EF-hand family are widely distributed in the vertebrate central nervous system. In the present study of the trout brain, immunocytochemistry with a monoclonal antibody against chick gut calbindin-28k and a polyclonal antibody against bovine S100 protein specifically stained ependymocytes and radial glia cells with identical patterns. Western blot analysis of trout brain extracts with the antibodies to S100 and calbindin stained the same low-molecular-weight (10 kDa) protein band. In rat brain extracts, however, the monoclonal antibody to calbindin recognized a major protein band with molecular weight corresponding to that of calbindin-28k. This indicates that the trout protein is a new calcium-binding-like (calbindin-like) molecule that is immunologically related to both S100 and calbindin. Immunocytochemical studies of the trout brain using the antibodies to CaB and S100 showed that ependymocytes were stained in most ventricular regions, except in a few specialized ependymal areas of the ventral telencephalon, epithalamus, hypothalamus (including the paraventricular organ and saccus vasculosus) and brain stem. Immunocytochemistry also indicated the presence of calbindin-like protein in radial glia cells of several regions of the brain (thalamus, pretectal region, optic tectum, and rhombencephalon). Differences in immunoreactivity between neighbouring ependymal areas suggest that this protein may be a useful marker of different territories. All immunoreactive glial cells were nicotin-adenin-dinucleotide-phosphate diaphorase-positive, although this enzymohistochemical reaction is not specific for these glial cells since it reveals oligodendrocytes and some neurons. Immunoreactivity appears at different developmental stages in the different brain regions, with a broadly caudorostral gradient, suggesting that the expression of this protein is developmentally regulated. Comparison of the distribution of the calbindin-like protein with

  15. A complete backbone spectral assignment of human apolipoprotein AI on a 38 kDa preβHDL (Lp1-AI) particle

    SciTech Connect

    Ren, Xuefeng; Yang, Yunhuang; Neville, T.; Hoyt, David W.; Sparks, Daniel L.; Wang, Jianjun

    2007-06-12

    Apolipoprotein A-I (apoAI, 243-residues) is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. This important property of apoAI is related to its roles in reverse cholesterol transport pathway. Upon lipid-binding, apoAI undergoes conformational changes from lipid-free to several different HDL-associated states (1). These different conformational states regulate HDL formation, maturation and transportation. Two initial conformational states of apoAI are lipid-free apoAI and apoAI/preβHDL that recruit phospholipids and cholesterol to form HDL particles. In particular, lipid-free apoAI specifically binds to phospholipids to form lipid-poor apoAI, including apoAI/preβ-HDL (~37 kDa). As a unique class of lipid poor HDL, both in vitro and in vivo evidence demonstrates that apoAI/preβ-HDLs are the most effective acceptors specifically for free cholesterol in human plasma and serves as the precursor of HDL particles (2). Here we report a complete backbone spectral assignment of human apoAI/preβHDL. Secondary structure prediction using backbone NMR parameters indicates that apoAI/preβHDL displays a two-domain structure: the N-terminal four helix-bundle domain (residues 1-186) and the C-terminal flexible domain (residues 187-243). A structure of apoAI/preβ-HDL is the first lipid-associated structure of apoAI and is critical for us to understand how apoAI recruits cholesterol to initialize HDL formation. BMRB deposit with accession number: 15093.

  16. Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2011-08-01

    Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells. PMID:21721021

  17. Characterization of a novel acetamidobenzoxazolone-based PET ligand for translocator protein (18 kDa) imaging of neuroinflammation in the brain.

    PubMed

    Tiwari, Anjani K; Yui, Joji; Fujinaga, Masayuki; Kumata, Katsushi; Shimoda, Yoko; Yamasaki, Tomoteru; Xie, Lin; Hatori, Akiko; Maeda, Jun; Nengaki, Nobuki; Zhang, Ming-Rong

    2014-05-01

    We developed the novel positron emission tomography (PET) ligand 2-[5-(4-[(11)C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide ([(11)C]MBMP) for translocator protein (18 kDa, TSPO) imaging and evaluated its efficacy in ischemic rat brains. [(11)C]MBMP was synthesized by reacting desmethyl precursor (1) with [(11)C]CH3 I in radiochemical purity of ≥ 98% and specific activity of 85 ± 30 GBq/μmol (n = 18) at the end of synthesis. Biodistribution study on mice showed high accumulation of radioactivity in the TSPO-rich organs, e.g., the lungs, heart, kidneys, and adrenal glands. The metabolite analysis in mice brain homogenate showed 80.1 ± 2.7% intact [(11)C]MBMP at 60 min after injection. To determine the specific binding of [(11)C]MBMP with TSPO in the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity between the ipsilateral and contralateral sides in PET images. Displacement experiments with unlabelled MBMP or PK11195 minimized the difference in uptake between the two sides. In summary, [(11)C]MBMP is a potential PET imaging agent for TSPO and, consequently, for the up-regulation of microglia during neuroinflammation.

  18. The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases.

    PubMed Central

    Stanton, Heather; Ung, Linh; Fosang, Amanda J

    2002-01-01

    Fragments of fibronectin occur naturally in vivo and are increased in the synovial fluid of arthritis patients. We have studied the 45 kDa fragment (Fn-f 45), representing the N-terminal collagen-binding domain of fibronectin, for its ability to modulate the expression of metalloproteinases by porcine articular chondrocytes in vitro. We report that stimulation of cultured chondrocytes, or cartilage explants, with Fn-f 45 increased the levels of matrix metalloproteinase-13 (MMP-13; collagenase-3) released into the conditioned medium in a dose-dependent manner. Increased levels of MMP-13 were due to stimulation of MMP-13 synthesis, rather than release of MMP-13 from accumulated matrix stores. Fn-f 45 also stimulated the synthesis of MMP-3 (stromelysin-1) from cultured chondrocytes and cartilage cultures. The Fn-f 45-induced increase in MMP-3 and MMP-13 synthesis occurred via an interleukin 1-independent mechanism, since the receptor antagonist of interleukin-1 was unable to block the increased synthesis. The gelatinases, MMP-2 and MMP-9, were not modulated by Fn-f 45 in these culture systems. Fn-f 45 also stimulated the release of aggrecan from cartilage explants into conditioned medium. Neoepitope antibodies specific for aggrecan fragments generated by MMPs or aggrecanases showed that the Fn-f 45-induced aggrecan loss was mediated by aggrecanases, and not by MMPs. Extracts of cultured cartilage contained elevated levels of the aggrecanase-derived ITEGE(373)-G1 domain, whereas levels of the matrix metalloproteinase-derived DIPEN(341)-G1 domain were unchanged. These studies show that Fn-f 45 can induce a catabolic phenotype in articular chondrocytes by up-regulating the expression of metalloproteinases specific for the degradation of collagen and aggrecan. PMID:11988091

  19. Role of the 52 KDa thioredoxin protein disulfide isomerase of Toxoplasma gondii during infection to human cells.

    PubMed

    Moncada, Diego; Arenas, Aylan; Acosta, Alejandro; Molina, Diego; Hernández, Alejandro; Cardona, Néstor; Gomez-Yepes, Mónica; Gomez-Marin, Jorge E

    2016-05-01

    Toxoplasma protein disulfide isomerase (PDI) is a 52 KDa thioredoxin of interest because have a great immunogenicity for humans. We cloned and produced a recombinant protein (recTgPDI) used to test its effect during infection to different human cell lines (epithelial and retinal). We also determine if there were differences in gen expression during in vitro infection. Expression of the gen was lower after entry into the host cells. PDI's inhibitors bacitracin and nitroblue tetrazolium reduced the percent of infected cells and small amounts of recTgPDI proteins interfered with the invasion step. All these results support a role of Toxoplasma PDI during the first steps of infection (adhesion and invasion). Toxoplasma PDI is a protein linked to early steps of invasion, it would be of importance to identify the host proteins substrates during invasion steps. PMID:26896642

  20. Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

    PubMed Central

    Acker, J; Wintzerith, M; Vigneron, M; Kedinger, C

    1993-01-01

    The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity. Images PMID:8265347

  1. Partial de novo sequencing and unusual CID fragmentation of a 7 kDa, disulfide-bridged toxin.

    PubMed

    Medzihradszky, Katalin F; Bohlen, Christopher J

    2012-05-01

    A 7 kDa toxin isolated from the venom of the Texas coral snake (Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

  2. ATPase active-site electrostatic interactions control the global conformation of the 100 kDa SecA translocase.

    PubMed

    Kim, Dorothy M; Zheng, Haiyan; Huang, Yuanpeng J; Montelione, Gaetano T; Hunt, John F

    2013-02-27

    SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes. PMID:23167435

  3. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10.

    PubMed

    Hoff, E F; Cook, S H; Sherman, G D; Harper, J M; Ferguson, D J; Dubremetz, J F; Carruthers, V B

    2001-02-01

    Hoff, E. F., Cook, S. H., Sherman, G. D., Harper, J. M., Ferguson, D. J. P., Dubremetz, J. F., and Carruthers, V. B. 2001. Toxoplasma gondii: Molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology, 97, 77-88. During host cell invasion, Toxoplasma gondii secretes proteins from specialized organelles (micronemes and rhoptries) located at the apical end of the parasite. The contents of the micronemes appear to be crucial to T. gondii invasion, as inhibition of microneme secretion prevents parasite entry into host cells. Here we describe a new T. gondii microneme protein, TgMIC10. Molecular characterization of a full-length TgMIC10 cDNA revealed that TgMIC10 lacks homology to any previously characterized proteins, although a homologue, NcMIC10, was identified in a closely related parasite, Neospora caninum. TgMIC10 has an unusually long secretory leader sequence of 58 amino acids; the mature TgMIC10 is 18 kDa, possesses nine diglutamic acid repeats and an imperfect repeat sequence (RK(R/Y)HEEL), and is entirely devoid of cysteines. Antibodies raised against recombinant TgMIC10 recognized the native TgMIC10 and localized the protein to the micronemes in indirect immunofluorescence and immunoEM experiments. Comparison of immunofluorescence images indicates that TgMIC10 expression is higher in T. gondii tachyzoites, which are responsible for active infection, than in bradyzoites, which are responsible for latent infection.

  4. A 170kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system.

    PubMed

    Geadkaew, Amornrat; Kosa, Nanthawat; Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

    2014-09-01

    Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization.

  5. A Novel Autoantibody Recognizing 200 and 100 kDa Proteins is Associated with an Immune-Mediated Necrotizing Myopathy

    PubMed Central

    Christopher-Stine, Lisa; Casciola-Rosen, Livia A.; Hong, Grace; Chung, Tae; Corse, Andrea M.; Mammen, Andrew L.

    2010-01-01

    Objective Myofiber necrosis without prominent inflammation is a non-specific finding seen in patients with dystrophies and toxic or immune-mediated myopathies. However, the etiology of a necrotizing myopathy is often obscure and which patients would benefit from immunosuppression remains uncertain. We sought to identify novel autoantibodies in necrotizing myopathy patients. Methods Muscle biopsy and serum were available for 225 myopathy patients. Antibody specificities were determined by performing immunoprecipitations from 35S-methionine-labeled HeLa cell lysates. Selected biopsies were stained for membrane attack complex (MAC), major histocompatability complex I (MHC I), and endothelial cell marker CD31. Results 38 of 225 patients had predominant myofiber necrosis on muscle biopsy. 12 of these had a known autoantibody association or other etiology for their myopathy. 16 of the remaining 26 sera immunoprecipitated 200 and 100 kDa proteins; this specificity was found in only 1/187 patients without necrotizing myopathy. Patients with anti-200/100 specificity had proximal weakness (100%), high creatine kinase (CK) levels (mean 10,333 IU/L), and an irritable myopathy on electromyography (EMG) (88%). 63% had exposure to statins prior to the onset of weakness. All patients responded to immunosuppressive therapy and many relapsed with medication tapering. Immunohistochemical studies showed MAC on small blood vessels in 6/8 and on the surface of non-necrotic myofibers in 4/8. 5/8 had abnormal capillary morphology and 4/8 expressed MHC I on the surface of non-necrotic myofibers. Conclusion An anti-200/100 kDa specificity defines a subgroup of necrotizing myopathy patients previously considered to be "autoantibody negative." We propose that these patients have an immune-mediated myopathy which is frequently associated with prior statin use and should be treated with immunosuppressive therapy. PMID:20496415

  6. Transforming growth factor beta 1 enhances expression of 50 kDa protein related to 2'-5' oligoadenylate synthetase in human sperm cells.

    PubMed

    Naz, R K; Kumar, R

    1991-01-01

    Human cellular polypeptide factors, namely interferon-alpha, interferon-gamma transforming growth factor (TGF)-alpha, and TGF-beta 1, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2'-5' oligoadenylate [2-5(A)]synthetase activity. TGF-alpha affected neither motility nor the levels of 2-5(A) synthetase in sperm cells. TGF-beta 1 had no affect on sperm motility, yet it caused an induction of 2-5(A)synthetase activity. Western immunoblot analysis of TGF-beta 1-treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30-92 kDa; the level of 50 kDa protein increased after treatment with TGF-beta 1. The treatment of sperm cells with TGF-beta 1 did not affect their penetration in zona-free hamster eggs (SPA). These results indicate that TGF-beta 1 enhances expression of a 50 kDa protein related to 2-5(A) synthetase in human sperm cells along with other minor proteins, and this increase does not affect sperm motility and SPA.

  7. Evolutionary analyses of the 12-kDa acidic ribosomal P-proteins reveal a distinct protein of higher plant ribosomes

    PubMed Central

    Szick, Kathleen; Springer, Mark; Bailey-Serres, Julia

    1998-01-01

    The P-protein complex of eukaryotic ribosomes forms a lateral stalk structure in the active site of the large ribosomal subunit and is thought to assist in the elongation phase of translation by stimulating GTPase activity of elongation factor-2 and removal of deacylated tRNA. The complex in animals, fungi, and protozoans is composed of the acidic phosphoproteins P0 (35 kDa), P1 (11–12 kDa), and P2 (11–12 kDa). Previously we demonstrated by protein purification and microsequencing that ribosomes of maize (Zea mays L.) contain P0, one type of P1, two types of P2, and a distinct P1/P2 type protein designated P3. Here we implemented distance matrices, maximum parsimony, and neighbor-joining analyses to assess the evolutionary relationships between the 12 kDa P-proteins of maize and representative eukaryotic species. The analyses identify P3, found to date only in mono- and dicotyledonous plants, as an evolutionarily distinct P-protein. Plants possess three distinct groups of 12 kDa P-proteins (P1, P2, and P3), whereas animals, fungi, and protozoans possess only two distinct groups (P1 and P2). These findings demonstrate that the P-protein complex has evolved into a highly divergent complex with respect to protein composition despite its critical position within the active site of the ribosome. PMID:9482893

  8. Exploring the size limit of protein diffusion through the periplasm in cyanobacterium Anabaena sp. PCC 7120 using the 13 kDa iLOV fluorescent protein.

    PubMed

    Zhang, Li-Chen; Risoul, Véronique; Latifi, Amel; Christie, John M; Zhang, Cheng-Cai

    2013-09-01

    In the filamentous heterocyst-forming cyanobacterium Anabaena PCC 7120, vegetative cells and heterocysts are interdependent on each other and engaged in exchanges of metabolites for survival when grown under diazotrophic conditions. In this organism, the periplasm appears to be continuous along each filament, with a shared outer membrane; however, barriers exist preventing free diffusion of the fluorescent protein GFP (27 kDa) targeted into the periplasmic space. Here we expressed a smaller fluorescent protein iLOV (≈ 13 kDa) fused to the All3333 (a putative homologue of NrtA) signal sequence corresponding to those recognized by the TAT protein translocation system, which exports iLOV to the periplasm of either heterocysts or vegetative cells. Fluorescence microscopy and immunoblot analysis indicated that the iLOV protein is translocated into the periplasm of the producing cell and properly processed, but does not diffuse to neighboring cells via the periplasm. Thus, periplasmic barriers appear to block diffusion of molecules with a size of 13 kDa, the minimum size tested thus far. Assuming that the physical barrier is the peptidoglycan sacculus, its pores might allow diffusion of molecules within the size range between the PatS pentapeptide and iLOV, thus between 0.53 kDa and 13 kDa.

  9. Exploring the size limit of protein diffusion through the periplasm in cyanobacterium Anabaena sp. PCC 7120 using the 13 kDa iLOV fluorescent protein.

    PubMed

    Zhang, Li-Chen; Risoul, Véronique; Latifi, Amel; Christie, John M; Zhang, Cheng-Cai

    2013-09-01

    In the filamentous heterocyst-forming cyanobacterium Anabaena PCC 7120, vegetative cells and heterocysts are interdependent on each other and engaged in exchanges of metabolites for survival when grown under diazotrophic conditions. In this organism, the periplasm appears to be continuous along each filament, with a shared outer membrane; however, barriers exist preventing free diffusion of the fluorescent protein GFP (27 kDa) targeted into the periplasmic space. Here we expressed a smaller fluorescent protein iLOV (≈ 13 kDa) fused to the All3333 (a putative homologue of NrtA) signal sequence corresponding to those recognized by the TAT protein translocation system, which exports iLOV to the periplasm of either heterocysts or vegetative cells. Fluorescence microscopy and immunoblot analysis indicated that the iLOV protein is translocated into the periplasm of the producing cell and properly processed, but does not diffuse to neighboring cells via the periplasm. Thus, periplasmic barriers appear to block diffusion of molecules with a size of 13 kDa, the minimum size tested thus far. Assuming that the physical barrier is the peptidoglycan sacculus, its pores might allow diffusion of molecules within the size range between the PatS pentapeptide and iLOV, thus between 0.53 kDa and 13 kDa. PMID:23748014

  10. Identification of the nuclear localisation signal of O-GlcNAc transferase and its nuclear import regulation

    PubMed Central

    Seo, Hyeon Gyu; Kim, Han Byeol; Kang, Min Jueng; Ryum, Joo Hwan; Yi, Eugene C.; Cho, Jin Won

    2016-01-01

    Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451–453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible β-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously. PMID:27713473

  11. Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.

    PubMed

    Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

    2015-12-01

    Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence.

  12. Regulation of Nrf2-Mediated Phase II Detoxification and Anti-oxidant Genes

    PubMed Central

    Keum, Young-Sam

    2012-01-01

    The molecular mechanisms by which a variety of naturally-occurring dietary compounds exert chemopreventive effects have been a subject of intense scientific investigations. Induction of phase II detoxification and anti-oxidant enzymes through activation of Nrf2/ARE-dependent gene is recognized as one of the major cellular defense mechanisms against oxidative or xenobiotic stresses and currently represents a critical chemopreventive mechanism of action. In the present review, the functional significance of Keap1/Nrf2 protein module in regulating ARE-dependent phase II detoxification and anti-oxidant gene expression is discussed. The biochemical mechanisms underlying the phosphorylation and expression of Keap1/Nrf2 proteins that are controlled by the intracellular signaling kinases and ubiquitin-mediated E3 ligase system as well as control of nucleocytoplasmic translocation of Nrf2 by its innate nuclear export signal (NES) are described. PMID:24116287

  13. Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration

    SciTech Connect

    Furneaux, H.M.; Dropcho, E.J.; Barbut, D.; Chen, Yaotseng; Rosenblum, M.K.; Old, L.J.; Posner, J.B. )

    1989-04-01

    Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. The authors have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.

  14. Sequence of the human 40-kDa keratin reveals an unusual structure with very high sequence identity to the corresponding bovine keratin.

    PubMed Central

    Eckert, R L

    1988-01-01

    The complete amino acid and DNA sequences of the human 40-kDa keratin are reported. The DNA sequence encodes a protein of 44,098 Da, which is unique in that it lacks the terminal non-alpha-helical tail segment found in all other keratins. When the human 40-kDa keratin amino acid sequence is compared to the corresponding bovine keratin, the overall identity is 89%. The coil-forming regions are 89% identical and the head regions are 88% identical. This similarity is also evident in the DNA sequence of the coding region, the 5' upstream sequences, and the 3' noncoding sequences. The high degree of cross-species identity between bovine and human 40-kDa keratins suggests that there is strong evolutionary pressure to conserve the structure of this keratin. This in turn suggests an important and universal role for this intermediate filament subunit in all species. Images PMID:2448790

  15. The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus.

    PubMed

    Perrot-Applanat, M; Cibert, C; Géraud, G; Renoir, J M; Baulieu, E E

    1995-05-01

    FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from

  16. Cultured human melanoma cells biosynthesize a novel form of laminin that lacks the M/sub r/ = 400 kDa subunit

    SciTech Connect

    Peters, B.P.; Kroll, T.G.; Ruddon, R.W.

    1987-05-01

    A human melanoma cell line (A375) was found to produce an unusual type of laminin composed exclusively of M/sub r/ = 200 kDa B subunits and lacking the M/sub r/ = 400 kDa A subunit. Detergent lysates of A375 cells pulsed with (/sup 35/S)methionine contained an immunoreactive form of laminin that migrated on nonreduced SDS-PAGE with an apparent M/sub r/ = 850 kDa. The 850 kDa laminin was clearly resolved from the typical M/sub r/ = 950 kDa laminin composed of A and B subunit types that is produced by a variety of cultured cells such as human choriocarcinoma (JAR) cells. Upon reduction of the intersubunit disulfide bonds of the A375 laminin, a pair of polypeptides co-migrated on SDS-PAGE with the M/sub r/ = 200 kDa B subunit doublet of JAR laminin. The A-deficient A375 laminin does not seem to be a result of the proteolysis of A subunit in cell lysates. No degradation of (/sup 35/S)methionine-labeled JAR laminin was observed upon incubation with nonradioactive A375 cell lysate supplemented in the usual manner with ten protease inhibitors. Furthermore, A subunit did not appear transiently in A375 cells at any chase time (0-4 hr) following a 10-min biosynthetic pulse with (/sup 35/S)methionine. The authors observations suggest that A375 cells biosynthesize principally the B subunits of laminin and assemble them to form a disulfide-linked macromolecule, possibly a B/sub 4/ tetramer.

  17. Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.

    PubMed Central

    Sidorenko, S P; Law, C L; Chandran, K A; Clark, E A

    1995-01-01

    The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7831290

  18. Evolution of a transcriptional regulator from a transmembrane nucleoporin.

    PubMed

    Franks, Tobias M; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C N; Young, Janet M; Malik, Harmit S; Gage, Fred H; Hetzer, Martin W

    2016-05-15

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components. PMID:27198230

  19. Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

    PubMed Central

    Okada, Naoyuki; Sato, Masamitsu

    2015-01-01

    Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

  20. Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

    PubMed

    Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

    2014-01-29

    After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

  1. Silencing the Nucleocytoplasmic O-GlcNAc Transferase Reduces Proliferation, Adhesion, and Migration of Cancer and Fetal Human Colon Cell Lines.

    PubMed

    Steenackers, Agata; Olivier-Van Stichelen, Stéphanie; Baldini, Steffi F; Dehennaut, Vanessa; Toillon, Robert-Alain; Le Bourhis, Xuefen; El Yazidi-Belkoura, Ikram; Lefebvre, Tony

    2016-01-01

    The post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is regulated by a unique couple of enzymes. O-GlcNAc transferase (OGT) transfers the GlcNAc residue from UDP-GlcNAc, the final product of the hexosamine biosynthetic pathway (HBP), whereas O-GlcNAcase (OGA) removes it. This study and others show that OGT and O-GlcNAcylation levels are increased in cancer cell lines. In that context, we studied the effect of OGT silencing in the colon cancer cell lines HT29 and HCT116 and the primary colon cell line CCD841CoN. Herein, we report that OGT silencing diminished proliferation, in vitro cell survival and adhesion of primary and cancer cell lines. SiOGT dramatically decreased HT29 and CCD841CoN migration, CCD841CoN harboring high capabilities of migration in Boyden chamber system when compared to HT29 and HCT116. The expression levels of actin and tubulin were unaffected by OGT knockdown but siOGT seemed to disorganize microfilament, microtubule, and vinculin networks in CCD841CoN. While cancer cell lines harbor higher levels of OGT and O-GlcNAcylation to fulfill their proliferative and migratory properties, in agreement with their higher consumption of HBP main substrates glucose and glutamine, our data demonstrate that OGT expression is not only necessary for the biological properties of cancer cell lines but also for normal cells.

  2. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.

    PubMed

    Mouilleron, Stéphane; Langer, Carola A; Guettler, Sebastian; McDonald, Neil Q; Treisman, Richard

    2011-06-14

    Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.

  3. Regulation of thymosin beta 4 in chicken macrophages by toll like receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thymosin beta 4 (TB4) is a 5kDa peptide that binds to the cytoskeletal protein actin and regulates cell motility. At the extracellular level it also promotes chemotaxis, wound healing and angiogenesis. However, the mechanism responsible for the regulation of TB4 production and its secretion to the e...

  4. Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro.

    PubMed

    Huang, Y S; Chuang, D T

    1999-04-01

    Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive

  5. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.

    PubMed Central

    Covacci, A; Censini, S; Bugnoli, M; Petracca, R; Burroni, D; Macchia, G; Massone, A; Papini, E; Xiang, Z; Figura, N

    1993-01-01

    Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease. Images Fig. 1 Fig. 4 Fig. 5 PMID:8516329

  6. Identification of three competitive inhibitors for membrane-associated, Mg2+-dependent and neutral 60 kDa sphingomyelinase activity.

    PubMed

    Kim, Seok Kyun; Jung, Sang Mi; Ahn, Kyong Hoon; Jeon, Hyung Jun; Lee, Dong Hun; Jung, Kwang Mook; Jung, Sung Yun; Kim, Dae Kyong

    2005-08-01

    Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 microg/ mL, 9.4 microg/mL, and 12.9 microg/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.

  7. Synthetic biology design to display an 18 kDa rotavirus large antigen on a modular virus-like particle.

    PubMed

    Lua, Linda H L; Fan, Yuanyuan; Chang, Cindy; Connors, Natalie K; Middelberg, Anton P J

    2015-11-01

    Virus-like particles are an established class of commercial vaccine possessing excellent function and proven stability. Exciting developments made possible by modern tools of synthetic biology has stimulated emergence of modular VLPs, whereby parts of one pathogen are by design integrated into a less harmful VLP which has preferential physical and manufacturing character. This strategy allows the immunologically protective parts of a pathogen to be displayed on the most-suitable VLP. However, the field of modular VLP design is immature, and robust design principles are yet to emerge, particularly for larger antigenic structures. Here we use a combination of molecular dynamic simulation and experiment to reveal two key design principles for VLPs. First, the linkers connecting the integrated antigenic module with the VLP-forming protein must be well designed to ensure structural separation and independence. Second, the number of antigenic domains on the VLP surface must be sufficiently below the maximum such that a "steric barrier" to VLP formation cannot exist. This second principle leads to designs whereby co-expression of modular protein with unmodified VLP-forming protein can titrate down the amount of antigen on the surface of the VLP, to the point where assembly can proceed. In this work we elucidate these principles by displaying the 18.1 kDa VP8* domain from rotavirus on the murine polyomavirus VLP, and show functional presentation of the antigenic structure.

  8. Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

    PubMed

    Peyretaillade, E; Broussolle, V; Peyret, P; Méténier, G; Gouy, M; Vivarès, C P

    1998-06-01

    An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated. PMID:9615449

  9. Ultrahigh-Resolution Differential Ion Mobility Separations of Conformers for Proteins above 10 kDa: Onset of Dipole Alignment?

    SciTech Connect

    Shvartsburg, Alexandre A.

    2014-11-04

    Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with same peak width and resolving power up to ~400 as for peptides. Present work explores the reach of this approach for larger proteins, exemplified by cytochrome c and myoglobin. Resolution similar to that for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of present technique to proteins under ~20 kDa. This capability may enable distinguishing whole proteins with differing residue sequences or localizations of posttranslational modifications. Small features at negative compensation voltages that markedly grow from cytochrome c to myoglobin indicate the dipole alignment of rare conformers in accord with theory, further supporting the concept of pendular macroions in FAIMS.

  10. Immunoreactivity for phosphorylated 200-kDa neurofilament subunit is heterogeneously expressed in human sympathetic and primary sensory neurons.

    PubMed

    Vega, J A; Humara, J M; Naves, F J; Esteban, I; Del Valle, M E

    1994-11-01

    This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.

  11. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  12. The 18-kDa Translocator Protein (TSPO) Disrupts Mammary Epithelial Morphogenesis and Promotes Breast Cancer Cell Migration

    PubMed Central

    Wu, Xiaoting; Gallo, Kathleen A.

    2013-01-01

    Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO), are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER)-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864) and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer. PMID:23967175

  13. Control of crystal polymorph in microfluidics using molluscan 28 kDa Ca²(+)-binding protein.

    PubMed

    Ji, Bozhi; Cusack, Maggie; Freer, Andy; Dobson, Phil S; Gadegaard, Nikolaj; Yin, Huabing

    2010-10-01

    Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery.

  14. A plastid-targeted heat shock cognate 70 kDa protein interacts with the Abutilon mosaic virus movement protein

    SciTech Connect

    Krenz, Bjoern; Windeisen, Volker; Wege, Christina; Jeske, Holger; Kleinow, Tatjana

    2010-05-25

    The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70 kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 gene of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.

  15. A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa.

    PubMed Central

    Rosa, A L; Alvarez, M E; Lawson, D; Maccioni, H J

    1990-01-01

    Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2141976

  16. Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface.

    PubMed

    Ahmad, Atta; Bhattacharya, Akash; McDonald, Ramsay A; Cordes, Melissa; Ellington, Benjamin; Bertelsen, Eric B; Zuiderweg, Erik R P

    2011-11-22

    The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].

  17. Reduction of Gap Junctional Conductance by Microinjection of Antibodies against the 27-kDa Liver Gap Junction Polypeptide

    NASA Astrophysics Data System (ADS)

    Hertzberg, E. L.; Spray, D. C.; Bennett, M. V. L.

    1985-04-01

    Antibody raised against isolated rat liver gap junctions was microinjected into coupled cells in culture to assess its influence on gap junctional conductance. A rapid inhibition of fluorescent dye transfer and electrical coupling was produced in pairs of freshly dissociated adult rat hepatocytes and myocardial cells as well as in pairs of superior cervical ganglion neurons from neonatal rats cultured under conditions in which electrotonic synapses form. The antibodies have been shown by indirect immunofluorescence to bind to punctate regions of the plasma membrane in liver. By immunoreplica analysis of rat liver homogenates, plasma membranes, and isolated gap junctions resolved on NaDodSO4/polyacrylamide gels, binding was shown to be specific for the 27-kDa major polypeptide of gap junctions. This and similar antibodies should provide a tool for further investigation of the role of cell-cell communication mediated by gap junctions and indicate that immunologically similar polypeptides comprise gap junctions in adult mammalian cells derived from all three germ layers.

  18. Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

    PubMed

    Peyretaillade, E; Broussolle, V; Peyret, P; Méténier, G; Gouy, M; Vivarès, C P

    1998-06-01

    An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

  19. Calcium-induced splitting of connectin filaments into beta-connectin and a 1,200-kDa subfragment.

    PubMed

    Takahashi, K; Hattori, A; Tatsumi, R; Takai, K

    1992-06-01

    When rabbit skeletal muscle myofibrils were treated with a solution containing 0.1 mM Ca2+ and 30 micrograms of leupeptin/ml, alpha-connectin, which forms very thin filaments in myofibrils, was split into beta-connectin and a 1,200-kDa subfragment. A part of beta-connectin located near the junction between beta-connectin and the subfragment seems to have an affinity for calcium ions and to be susceptible to the binding of large amounts of calcium ions. The calcium-binding site on beta-connectin is localized near the N2 line in the I band, and the subfragment is localized adjacent to the Z disk. It is possible that connectin filaments change their elasticity during the contraction-relaxation cycle of skeletal muscle at the physiological concentration of calcium ions. Because postmortem skeletal muscles lose their elasticity and become plastic in association with the calcium-specific splitting of connectin filaments, the splitting is considered to be a factor in meat tenderization during postrigor ageing.

  20. A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen.

    PubMed

    Xue, Dahai; Shi, Hong; Smith, James D; Chen, Xinguo; Noe, Dennis A; Cedervall, Tommy; Yang, Derek D; Eynon, Elizabeth; Brash, Douglas E; Kashgarian, Michael; Flavell, Richard A; Wolin, Sandra L

    2003-06-24

    Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24-60% of all patients with systemic lupus erythematosus. Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions. In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs. Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis. Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation. Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis. Moreover, in one strain background, Ro-/- mice display increased sensitivity to irradiation with UV light. Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance. Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans.

  1. Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor.

    PubMed Central

    Denzer, K; Weber, B; Hille-Rehfeld, A; Figura, K V; Pohlmann, R

    1997-01-01

    The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant. PMID:9291124

  2. A novel ∼34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: production, purification, and characterization.

    PubMed

    Mojallali, Leila; Shahbani Zahiri, Hossein; Rajaei, Sarah; Akbari Noghabi, Kambiz; Haghbeen, Kamahldin

    2014-01-01

    An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²⁺ but decreased by Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺, and Ca²⁺. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.

  3. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    SciTech Connect

    Chen, J.; Varner, J.E.

    1985-07-01

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.

  4. In vitro transcription directed from the somatostatin promoter is dependent upon a purified 43-kDa DNA-binding protein.

    PubMed Central

    Andrisani, O M; Zhu, Z N; Pot, D A; Dixon, J E

    1989-01-01

    In vitro transcription analyses were used to establish the biological function of a 43-kDa affinity-purified DNA-binding protein. The 43-kDa affinity-purified protein protects the region from position -59 to position -35 of the somatostatin promoter from DNase I digestion. This region of the somatostatin promoter harbors the TGACGTCA motif, also found and required for function in a number of other cAMP-responsive and adenovirus E1A-inducible promoters. Efficient and authentic transcription in vitro directed from the somatostatin promoter requires the TGACGTCA promoter element. In vitro transcription assays performed in the presence of somatostatin (positions -60 to -29), enkephalin (positions -105 to -71), and adenovirus type 5 E3 gene (positions -72 to -42) competitor fragments, harboring similar TGACGTCA motifs, selectively inhibit transcription directed from the somatostatin promoter, suggesting that the TGACGTCA element is the site of interaction of a somatostatin gene transactivator. Furthermore, extracts depleted of the TGACGTCA-binding activities by affinity chromatography utilizing a biotinylated oligonucleotide-avidin resin, are incapable of directing transcription from the somatostatin but not from the adenovirus major late promoter. Addition of the purified 43-kDa protein to the affinity-depleted extract restores transcription from the somatostatin promoter. These results are consistent with the 43-kDa protein being a trans-activator of the somatostatin gene. Images PMID:2564679

  5. A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

    EPA Science Inventory

    Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

  6. The 42.1 and 53.7 kDa bands in SDS-PAGE of R-phycoerythrin from Polysiphonia urceolata.

    PubMed

    Zhao, Mingri; Sun, Li; Sun, Shichun; Gong, Xueqin; Fu, Xuejun; Chen, Min

    2013-09-01

    In SDS-PAGE gels of three purified R-phycoerythrins (R-PEs) isolated from three species of red algae, two bands whose molecular weights were about 40 kDa and 50 kDa can stably be found when the sample loading amount was enough. It is important for structure study of R-PE to clarify what these bands represent and how they are formed. According to results of the second SDS-PAGE, as well as molecular weights, fluorescences under UV and abundance, the 42.1 kDa and 53.7 kDa bands in SDS-PAGE gels of R-PE from Polysiphonia urceolata were believed to be complexes of αβ and βγ1, respectively. Formation of these bands may be related to light and phycourobilins (PUB) in subunits; and appearance of these two bands provided some proofs on position of chromophores and directions of energy transfer in R-PE. R-PE containing γ1 subunit was obviously more stable than R-PE containing γ2 subunit when they were exposed to protein denaturants, so γ subunits of R-PE may play important roles in structural stability of R-PE aggregates and the main forces that maintain the stability of R-PE may be interactions between γ subunit and β subunits.

  7. Characterization of a 60-kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2015-08-01

    A sandwich enzyme-linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60-kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60-kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma-derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60-kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2-dimensional gel electrophoresis (2-D GE) and amino acid sequencing revealed the 60-kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons. PMID:26172875

  8. An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: Identification by photoaffinity labeling and purification of a 23-kDa polypeptide

    SciTech Connect

    Feldwisch, J.; Zettl, R.; Hesse, F.; Schell, J.; Palme, K. )

    1992-01-15

    Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-(7-{sup 3}H)indole-3-acetic acid (({sup 3}H)N{sub 3}IAA), the authors identified several IAA-binding proteins with the molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, they were able to selectively extract pm23 from the plasma membrane. They show that auxins and functional analogues compete with ({sup 3}H)N{sub 3}IAA for binding to pm23. They found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, they identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. They designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7,000-fold purification.

  9. Prominent 85-kDa oligomannosidic glycoproteins of rat brain are signal regulatory proteins and include the SHP substrate-1 for tyrosine phosphatases.

    PubMed

    Bartoszewicz, Z P; Jaffe, H; Sasaki, M; Möller, J R; Stebbins, J W; Gebrekristos, H; Quarles, R H

    1999-04-01

    The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.

  10. Looking into laminin receptor: critical discussion regarding the non-integrin 37/67-kDa laminin receptor/RPSA protein.

    PubMed

    DiGiacomo, Vincent; Meruelo, Daniel

    2016-05-01

    The 37/67-kDa laminin receptor (LAMR/RPSA) was originally identified as a 67-kDa binding protein for laminin, an extracellular matrix glycoprotein that provides cellular adhesion to the basement membrane. LAMR has evolutionary origins, however, as a 37-kDa RPS2 family ribosomal component. Expressed in all domains of life, RPS2 proteins have been shown to have remarkably diverse physiological roles that vary across species. Contributing to laminin binding, ribosome biogenesis, cytoskeletal organization, and nuclear functions, this protein governs critical cellular processes including growth, survival, migration, protein synthesis, development, and differentiation. Unsurprisingly given its purview, LAMR has been associated with metastatic cancer, neurodegenerative disease and developmental abnormalities. Functioning in a receptor capacity, this protein also confers susceptibility to bacterial and viral infection. LAMR is clearly a molecule of consequence in human disease, directly mediating pathological events that make it a prime target for therapeutic interventions. Despite decades of research, there are still a large number of open questions regarding the cellular biology of LAMR, the nature of its ability to bind laminin, the function of its intrinsically disordered C-terminal region and its conversion from 37 to 67 kDa. This review attempts to convey an in-depth description of the complexity surrounding this multifaceted protein across functional, structural and pathological aspects.

  11. Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

    PubMed Central

    Kim, Won-Tae; Kong, Hyun-Hee; Ha, Young-Ran; Hong, Yeon-Chul; Jeong, Hae Jin; Yu, Hak Sun

    2006-01-01

    The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba. PMID:17170574

  12. Characterization of a 60-kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2015-08-01

    A sandwich enzyme-linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60-kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60-kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma-derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60-kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2-dimensional gel electrophoresis (2-D GE) and amino acid sequencing revealed the 60-kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons.

  13. The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

    SciTech Connect

    Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.; Harauz, George

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

  14. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    SciTech Connect

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  15. Class IIa Histone Deacetylases Are Conserved Regulators of Circadian Function*

    PubMed Central

    Fogg, Paul C. M.; O'Neill, John S.; Dobrzycki, Tomasz; Calvert, Shaun; Lord, Emma C.; McIntosh, Rebecca L. L.; Elliott, Christopher J. H.; Sweeney, Sean T.; Hastings, Michael H.; Chawla, Sangeeta

    2014-01-01

    Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca2+ and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery. PMID:25271152

  16. Polymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer

    PubMed Central

    Sutherland, Alison; Kim, Dong-Hyun; Relton, Caroline; Ahn, Yoon-Ok

    2010-01-01

    Selenium (Se), a dietary trace metal essential for human health, is incorporated into ~25 selenoproteins including selenoprotein S (SelS) and the 15-kDa selenoprotein (Sep15) both of which have functions in the endoplasmic reticulum protein unfolding response. The aim of this study was to investigate whether genetic variants in such selenoprotein genes are associated with altered risk of colorectal cancer (CRC). A Korean population of 827 patients with CRC and 733 healthy controls was genotyped for 7 SNPs in selenoprotein genes and one SNP in the gene encoding manganese superoxide dismutase using Sequenom technology. Multivariate logistic regression analysis showed that after adjustment for lifestyle factors three SNP variants were associated with altered disease risk. There was a mean odds ratio of 2.25 [95% CI 1.13,4.48] in females homozygous TT for rs34713741 in SELS with the T variant being associated with higher risk of rectal cancer, and odds ratios of 2.47 and 2.51, respectively, for rs5845 and rs5859 in SEP15 with the minor A and T alleles being associated with increased risk of male rectal cancer. The data indicate that the minor alleles for rs5845, rs5859 and rs34713741 are associated with increased rectal cancer risk and that the effects of the three SNPs are dependent on gender. The results highlight potential links between Se, the function of two selenoproteins involved in the protein unfolding response and CRC risk. Further studies are required to investigate whether the effects of the variants on CRC risk are also modulated by dietary Se intake. PMID:21052528

  17. Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp.

    PubMed

    Levy-Ontman, Oshrat; Arad, Shoshana Malis; Harvey, David J; Parsons, Thomas B; Fairbanks, Antony; Tekoah, Yoram

    2011-06-17

    We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2). The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.

  18. Expression of 32/67-kDa laminin receptor in laminin adhesion-selected human colon cancer cell lines.

    PubMed Central

    Kim, W. H.; Lee, B. L.; Jun, S. H.; Song, S. Y.; Kleinman, H. K.

    1998-01-01

    Laminin promotes the malignant phenotype, and the expression of certain laminin receptors is increased in malignancy. Previously, we demonstrated that a laminin-adhesive subclone of a human colon cancer cell line showed increased tumorigenicity in nude mice and increased affinity of the beta1 integrin for laminin relative to the laminin-non-adhesive subclone. The total amount of either beta1 integrin protein or mRNA did not increase. As levels of the 32/67-kDa laminin receptor (67LR) correlate with malignancy, we examined 67LR expression in the laminin adhesion-selected human colon cancer cells. The laminin-adhesive subclone, which was more tumorigenic in both heterotopic and orthotopic locations than in a laminin-non-adhesive subclone, showed cell-surface membrane staining of 67LR, whereas the laminin-non-adhesive subclone showed cytoplasmic staining of 67LR. No difference in either the amount of 67LR mRNA or the amount of protein was observed in the parental cells than in the laminin-adhesive and non-adhesive subclones. When assayed on a laminin affinity column, more 67LR molecules bound to the column with cell extracts from the laminin-adhesive subclone than was observed with the non-adhesive subclone. These findings suggest that the increased tumorigenicity of laminin adhesion-selected tumour cells might be due to an alteration in the distribution and/or adhesiveness of multiple receptors including 67LR and beta1 integrin. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9459140

  19. A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV.

    PubMed Central

    Santana, J M; Grellier, P; Schrével, J; Teixeira, A R

    1997-01-01

    Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy. PMID:9224638

  20. Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications.

    PubMed

    Song, Jikui; Lee, Min S; Carlberg, Inger; Vener, Alexander V; Markley, John L

    2006-12-26

    Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal alpha-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal alpha-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function. PMID:17176085

  1. Micelle-induced Folding of Spinach Thylakoid Soluble Phosphoprotein of 9 kDa and its Functional Implications †, ‡

    PubMed Central

    Song, Jikui; Lee, Min S.; Carlberg, Inger; Vener, Alexander V.; Markley, John L

    2008-01-01

    Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al., Proc. Natl. Acad. Sci. 2003, 100, 757–762). Non-phosphorylated TSP9 is associated with the membrane, whereas after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of non-phosphorylated TSP9 in both aqueous solution and in membrane mimetic micelles. The results show that both wild type non-phosphorylated TSP9 and a triple-mutant (T46E+T53E+T60E) mimic of the tri-phosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal α-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for non-phosphorylated TSP9 in DPC micelles indicated that the molecule is highly flexible with the highest order in the N-terminal α-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function. PMID:17176085

  2. Cloning and characterization of a novel gene encoding 16 kDa protein (Ac16) from Angiostrongylus cantonensis.

    PubMed

    Li, Zheng-Yu; Lv, Zhi-Yue; Wei, Jie; Liao, Qi; Zheng, Huan-Qin; Wu, Zhong-Dao

    2012-06-01

    Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis or meningoencephalitis. A novel gene (AC16) was isolated from a cDNA library of A. cantonensis fourth-stage larvae. The putative 16-kDa protein has 149 amino acids and is homologous to an immunodominant hypodermal antigen (IHA16) from Ancylostoma caninum (identities = 57%). In this paper, we cloned the gene and purified the recombinant Ac16 (rAC16) protein. Real-time quantitative PCR revealed that Ac16 was expressed significantly higher in the fourth-stage larvae and adult worms derived from rats than that in the fourth-stage larvae derived from mice. Moreover, sera from rat (permissive host) infected with A. cantonensis detected Ac16 by Western blot, while sera from infected mouse (non-permissive host) could not. The results implied that Ac16 was related to the parasitic adaptation of A. cantonensis in different hosts and non-permissive host mouse had no circulating antibody to the antigen Ac16 from A. cantonensis and thus might contribute to understanding the mechanism of parasite immune evasion. Furthermore, we evaluated the ability of Ac16 antibody diagnosing A. cantonensis infection by an indirect enzyme-linked immunosorbent assay. The results showed that the Ac16 antibody had a 79.17% sensitivity to rAC16 and 83.33% to crude adult worm antigens (CA) (P > 0.05), while the specificity to rAC16 and to CA were 95.89% and 86.30% respectively (P < 0.05), thus implying that rAc16 may constitute a putative serodiagnostic antigen for Angiostrongyliasis cantonensis. PMID:22146998

  3. Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme

    PubMed Central

    Gumbs, Orlando H.; Padgett, Richard A.; Dayie, Kwaku T.

    2006-01-01

    We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1–3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1–10 mM MgCl2). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K d of 650 ± 250 nM, a K m of ∼300 nM, and a K cat of 0.02 min−1 under single turnover conditions. Within the ∼160-kDa D123–D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a well-behaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies. PMID:16894219

  4. Purification and properties of a self-associating, 50-kDa copper-binding protein from brindled mouse livers.

    PubMed

    Seo, H C; Ettinger, M J

    1993-01-15

    The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal 64Cu binding was detected in both the fraction which contained the tetramer plus dimer (approximately 26% less) and the fraction containing the monomer of CuBP (approximately 37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at approximately 20 min after starting the final water elution step, the brindled mouse protein eluted by approximately 5 min. This seemed to be due to abnormal self-association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consistent with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease.

  5. A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa.

    PubMed

    Tabuchi, Tomohito; Shidara, Osamu; Harayama, Hiroshi

    2008-12-01

    Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation. PMID:18787309

  6. Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

    PubMed

    Hatty, Claire R; Le Brun, Anton P; Lake, Vanessa; Clifton, Luke A; Liu, Guo Jun; James, Michael; Banati, Richard B

    2014-03-01

    The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

  7. Cloning and expression of the translocator protein (18 kDa), voltage-dependent anion channel, and diazepam binding inhibitor in the gonad of largemouth bass (Micropterus salmoides) across the reproductive cycle.

    PubMed

    Doperalski, Nicholas J; Martyniuk, Christopher J; Prucha, Melinda S; Kroll, Kevin J; Denslow, Nancy D; Barber, David S

    2011-08-01

    Cholesterol transport across the mitochondrial membrane is rate-limiting for steroidogenesis in vertebrates. Previous studies in fish have characterized expression of the steroidogenic acute regulatory protein, however the function and regulation of other genes and proteins involved in piscine cholesterol transport have not been evaluated. In the current study, mRNA sequences of the 18 kDa translocator protein (tspo; formerly peripheral benzodiazepine receptor), voltage-dependent anion channel (vdac), and diazepam binding inhibitor (dbi; also acyl-CoA binding protein) were cloned from largemouth bass. Gonadal expression was examined across reproductive stages to determine if expression is correlated with changes in steroid levels and with indicators of reproductive maturation. In testis, transcript abundance of tspo and dbi increased with reproductive maturation (6- and 23-fold maximal increase, respectively) and expression of tspo and dbi was positively correlated with reproductive stage, gonadosomatic index (GSI), and circulating levels of testosterone. Testis vdac expression was positively correlated with reproductive stage and GSI. In females, gonadal tspo and vdac expression was negatively correlated with GSI and levels of plasma testosterone and 17β-estradiol. Ovarian dbi expression was not correlated with indicators of reproductive maturation. These studies represent the first investigation of the steroidogenic role of tspo, vdac, and dbi in fish. Findings suggest that cholesterol transport in largemouth bass testis, but not in ovary, may be transcriptionally-regulated, however further investigation will be necessary to fully elucidate the role of these genes in largemouth bass steroidogenesis.

  8. The small 11kDa nonstructural protein of human parvovirus B19 plays a key role in inducing apoptosis during B19 virus infection of primary erythroid progenitor cells

    PubMed Central

    Chen, Aaron Yun; Zhang, Elizabeth Yan; Guan, Wuxiang; Cheng, Fang; Kleiboeker, Steve; Yankee, Thomas M.

    2010-01-01

    Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5kDa, 11kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo–expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11kDa is a more significant inducer of apoptosis than NS1, whereas 7.5kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11kDa expression using antisense oligos targeting specifically to the 11kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11kDa protein contributes to erythroid progenitor cell death during B19V infection. PMID:19861680

  9. Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.

    PubMed

    Dizhoor, A M; Olshevskaya, E V; Henzel, W J; Wong, S C; Stults, J T; Ankoudinova, I; Hurley, J B

    1995-10-20

    Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+)-sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, E.V., Laura, R.P., and Hurley, J.B. (1994) Neuron 12, 1345-1352; Lowe, D.G., Dizhoor, A.M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as

  10. Field assessment of recombinant Schistosoma japonicum 26 kDa glutathione S-transferase in Chinese water buffaloes.

    PubMed

    He, Y K; Liu, S X; Zhang, X Y; Song, G C; Luo, X S; Li, Y S; Xu, Y X; Yu, X L; Li, Y; Hou, X Y; McManus, D P

    2003-09-01

    We have shown previously that anti-fecundity immunity can be induced experimentally against recombinant 26 kDa glutathione S-transferase (reSjc26GST) in Chinese water buffaloes (Bos buffelus), important reservoir hosts for Schistosoma japonicum in China. In the field study described here, we immunized buffaloes with reSjc26GST to induce protective immunity against S. japonicum and to evaluate its effectiveness in controlling schistosomiasis japonica. We selected two villages as test and control groups in inside-embankment areas endemic for schistosomiasis japonica. The buffaloes in the test village were vaccinated with reSjc26GST, whereas those in the control village were not. The indicators of the effect of the vaccine included the generation of specific IgG antibodies in the vaccinated buffaloes, changes in the prevalence and infection intensity in buffaloes and village children, changes in the density of infected snails, and changes in the infectivity of water bodies (assessed by sentinel mice) in transmission areas adjacent to both villages. Twenty months after vaccination, the infection rate of buffaloes in the test village was decreased by 60.4% (from an initial prevalence of 13.5% to 5.4%), and 67.9% when compared with that in the control village (initial prevalence of 16.7%). However, the infection rate in village children remained unchanged. The density of infected snails decreased by 71.4%, from 0.0049/0.11 m2 to 0.0014/0.11m2 in the high transmission area outside the embankment in the test village. There was no change in the infectivity of the water body transmission areas between the test and control villages. The levels of specific antibodies to reSjc26GST showed a continuous increase after vaccination. These results indicate that protective immunity was induced and maintained in buffaloes after vaccination with reSjc26GST. The vaccine could thus play a significant role in reducing S. japonicum transmission caused by water buffaloes in the Lake region

  11. Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H(+)-ATPase.

    PubMed

    Finbow, M E; Eliopoulos, E E; Jackson, P J; Keen, J N; Meagher, L; Thompson, P; Jones, P; Findlay, J B

    1992-01-01

    A 16 kDa protein has been isolated in a homogeneous form as the major component of a paracrystalline paired membrane structure closely resembling the gap junction. The primary structure of this protein from arthropod and vertebrate species has been determined by protein and cDNA sequencing. The amino acid sequences are highly conserved and virtually identical to the amino acid sequence of the proteolipid subunit of the vacuolar H(+)-ATPases. The disposition of the protein in the membrane has been studied using proteases and the N,N'-dicyclohexylcarbodiimide reactive site identified. These data, together with secondary structure predictions, suggest that the 16 kDa protein is for the most part buried in the membrane, arranged in a bundle of four hydrophobic alpha-helices. Using computer graphics, a model has been constructed based on this arrangement and on the electron microscopic images of the paracrystalline arrays.

  12. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    PubMed

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN. PMID:9931470

  13. Lysine-acetylation as a fundamental regulator of Ran function: Implications for signaling of proteins of the Ras-superfamily

    PubMed Central

    Knyphausen, Philipp; Kuhlmann, Nora; de Boor, Susanne; Lammers, Michael

    2015-01-01

    The small GTP-binding protein Ran is involved in the regulation of essential cellular processes in interphase but also in mitotic cells: Ran controls the nucleocytoplasmic transport of proteins and RNA, it regulates mitotic spindle formation and nuclear envelope assembly. Deregulations in Ran dependent processes were implicated in the development of severe diseases such as cancer and neurodegenerative disorders. To understand how Ran-function is regulated is therefore of highest importance. Recently, several lysine-acetylation sites in Ran were identified by quantitative mass-spectrometry, some being located in highly important regions such as the P-loop, switch I, switch II and the G5/SAK motif. We recently reported that lysine-acetylation regulates nearly all aspects of Ran-function such as RCC1 catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, its interaction with NTF2 and the formation of import- and export-complexes. As a hint for its biological importance, we identified Ran-specific lysine-deacetylases (KDACs) and -acetyltransferases (KATs). Also for other small GTPases such as Ras, Rho, Cdc42, and for many effectors and regulators thereof, lysine-acetylation sites were discovered. However, the functional impact of lysine-acetylation as a regulator of protein function has only been marginally investigated so far. We will discuss recent findings of lysine-acetylation as a novel modification to regulate Ras-protein signaling. PMID:26507377

  14. Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

    PubMed

    Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

    2016-04-01

    The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

  15. Reverse immunoaffinity chromatography: application to the purification of the 60- to 90-kDa gastric parietal cell autoantigen associated with autoimmune gastritis.

    PubMed

    Goldkorn, I; Gleeson, P A; Toh, B H

    1991-05-01

    Previous attempts to purify autoantigens by affinity chromatography using the total IgG from autoantibody-positive sera have not been very successful. We describe here a novel method using parietal cell autoantibodies, isolated on a crude autoantigen immunoabsorbent column, for the purification of the 60- to 90-kDa gastric parietal cell antigen targeted in autoimmune gastritis. The 60- to 90-kDa parietal cell antigen was first enriched 2.5-fold from a 0.5% Triton X-100 extract of total pig gastric mucosal membranes by DEAE-Sepharose 4B chromatography. The fraction containing the 60- to 90-kDa antigen was directly coupled to CNBr-activated Sepharose 4B to produce an immunoadsorbent column for the "reverse" purification of the specific parietal cell autoantibodies from pooled gastric parietal cell autoantibody-positive human sera. A 0.5% Triton X-100 extract of pig gastric mucosal membranes was initially precleared by sequential passage through Protein A-Sepharose 4B and normal human Ig-Sepharose 4B columns. The 60- to 90-kDa gastric parietal cell antigen (approximately 10 micrograms) was then purified from this precleared Triton X-100 membrane extract by affinity chromatography on a 1-ml column of purified autoantibody-Sepharose 4B, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophonesis. This method, incorporating a "reverse" immunoaffinity chromatography step, has general applicability for the purification of tissue autoantigens using highly specific autoantibodies present in the sera of humans and experimental animals with a variety of other autoimmune diseases.

  16. Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome).

    PubMed Central

    Lazarow, P B; Fujiki, Y; Small, G M; Watkins, P; Moser, H

    1986-01-01

    Peroxisomes have not been detected in liver and kidney of patients with Zellweger syndrome. Some peroxisome proteins are missing; others are present in normal amounts but are located in the cytosol. We have prepared an antiserum against the 22-kDa integral membrane protein characteristic of rat liver peroxisomes. The antiserum crossreacts with the human liver counterpart, which likewise has a mass of 22 kDa. By immunoblot analysis, we demonstrate that the 22-kDa protein is present in normal amount in Zellweger liver and is integral to a membrane. The result suggests that peroxisome membranes are assembled in Zellweger syndrome but may be defective for the import of matrix proteins. As a result, newly synthesized proteins are left in the cytosol, where some persist and others are degraded. Lacking their usual content, such aberrant peroxisomal membranes would be unrecognizable morphologically. Immunoblot analyses also showed that the peroxisomal hydratase-dehydrogenase is deficient in Zellweger kidney as well as liver, but catalase is present in both organs. Images PMID:3538019

  17. A 70 kDa MHC class I associated protein (MAP-70) identified as a receptor molecule for Coxsackievirus A9 cell attachment.

    PubMed

    Triantafilou, M; Triantafilou, K; Wilson, K M

    2000-09-01

    One of the major categories of disease-causing micro-organisms are viruses. New studies on many different viruses have shown that virus attachment and cell entry is often a multistep process, requiring many interactions between the virus and cell surface molecules. In this study, we have attempted to identify the cell surface molecules involved in Coxsackievirus A9 (CAV-9), a common human pathogen and a member of the Picornavirus family, infectious process. GMK cells susceptible to virus infection were surfaced labeled with biotin and then solubilized in non-ionic and zwiterionic detergents. Free CAV-9 virions were used as an affinity surface, allowing the virus to bind to the solubilized receptors. The virus-receptor complexes were then immunoprecipitated by an anti CAV-9 serum and protein-A sepharose beads. SDS-PAGE and two-dimensional electrophoresis revealed the presence of integrin alpha v beta 3 molecules and a 70 kDa protein with apparent isoelectric point (pI) 5.5. The identity of the integrin alpha v beta 3 molecules was confirmed by immunoprecipitation and Western blotting; whereas the 70 kDa protein was also found to co-immunoprecipitate with MHC class I molecules in non-stringent conditions. Sequential immunoprecipitation experiments confirmed that the MHC class I associated protein (MAP-70) and the 70 kDa protein utilized by CAV-9 were identical. The role of MAP-70 in CAV-9 infectious process is discussed.

  18. In vitro galactosylation of a 110-kDa glycoprotein by an endogenous cell surface galactosyltransferase correlates with the invasiveness of adrenal carcinoma cells

    SciTech Connect

    Penno, M.B.; Passaniti, A.; Hart, G.W.; Jordan, C.; Kumar, S.; Scott, A.F. ); Fridman, R. )

    1989-08-01

    The authors have examined the role of a cell surface galactosyltransferase, laminin, and laminin-binding protein (receptor) in the invasion of clonal derivatives of a murine adrenal carcinoma cell line. Although a 10-fold variation was found in the ability to invade a reconstituted basement membrane matrix, levels of intracellular laminin and the laminin-binding protein were shown to be present and secreted equally in all lines. Of the eight lines tested, seven showed a correlation between invasion and the incorporation of ({sup 3}H)galactose from UDP-({sup 3}H)galactose into a 90- to 110-kDa protein. One noninvasive line (clone HSR), however, retained high galactosyltransferase activity yet could not galactosylate the endogenous 90- to 110-kDa substrate. Interestingly, this clone was unable to attach to laminin. Although high galactosyltransferase activity can be consistent with cells of high invasiveness, the results suggest that the galactosylation status of a 90- to 110-kDa Y1 cell surface glycoprotein is most indicative of invasion potential.

  19. The location of a disease-associated polymorphism and genomic structure of the human 52-kDa Ro/SSA locus (SSA1)

    SciTech Connect

    Tsugu, H.; Horowitz, R.; Gibson, N.

    1994-12-01

    Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exons were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race. 41 refs., 4 figs., 2 tabs.

  20. 40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle.

    PubMed

    Sirenko, V V; Simonyan, A H; Dobrzhanskaya, A V; Shelud'ko, N S; Borovikov, Y S

    2013-03-01

    Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

  1. Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form.

    PubMed

    Martín, Mariana; Rius, Sebastián Pablo; Podestá, Florencio Esteban

    2011-06-01

    Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.

  2. Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland.

    PubMed

    Abe, T; Nakabayashi, H; Tamada, H; Takagi, T; Sakuragi, S; Yamaki, K; Shinohara, T

    1990-07-16

    A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland.

  3. Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores.

    PubMed Central

    Remes, J J; Petäjä-Repo, U E; Rajaniemi, H J

    1991-01-01

    Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of

  4. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  5. Vaccinia virus kelch protein A55 is a 64 kDa intracellular factor that affects virus-induced cytopathic effect and the outcome of infection in a murine intradermal model.

    PubMed

    Beard, Philippa M; Froggatt, Graham C; Smith, Geoffrey L

    2006-06-01

    The vaccinia virus (VACV) protein A55 is a BTB/kelch protein with a broad-complex, tramtrack and bric-a-brac (BTB) domain in the N-terminal region and five kelch repeats in the C-terminal half. The BTB/kelch subgroup of the kelch superfamily of proteins has been associated with a wide variety of functions including regulation of the cytoskeleton. VACV contains three genes predicted to encode BTB/kelch proteins: A55R, F3L and C2L. The A55R gene product has been identified as an intracellular protein of 64 kDa that is expressed late in infection. A VACV strain lacking 93.6% of the A55R open reading frame (vdeltaA55) was constructed and found to have an unaltered growth rate in vivo but a different plaque morphology and cytopathic effect, as well as reduced development of VACV-induced Ca2+-independent cell/extracellular matrix adhesion. In a murine intradermal model of VACV infection, a virus lacking the A55R gene induced larger lesions than wild-type and revertant control viruses.

  6. Further Characterization of Complement Regulator-Acquiring Surface Proteins of Borrelia burgdorferi

    PubMed Central

    Kraiczy, Peter; Skerka, Christine; Brade, Volker; Zipfel, Peter F.

    2001-01-01

    The three genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B. afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674–1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived from B. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37°C compared with those cultured at 20°C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates. PMID:11705962

  7. Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse.

    PubMed

    Herrera-Herrera, Jesús Antonio; Pérez-Avalos, Odilia; Salgado, Luis M; Ponce-Noyola, Teresa

    2009-10-01

    Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.

  8. Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

    PubMed Central

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

    2014-01-01

    In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. PMID:24784323

  9. Major isoform of zebrafish P0 is a 23.5 kDa myelin glycoprotein expressed in selected white matter tracts of the central nervous system.

    PubMed

    Bai, Qing; Sun, Ming; Stolz, Donna B; Burton, Edward A

    2011-06-01

    The zebrafish mpz gene, encoding the ortholog of mammalian myelin protein zero, is expressed in oligodendrocytes of the zebrafish central nervous system (CNS). The putative gene product, P0, has been implicated in promoting axonal regeneration in addition to its proposed structural functions in compact myelin. We raised novel zebrafish P0-specific antibodies and established that P0 is a 23.5 kDa glycoprotein containing a 3 kDa N-linked carbohydrate moiety. P0 was localized to myelin sheaths surrounding axons, but was not detected in the cell bodies or proximal processes of oligodendrocytes. Many white matter tracts in the adult zebrafish CNS were robustly immunoreactive for P0, including afferent visual and olfactory pathways, commissural and longitudinal tracts of the brain, and selected ascending and descending tracts of the spinal cord. P0 was first detected during development in premyelinating oligodendrocytes of the ventral hindbrain at 48 hours postfertilization (hpf). By 72 hpf, short segments of longitudinally oriented P0-immunoreactive myelinating axons were seen in the hindbrain; expression in the spinal cord, optic pathways, hindbrain commissures, midbrain, and peripheral nervous system followed. The mpz transcript was found to be alternatively spliced, giving rise to P0 isoforms with alternative C-termini. The 23.5 kDa isoform was most abundant in the CNS, but other isoforms predominated in the myelin sheath surrounding the Mauthner axon. These data provide a detailed account of P0 expression and demonstrate novel P0 isoforms, which may have discrete functional properties. The restriction of P0 immunoreactivity to myelin sheaths indicates that the protein is subject to stringent intracellular compartmentalization, which likely occurs through posttranslational mechanisms.

  10. Overexpression in E. coli of the complete petH gene product from Anabaena: purification and properties of a 49 kDa ferredoxin-NADP+ reductase.

    PubMed

    Martínez-Júlvez, M; Hurley, J K; Tollin, G; Gómez-Moreno, C; Fillat, M F

    1996-10-17

    The complete petH gene product from Anabaena PCC 7119 has been overexpressed in E. coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP+ reductase with its substrates. The intact 49 kDa FNR can be easily purified in a two-step procedure using batch extraction with DEAE-cellulose followed by Cibacron blue-Sepharose chromatography of the proteins unbound to DEAE. Isoelectric focusing of FNR shows several forms, with the major band at pH 6.26. The presence of the N-terminal extension increases the K(m) of FNR for NADPH by 4-fold and by 16.4-fold in the reduction reactions of DCPIP and cytochrome c. However, the K(m) for ferredoxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR than with the 36 kDa protein. This indicates that the presence of the third domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension. Comparable rate constants for both enzymes, were obtained for the photoreduction of NADP+ using photosynthetic membranes and also using rapid kinetic techniques. Slightly different ionic strength dependences of the rate constants were obtained, nevertheless, for both forms of the enzyme. These are a consequence of the structural differences that the proteins show at the N-terminal and of their effect on the interaction with ferredoxin.

  11. Draft genome sequences of two Bacillus thuringiensis strains and characterization of a putative 41.9-kDa insecticidal toxin.

    PubMed

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

    2014-04-30

    In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein's target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.

  12. A single residue in the 126-kDa protein of pepper mild mottle virus controls the severity of symptoms on infected green bell pepper plants.

    PubMed

    Ichiki, T U; Nagaoka, E N; Hagiwara, K; Sasaya, T; Omura, T

    2009-01-01

    Infectious cDNA clones originally derived from a mild strain of Pepper mild mottle virus were constructed by replacing residue 649, a critical point for attenuation of this virus, with all possible amino acids. All clones were infectious to pepper plants and induced a variety of symptoms, including no visible symptoms. The results of this study showed that a single amino acid mutation at residue 649 could control the function of the 126- and 183-kDa proteins, replicases with multiple roles in the life cycle of this virus.

  13. Enhancement of angiogenesis by a 27 kDa lectin from perivitelline fluid of horseshoe crab embryos through upregulation of VEGF and its receptor.

    PubMed

    Surekha, K L; Waghchoude, Meenal; Ghaskadbi, Surendra

    2013-01-25

    Angiogenesis, the expansion of a capillary network, is implicated in several pathological conditions. Drug-based inhibition of angiogenesis is being explored as therapy. Conversely, therapeutic angiogenesis contributes to control conditions such as ischemia. Here we report pro-angiogenic activity of perivitelline fluid (PVF) from Indian horseshoe crab embryos and one of its purified fractions, a 27 kDa lectin, using the chick embryonic chorioallantoic membrane assay. Enhancement in number and diameter of blood vessels after treatment with PVF and lectin suggested their pro-angiogenic effect. Quantitative RT-PCR showed that this effect is mediated through modulation of expression of VEGF and VEGFR-2/kinase domain receptor genes.

  14. Nucleoporin 35 regulates cardiomyocyte pH homeostasis by controlling Na+-H+ exchanger-1 expression.

    PubMed

    Xu, Liang; Pan, Lei; Li, Jun; Huang, Bijun; Feng, Jing; Li, Changming; Wang, Shiyi; The, Erlinda; Liu, Yuan; Yuan, Tianyou; Zhen, Lixiao; Liang, Dandan; Liu, Yi; Li, Li; Cui, Yingyu; Jiang, Xiaoyan; Peng, Luying; Chen, Yi-Han

    2015-10-01

    The mammalian nuclear pore complex is comprised of ∼ 30 different nucleoporins (Nups). It governs the nuclear import of gene expression modulators and the export of mRNAs. In cardiomyocytes, Na(+)-H(+) exchanger-1 (NHE1) is an integral membrane protein that exclusively regulates intracellular pH (pHi) by exchanging one intracellular H(+) for one extracellular Na(+). However, the role of Nups in cardiac NHE1 expression remains unknown. We herein report that Nup35 regulates cardiomyocyte NHE1 expression by controlling the nucleo-cytoplasmic trafficking of nhe1 mRNA. The N-terminal domain of Nup35 determines nhe1 mRNA nuclear export by targeting the 5'-UTR (-412 to -213 nt) of nhe1 mRNA. Nup35 ablation weakens the resistance of cardiomyocytes to an acid challenge by depressing NHE1 expression. Moreover, we identify that Nup35 and NHE1 are simultaneously downregulated in ischemic cardiomyocytes both in vivo and in vitro. Enforced expression of Nup35 effectively counteracts the anoxia-induced intracellular acidification. We conclude that Nup35 selectively regulates cardiomyocyte pHi homeostasis by posttranscriptionally controlling NHE1 expression. This finding reveals a novel regulatory mechanism of cardiomyocyte pHi, and may provide insight into the therapeutic strategy for ischemic cardiac diseases.

  15. Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression

    PubMed Central

    Wang, Peng; Galan, Jacob A.; Normandin, Karine; Bonneil, Éric; Hickson, Gilles R.; Roux, Philippe P.; Thibault, Pierre

    2013-01-01

    Cell division requires the coordination of critical protein kinases and phosphatases. Greatwall (Gwl) kinase activity inactivates PP2A-B55 at mitotic entry to promote the phosphorylation of cyclin B–Cdk1 substrates, but how Gwl is regulated is poorly understood. We found that the subcellular localization of Gwl changed dramatically during the cell cycle in Drosophila. Gwl translocated from the nucleus to the cytoplasm in prophase. We identified two critical nuclear localization signals in the central, poorly characterized region of Gwl, which are required for its function. The Polo kinase associated with and phosphorylated Gwl in this region, promoting its binding to 14-3-3ε and its localization to the cytoplasm in prophase. Our results suggest that cyclin B–Cdk1 phosphorylation of Gwl is also required for its nuclear exclusion by a distinct mechanism. We show that the nucleo-cytoplasmic regulation of Gwl is essential for its functions in vivo and propose that the spatial regulation of Gwl at mitotic entry contributes to the mitotic switch. PMID:23857770

  16. Genetic Regulation of Acquired Immune Responses to Antigens of Mycobacterium tuberculosis: a Study of Twins in West Africa

    PubMed Central

    Jepson, Annette; Fowler, Amanda; Banya, Winston; Singh, Mahavir; Bennett, Steve; Whittle, Hilton; Hill, Adrian V. S.

    2001-01-01

    The role of genetic factors in clinical tuberculosis is increasingly recognized; how such factors regulate the immune response to Mycobacterium tuberculosis in healthy individuals is unclear. In this study of 255 adult twin pairs residing in The Gambia, West Africa, it is apparent that memory T-cell responses to secreted mycobacterial antigens (85-kDa antigen complex, “short-term culture filtrate,” and peptides from the ESAT-6 protein), as well as to the 65-kDa heat shock protein, are subject to effective genetic regulation. The delayed hypersensitivity response to intradermal tuberculin also demonstrates significant genetic variance, while quantitative T-cell and antibody responses to the 38-kDa cell membrane protein appear to be determined largely by environmental factors. Such findings have implications for vaccine development. PMID:11349068

  17. Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ

    PubMed Central

    Cuéllar, Jorge; Perales-Calvo, Judit; Muga, Arturo; Valpuesta, José María; Moro, Fernando

    2013-01-01

    Hsp40 chaperones bind and transfer substrate proteins to Hsp70s and regulate their ATPase activity. The interaction of Hsp40s with native proteins modifies their structure and function. A good model for this function is DnaJ, the bacterial Hsp40 that interacts with RepE, the repressor/activator of plasmid F replication, and together with DnaK regulates its function. We characterize here the structure of the DnaJ-RepE complex by electron microscopy, the first described structure of a complex between an Hsp40 and a client protein. The comparison of the complexes of DnaJ with two RepE mutants reveals an intrinsic plasticity of the DnaJ dimer that allows the chaperone to adapt to different substrates. We also show that DnaJ induces conformational changes in dimeric RepE, which increase the intermonomeric distance and remodel both RepE domains enhancing its affinity for DNA. PMID:23580641

  18. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

    PubMed Central

    Ramprasad, M P; Fischer, W; Witztum, J L; Sambrano, G R; Quehenberger, O; Steinberg, D

    1995-01-01

    We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568176

  19. Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues

    SciTech Connect

    Ota, I.M.; Clarke, S. )

    1989-08-05

    A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase. To determine the site(s) of methylation, {sup 3}H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins.

  20. Activated Ki-Ras complements erythropoietin signaling in CTLL-2 cells, inducing tyrosine phosphorylation of a 160-kDa protein.

    PubMed Central

    Yamamura, Y; Noda, M; Ikawa, Y

    1994-01-01

    We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3-dependent pro-B-cell lines, such as BAF-B03, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth. Images PMID:7522324

  1. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    SciTech Connect

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated /sup 32/P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. /sup 32/P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.

  2. Six-months pain relief and functional recovery after intra-articular injections with hyaluronic acid (mw 500–730 KDa) in trapeziometacarpal osteoarthritis

    PubMed Central

    Frizziero, Antonio; Maffulli, Nicola; Masiero, Stefano; Frizziero, Luigi

    2014-01-01

    Summary Background: this retrospective open label study evaluates the efficacy and tolerability of intra-articular injections of Hyaluronic Acid (HA) (MW 500–730 KDa - Hyalgan®) for the treatment of pain and disability of trapeziometacarpal joint osteoarthritis (TMCJ OA). Methods: fifty eight patients, 50 females (86%) and 8 male (14%), aged between 40–75 years, suffering from TMCJ OA according to Kellgren-Laurence grades 2–3 on standard plain radiography, were included. Patients with known inflammatory arthritis, previous thumb trauma and intra-articular (i.a.) injections with corticosteroids were excluded. Primary endpoints were: pain (VAS), NSAID intake, radial and palmar ab-/adduction, pinch strength. All patients received an i.a. injection of 0.8 mL of HA (MW 500–730 KDa) once weekly for three weeks. Control examinations were carried out at 1, 3, and 6 months. Results: intra-articular HA injections have significantly reduced spontaneous and provoked pain and improved hand mobility. At 1,3, and 6 months from baseline, the spontaneous and provoked pain revealed a statistically significant improvement (p<0,0001). NSAID’s intake evidenced a statistically significant reduction against baseline (p<0.017). The adverse events (21%) were related to local symptoms such as pain during or following HA administration. Conclusions: this study shows that i.a. HA injections for TMCJ OA can induce a significant improvement of function associated to stiffness decrease and pain relief. PMID:25332944

  3. Selective response of dermal Th-1 cells to 20-50 kDa streptococcal cell-wall proteins in chronic plaque psoriasis.

    PubMed

    Baker, B S; Ovigne, J-M; Fischetti, V A; Powles, A; Fry, L

    2003-09-01

    We have recently described a dermal Th-1 subset in skin lesions of psoriasis which recognizes cell-wall extract isolated from group A streptococci (GAS). As a first step in the identification of the streptococcal proteins involved, dermal T-cell lines (TCL) cultured from the lesional skin of 12 human leucocyte antigen (HLA)-typed psoriasis patients were stimulated with GAS cell-wall extract and 14 fractions (MWt approximately 20-100 kDa) separated from the cell-wall extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution, stained for intracellular interferon-gamma(IFN-gamma) expression and analysed by flow cytometry. All the TCL responded to GAS cell-wall extract to varying extents (3.5-27.6% IFN-gamma+). This response was consistently directed against 20-50 kDa cell-wall fractions and inhibited by anti-HLA-DR antibody. TCL with higher responses to GAS cell-wall extract recognized a larger number of fractions within this range than the lower responder TCL. No difference between the level and pattern of response to the fractions was observed for TCL from HLA-DR7+ (n = 6) and HLA-DR7- (n = 6) individuals. This preliminary study has shown a selective response to lower MWt proteins expressed on GAS cell wall by skin Th-1 cells in psoriasis. Further studies are required to identify the proteins involved.

  4. Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

    PubMed

    Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

    2012-01-01

    Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

  5. Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein.

    PubMed Central

    Flaherty, K M; McKay, D B; Kabsch, W; Holmes, K C

    1991-01-01

    Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction. Images PMID:1828889

  6. Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55).

    PubMed

    Govind, Anitha P; Sreeja, S; Thampan, Raghava Varman

    2003-05-01

    The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein. A conformational change induced by estrogen binding is thought to favor the anchoring process. (2) The anchoring of E-RAF by ap55 is mediated by yet another protein. This is the 66 kDa transport protein (tp66) which recognizes ap55 on the one hand and E-RAF on the other. The presence of estradiol that saturates the hormone binding sites on ap55 appears to favor the anchoring of tp66-E-RAF complex to ap55. This interaction appears to be weakened by levels of estradiol below 7 nM concentration leading to the dissociation of the tp66-E-RAF complex from ap55. The tp66-E-RAF complex moves towards the nucleus. PMID:12682911

  7. A Trichomonas vaginalis 120 kDa protein with identity to hydrogenosome pyruvate:ferredoxin oxidoreductase is a surface adhesin induced by iron.

    PubMed

    Moreno-Brito, Verónica; Yáñez-Gómez, Carmina; Meza-Cervantez, Patricia; Avila-González, Leticia; Rodríguez, Mario Alberto; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2005-02-01

    Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 microM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.

  8. Structural and electron-microscopic studies of jacalin from jackfruit (Artocarpus integrifolia) show that this lectin is a 65 kDa tetramer.

    PubMed

    Ruffet, E; Paquet, N; Frutiger, S; Hughes, G J; Jaton, J C

    1992-08-15

    The 133-amino-acid sequences of the alpha-subunit of jacalin (a lectin from Artocarpus integrifolia) and of the slightly larger alpha'-subunit were determined. The alpha'- and alpha-subunits, in the approximate ratio of 1:3, were found to be virtually identical in their primary structures, except for one valine for isoleucine substitution at position 113. Although both alpha'- and alpha-chains were glycosylated, the extent of glycosylation in the alpha'-chain was much greater than that in the alpha-subunit. In the alpha'-polypeptide, all molecules contained an N-linked oligosaccharide at position 74 and some contained sugar at position 43. The alpha- and alpha'-subunits were found to be strongly non-covalently associated with three distinct beta-subunits containing 20 amino acids each. Electron-microscopic visualization of native jacalin disclosed a structure composed of four alpha-type subunits with a clear-cut 4-fold symmetry. Analytical-ultracentrifugation studies of jacalin revealed an average molecular mass of 65 kDa, a value compatible with a tetrameric structure of the alpha(alpha')-subunits. The recalculated number of sugar-binding sites per jacalin molecule, given a molecular mass of 65 kDa, would yield 0.8 sites per alpha(alpha')-promoter, i.e. about twice the value previously determined [Appukutan & Basu (1985) FEBS Lett. 180, 331-334; Ahmed & Chatterjee (1989) J. Biol. Chem. 264, 9365-9372].

  9. Characterization and chromosomal assignment of a human cDNA encoding a protein related to the murine 102-kDa cadherin-associated protein ([alpha]-catenin)

    SciTech Connect

    Claverie, J.M. ); Hardelin, J.P.; Legouis, R.; Levilliers, J.; Petit, C. ); Bougueleret, L. ); Mattei, M.G. )

    1993-01-01

    We report the characterization of a human cDNA encompassing the complete coding region of a 945-residue putative protein (CAP-R) 80% identical to the recently described murine 102-kDa [alpha]-catenin (CAP102). The CAP-R protein mostly differs from CAP102 by the presence of a 48-residue insert. This insert exhibits similarity with a segment of the type 1 neurofibromatosis gene product. The analysis of a publicly available human [open quote]expressed sequence tag[close quotes] collection revealed the existence of another human cDNA more closely related (89% identical) to CAP 102. This strongly suggests that CAP-R is not the human homologue of the murine 102- kDa [alpha]-catenin but a new closely related gene of the vinculin family. This is further supported by the computed mutation rates falling outside the range observed for mammalian orthologous genes. Using in situ hybridization, the CAP-R gene could be mapped to the pll.l-pl2 region of human chromosome 2 and to the homologous B3-D region of mouse chromosome 6. 32 refs., 4 fig.

  10. A novel type of crystallin in the frog eye lens. 35-kDa polypeptide is not homologous to any of the major classes of lens crystallins.

    PubMed

    Tomarev, S I; Zinovieva, R D; Dolgilevich, S M; Luchin, S V; Krayev, A S; Skryabin, K G; Gause, G G

    1984-06-11

    The nucleotide sequence of a cloned DNA coding for the 35-kDa polypeptide of the eye lens of the frog (Rana temporaria) has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins: alpha-, beta-, gamma- or delta-crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3'-non-translated region with the polyadenylation signal sequence and a poly(A) tract; the 5'-non-translated region is lost along with part of the coding region; this accounts for about 1/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin - Finkelstein method shows the presence of predominantly beta-strands with only a few alpha-helical regions. We conclude that the 35-kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name epsilon-crystallin.

  11. Diagnostic potential of Fasciola gigantica-derived 14.5 kDa fatty acid binding protein in the immunodiagnosis of bubaline fascioliasis.

    PubMed

    Allam, G; Bauomy, I R; Hemyeda, Z M; Diab, T M; Sakran, T F

    2013-06-01

    The 14.5 kDa fatty acid binding protein (FABP) was isolated from the crude extract of adult Fasciola gigantica worms. Polyclonal anti-FABP IgG was generated in rabbits immunized with prepared FABP antigen. Sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect coproantigen in stools and circulating Fasciola antigen (CA) in sera of 126 water buffaloes by using purified and horseradish peroxidase (HRP)-conjugated anti-FABP IgG. Sandwich ELISA sensitivity was 96.97% and 94.95%; while specificity was 94.12% and 82.35% for coproantigen and CA detection, respectively. However, sensitivity and specificity of the Kato-Katz technique was 73.74% and 100%, respectively. The diagnostic efficacy of sandwich ELISA was 96.55% and 93.1% for coproantigen and CA detection, respectively. In contrast, the diagnostic efficacy of the Kato-Katz technique was 77.59%. In conclusion, these results demonstrate that the purified 14.5 kDa FABP provides a more suitable antigen for immunodiagnosis of early and current bubaline fascioliasis by using sandwich ELISA.

  12. Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

    SciTech Connect

    Nakayama, Tokiko; Nishizawa, Kimiko; Sato, Chicako )

    1988-08-01

    The authors previously reported that treatment of SV-3Y1 cells in an exponential growth state with db-cAMP plus theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody. In the present study, the authors examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-Map-1 and -2 antibodies. Treatment with db-cAMP plus theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with colchicine, colcemid, putrescine, or {alpha}-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. They examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and {sup 125}I-protein A with anti-MAP-1 and -2 antibodies. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

  13. Regulation of gene expression by the RNA-binding protein Sam68 in cancer.

    PubMed

    Rajan, Prabhakar; Gaughan, Luke; Dalgliesh, Caroline; El-Sherif, Amira; Robson, Craig N; Leung, Hing Y; Elliott, David J

    2008-06-01

    Sam68 (Src-associated in mitosis 68 kDa) is the prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. Sam68 is implicated in a number of cellular processes including signal transduction, transcription, RNA metabolism, cell cycle regulation and apoptosis. In the present review, we summarize the functions of Sam68 as a transcriptional and post-transcriptional regulator of gene expression, with particular relevance to cancer. PMID:18481990

  14. The 37/67kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67kDa LR cell surface localization and interaction with the cellular prion protein

    PubMed Central

    Sarnataro, Daniela; Pepe, Anna; Altamura, Gennaro; De Simone, Imma; Pesapane, Ada; Nitsch, Lucio; Montuori, Nunzia; Lavecchia, Antonio; Zurzolo, Chiara

    2016-01-01

    The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrPC) and to be required for pathological PrP (PrPSc) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrPC interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrPC in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrPC. Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrPC interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrPC on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrPC and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases. PMID:27071549

  15. Cell cycle regulation of human WEE1.

    PubMed Central

    McGowan, C H; Russell, P

    1995-01-01

    WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. WEE1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating WEE1. WEE1 activity is strongly suppressed during M phase, suggesting that negative regulation of WEE1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase WEE1 is re-activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that WEE1 is inhibited by a mechanism that requires protein phosphorylation. Images PMID:7774574

  16. An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis.

    PubMed

    Sugliani, Matteo; Abdelkefi, Hela; Ke, Hang; Bouveret, Emmanuelle; Robaglia, Christophe; Caffarri, Stefano; Field, Ben

    2016-03-01

    The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development.

  17. The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei.

    PubMed

    Brasseur, Anaïs; Bayat, Shima; Chua, Xiu Ling; Zhang, Yu; Zhou, Qing; Low, Boon Chuan; He, Cynthia Y

    2014-11-15

    Cilia and flagella are conserved eukaryotic organelles important for motility and sensory. The RanGTPase, best known for nucleocytoplasmic transport functions, may also play a role in protein trafficking into the specialized flagellar/ciliary compartments, although the regulatory mechanisms controlling Ran activity at the flagellum remain unclear. The unicellular parasite Trypanosoma brucei contains a single flagellum necessary for cell movement, division and morphogenesis. Correct flagellum functions require flagellar attachment to the cell body, which is mediated by a specialized flagellum attachment zone (FAZ) complex that is assembled together with the flagellum during the cell cycle. We have previously identified the leucine-rich-repeat protein 1 LRRP1 on a bi-lobe structure at the proximal base of flagellum and FAZ. LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently affecting flagellum-driven cell motility and division. Here, we show that LRRP1 forms a complex with Ran and a Ran-binding protein, and regulates Ran-GTP hydrolysis in T. brucei. In addition to mitotic inhibition, depletion of Ran inhibits FAZ assembly in T. brucei, supporting the presence of a conserved mechanism that involves Ran in the regulation of flagellum functions in an early divergent eukaryote. PMID:25217630

  18. The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei.

    PubMed

    Brasseur, Anaïs; Bayat, Shima; Chua, Xiu Ling; Zhang, Yu; Zhou, Qing; Low, Boon Chuan; He, Cynthia Y

    2014-11-15

    Cilia and flagella are conserved eukaryotic organelles important for motility and sensory. The RanGTPase, best known for nucleocytoplasmic transport functions, may also play a role in protein trafficking into the specialized flagellar/ciliary compartments, although the regulatory mechanisms controlling Ran activity at the flagellum remain unclear. The unicellular parasite Trypanosoma brucei contains a single flagellum necessary for cell movement, division and morphogenesis. Correct flagellum functions require flagellar attachment to the cell body, which is mediated by a specialized flagellum attachment zone (FAZ) complex that is assembled together with the flagellum during the cell cycle. We have previously identified the leucine-rich-repeat protein 1 LRRP1 on a bi-lobe structure at the proximal base of flagellum and FAZ. LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently affecting flagellum-driven cell motility and division. Here, we show that LRRP1 forms a complex with Ran and a Ran-binding protein, and regulates Ran-GTP hydrolysis in T. brucei. In addition to mitotic inhibition, depletion of Ran inhibits FAZ assembly in T. brucei, supporting the presence of a conserved mechanism that involves Ran in the regulation of flagellum functions in an early divergent eukaryote.

  19. Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity.

    PubMed

    Boal, Frederic; Roumegoux, Jessica; Alfarano, Chiara; Timotin, Andrei; Calise, Denis; Anesia, Rodica; Drougard, Anne; Knauf, Claude; Lagente, Christine; Roncalli, Jerome; Desmoulin, Franck; Tronchere, Helene; Valet, Philippe; Parini, Angelo; Kunduzova, Oksana

    2015-11-06

    The increasing incidence of obesity accentuates the importance of identifying mechanisms and optimal therapeutic strategies for patients with heart failure (HF) in relation to obesity status. Here, we investigated the association between plasma level of apelin, an adipocyte-derived factor, and clinicopathological features of obese and non-obese patients with HF. We further explored potential regulatory mechanisms of cardiac cell fate responses in conditions combining myocardial injury and obesity. In a prospective, cross-sectional study involving patients with HF we show that obese patients (BMI ≥ 30 kg/m(2)) have higher left ventricular ejection fraction (LVEF) and greater levels of plasma apelin (p < 0.005) than non-obese patients (< 30 kg/m(2)), independently of ischemic etiology. In a mouse model combining ischemia-reperfusion (I/R) injury and high-fat diet (HFD)-induced obesity, we identify apelin as a novel regulator of FoxO3 trafficking in cardiomyocytes. Confocal microscopy analysis of cardiac cells revealed that apelin prevents nuclear translocation of FoxO3 in response to oxygen deprivation through a PI3K pathway. These findings uncover apelin as a novel regulator of FoxO3 nucleocytoplasmic trafficking in cardiac cells in response to stress and provide insight into its potential clinical relevance in obese patients with HF.

  20. Characterization of the binding between a 70-kDa heat shock protein, HspA1A, and phosphoinositides.

    PubMed

    McCallister, Chelsea; Kdeiss, Brianna; Oliverio, Ryan; Nikolaidis, Nikolas

    2016-03-25

    HspA1A, a seventy-kilodalton heat shock protein, binds to specific anionic lipids and this interaction regulates important physiological phenomena like apoptosis, tumor growth, and lysosomal rescue. However, whether HspA1A binds to phosphoinositides has yet to be established and quantified. Therefore, in this study, we determined the binding affinity of HspA1A to several phosphoinositides and characterized five aspects of their molecular interaction. First, we established that HspA1A binds phosphatidylinositol monophosphates with higher affinity than di- and triphosphorylated inositides. Second, using high concentrations of potassium we found that HSPA1A embeds within the lipid bilayer of all phosphoinositides tested. However, the effects of the high salt concentrations were significantly different between the different phosphoinositides. Third, using calcium and reaction buffers equilibrated at different pH values we found that these differentially affected HspA1A-phosphoinositide binding, revealing a lipid-specific pattern of binding. Fourth, by assessing the binding properties of the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, we determined that in most cases the full-length protein is necessary for binding to phosphoinositides. Fifth, by including in the reactions nucleotides and protein substrates we determined that they minimally and differentially affected phosphoinositide-binding. Collectively, these findings strongly suggest that the HspA1A-phosphoinositide binding is complex yet specific, is mediated by both electrostatic and hydrophobic interactions, is not related to the lipid-head charge, and depends on the physicochemical properties of the lipid. PMID:26923070

  1. Characterization of the binding between a 70-kDa heat shock protein, HspA1A, and phosphoinositides.

    PubMed

    McCallister, Chelsea; Kdeiss, Brianna; Oliverio, Ryan; Nikolaidis, Nikolas

    2016-03-25

    HspA1A, a seventy-kilodalton heat shock protein, binds to specific anionic lipids and this interaction regulates important physiological phenomena like apoptosis, tumor growth, and lysosomal rescue. However, whether HspA1A binds to phosphoinositides has yet to be established and quantified. Therefore, in this study, we determined the binding affinity of HspA1A to several phosphoinositides and characterized five aspects of their molecular interaction. First, we established that HspA1A binds phosphatidylinositol monophosphates with higher affinity than di- and triphosphorylated inositides. Second, using high concentrations of potassium we found that HSPA1A embeds within the lipid bilayer of all phosphoinositides tested. However, the effects of the high salt concentrations were significantly different between the different phosphoinositides. Third, using calcium and reaction buffers equilibrated at different pH values we found that these differentially affected HspA1A-phosphoinositide binding, revealing a lipid-specific pattern of binding. Fourth, by assessing the binding properties of the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, we determined that in most cases the full-length protein is necessary for binding to phosphoinositides. Fifth, by including in the reactions nucleotides and protein substrates we determined that they minimally and differentially affected phosphoinositide-binding. Collectively, these findings strongly suggest that the HspA1A-phosphoinositide binding is complex yet specific, is mediated by both electrostatic and hydrophobic interactions, is not related to the lipid-head charge, and depends on the physicochemical properties of the lipid.

  2. Tissue distribution of HSPA9/mortalin in avian species and its regulation by gender, genotype and heat stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat shock 70kDa protein 9 (HSPA9)/mortalin is a multipotent chaperone regulating cellular processes ranging from stress response to energy homeostasis. HSPA9 has been extensively studied in mammals however there is a paucity of information in avian species. The present study aimed to characterize H...

  3. The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

    PubMed

    Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H; Dickson, Claire F; Fabian, Marian; Olson, John S; Gell, David A; Clubb, Robert T

    2016-03-27

    Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

  4. Enhancement of angiogenesis by a 27 kDa lectin from perivitelline fluid of horseshoe crab embryos through upregulation of VEGF and its receptor.

    PubMed

    Surekha, K L; Waghchoude, Meenal; Ghaskadbi, Surendra

    2013-01-25

    Angiogenesis, the expansion of a capillary network, is implicated in several pathological conditions. Drug-based inhibition of angiogenesis is being explored as therapy. Conversely, therapeutic angiogenesis contributes to control conditions such as ischemia. Here we report pro-angiogenic activity of perivitelline fluid (PVF) from Indian horseshoe crab embryos and one of its purified fractions, a 27 kDa lectin, using the chick embryonic chorioallantoic membrane assay. Enhancement in number and diameter of blood vessels after treatment with PVF and lectin suggested their pro-angiogenic effect. Quantitative RT-PCR showed that this effect is mediated through modulation of expression of VEGF and VEGFR-2/kinase domain receptor genes. PMID:23316979

  5. Antibody against the C-terminal portion of dystrophin crossreacts with the 400 kDa protein in the pia mater of dystrophin-deficient mdx mouse brain.

    PubMed

    Ishiura, S; Arahata, K; Tsukahara, T; Koga, R; Anraku, H; Yamaguchi, M; Kikuchi, T; Nonaka, I; Sugita, H

    1990-04-01

    The mdx mouse is an animal model for X-linked Duchenne muscular dystrophy. A polyclonal antibody against a synthetic peptide IV equivalent to the C-terminal portion (amino acids 3495-3544) of dystrophin crossreacted with a 400 kDa protein in the brain and the spinal cord of mdx mouse, as well as in the control B10 mouse. However, the protein did not crossreact with the polyclonal antibody raised against the N-terminal portion of dystrophin peptide I (amino acids 215-264). Immunofluorescent micrography revealed that the outside of the small arteries and the pia mater of the brain strongly reacted with the anti-peptide IV antibody. These results strongly suggest the presence of a crossreactive protein other than dystrophin, possibly a dystrophin-related autosomal gene product, in the pia mater.

  6. Expression and characterization of a new isoform of the 9 kDa allergenic lipid transfer protein from tomato (variety San Marzano).

    PubMed

    Volpicella, Mariateresa; Leoni, Claudia; Fanizza, Immacolata; Rinalducci, Sara; Placido, Antonio; Ceci, Luigi R

    2015-11-01

    Lipid transfer proteins (LTPs) are food allergens found first in fruits of the Rosaceae family and later identified in other food plants. Their high structural stability causes them to behave as allergens in cooked and processed foods. Allergenic LTPs have been identified in tomato fruits as well, but studies of their thermal stability and structural characteristics are limited. In this article we report the identification of the coding region for a novel 9 kDa LTP isoform in the tomato variety San Marzano, together with the expression of the recombinant mature protein. The purified recombinant protein was further characterized for its thermal stability and was found to bind 1-palmitoil-2-lysophosphatidylcholine (Lyso-C16) after thermal treatments up to 105 °C. Analysis of a modeling derived structure of the protein allowed the identification of possible epitope regions on the molecular surface. PMID:26232648

  7. Eosinophilic granuloma of bone and biochemical demonstration of 49-kDa CD1a molecule expression by Langerhans-cell histiocytosis.

    PubMed

    Cambazard, F; Dezutter-Dambuyant, C; Staquet, M J; Schmitt, D; Thivolet, J

    1991-09-01

    Histiocytic cells infiltrating the lesions in eosinophilic granuloma of bone as well as in cutaneous histiocytosis X were studied using a murine monoclonal antibody (MA) produced with proliferating cells from an eosinophilic granuloma of bone. This MA reacts with Langerhans cells (LC) of normal human skin or mucous membranes and with proliferating cells of eosinophilic granuloma of bone and skin lesions of Letter-Siwe disease, as shown by immunohistochemistry and immunogold labelling. As other murine MA's obtained after immunization with human cortical thymocytes, this MA immunoprecipitates the 49-kDa CD1a antigen found on human LC and thymic-cell surfaces but not its breakdown product after treatment with trypsin, as demonstrated by analysis of immunoelectron labelling, cytofluorometry and gel electrophoresis. This first production of a CD1a MA from an eosinophilic granuloma supports the concept of Langerhans-cell histiocytosis.

  8. [Effects of noopept and cortexin on the behavior of matured rats treated with corticoliberin or 70-kDa heat shock proteins in early ontogeny].

    PubMed

    Shabanov, P D; Lebedev, A A; Stetsenko, V P; Lavrov, N V; Sablina, G V; Gudasheva, T A; Ostrovaskaia, R U

    2007-01-01

    Young Wistar rats aged 4 days were injected intraperitoneally with corticotropin releasing hormone (CRH), which is an agent activating the stress system, or 70-kDa heat shock proteins (HSP-70)--intracellular shaperons, possessing antistress properties. In grown adult rats aged 90-100 days, the effects of nootropic drugs noopept and cortexin (1 mg/kg, i.p.) were assessed. The activation of stress or antistress systems with CRH or HSP-70 significantly altered the drug action. The effects were different in males and females and depended on animal gender. The spectrum of pharmacological activity of noopept and cortexin changed: noopept demonstrated preferable psychoactivating and antiaggressive effects, whereas cortexin showed mild anxiolytic and antidepressant activity. It is suggested that the behavioral effects of nootropes depend on the conditions of the stress system formation in early ontogeny. PMID:17402584

  9. The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein

    PubMed Central

    Park, Eunkyung; Choi, Youngbong; Ahn, Eunseon; Park, Inyoung

    2009-01-01

    The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin α6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration. PMID:19561400

  10. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    SciTech Connect

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P.

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  11. Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104

    SciTech Connect

    Lee,S.; Tsai, F.

    2007-01-01

    A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 {angstrom} resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 {angstrom}. Native 2 diffracted to 2.9 {angstrom} resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 {angstrom}. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.

  12. Crystallization and preliminary X-ray crystallographic analysis of a 40 kDa N-terminal fragment of the yeast prion-remodeling factor Hsp104

    SciTech Connect

    Lee, Sukyeong; Tsai, Francis T. F.

    2007-09-01

    An N-terminal fragment of S. cerevisiae Hsp104 has been crystallized. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones. A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 Å. Native 2 diffracted to 2.9 Å resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 Å. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.

  13. Characterization Based on the 56-Kda Type-Specific Antigen Gene of Orientia tsutsugamushi Genotypes Isolated from Leptotrombidium Mites and the Rodent Host Post-Infection

    PubMed Central

    Takhampunya, Ratree; Tippayachai, Bousaraporn; Promsathaporn, Sommai; Leepitakrat, Surachai; Monkanna, Taweesak; Schuster, Anthony L.; Melendrez, Melanie C.; Paris, Daniel H.; Richards, Allen L.; Richardson, Jason H.

    2014-01-01

    Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector. PMID:24297814

  14. Purification of a 240 kDa protein from serum and follicular fluid of water buffalo and its identification as haptoglobin.

    PubMed

    Bergamo, P; Balestrieri, M; Carratore, V; Abrescia, P

    1995-04-15

    The fluids from healthy growing follicles of water buffalo were previously found free of the polypeptides H (M(r) 36,000) and L (M(r) 21,000) which were instead detected in fluids from atretic follicles and blood. Here we report evidence that these two polypeptides, as selected from serum by specific anti-L antibodies, are the subunits of an oligomeric protein. The protein was purified from serum or follicular fluid, and its molecular weight (240 kDa), isoelectric point (6.5), and amino acid composition were determined. The NH2-terminal sequences of the subunits L and H were analyzed: 100% and 90% homology with alpha and beta chains of bovine haptoglobin, respectively, was found. Thus, haptoglobin can be used as a novel molecular marker to assess the physiological state of the blood-follicle barrier or discriminate between atretic and healthy follicles.

  15. VOLTAGE REGULATOR

    DOEpatents

    Von Eschen, R.L.; Scheele, P.F.

    1962-04-24

    A transistorized voltage regulator which provides very close voitage regulation up to about 180 deg F is described. A diode in the positive line provides a constant voltage drop from the input to a regulating transistor emitter. An amplifier is coupled to the positive line through a resistor and is connected between a difference circuit and the regulating transistor base which is negative due to the difference in voltage drop across thc diode and the resistor so that a change in the regulator output causes the amplifier to increase or decrease the base voltage and current and incrcase or decrease the transistor impedance to return the regulator output to normal. (AEC)

  16. Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

    PubMed

    Tsai, S C; Adamik, R; Tsuchiya, M; Chang, P P; Moss, J; Vaughan, M

    1991-05-01

    Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.

  17. Ion Mobility Measurements of Nondenatured 12-150 kDa Proteins and Protein Multimers by Tandem Differential Mobility Analysis-Mass Spectrometry (DMA-MS)

    NASA Astrophysics Data System (ADS)

    Hogan, Christopher J.; de la Mora, Juan Fernández

    2011-01-01

    The mobilities of electrosprayed proteins and protein multimers with molecular weights ranging from 12.4 kDa (cytochrome C monomers) to 154 kDa (nonspecific concanavalin A hexamers) were measured in dry air by a planar differential mobility analyzer (DMA) coupled to a time-of-flight mass spectrometer (TOF-MS). The DMA determines true mobility at atmospheric pressure, without perturbing ion structure from that delivered by the electrospray. A nondenaturing aqueous 20 mM triethylammonium formate buffer yields compact ions with low charge states, moderating polarization effects on ion mobility. Conversion of mobilities into cross-sections involves a reduction factor ξ for the actual mobility relative to that associated with elastic specular collisions with smooth surfaces. ξ is known to be 1.36 in air from Millikan's oil drop experiments. A similar enhancement effect ascribed to atomic-scale surface roughness has been found in numerical simulations. Adopting Millikan's value ξ = 1.36 and assuming a spherical geometry yields a gas-phase protein density ρ p = 0.949 ± 0.053 g cm-3 for all our protein data. This is substantially higher than the 0.67 g cm-3 found in recent low-resolution DMA measurements of singly charged proteins. DMA-MS can distinguish nonspecific protein aggregates formed during the electrospray process from those formed preferentially in solution. The observed charge versus diameter relation is compatible with a protein charge reduction mechanism based on the evaporation of triethylammonium ions from electrosprayed drops.

  18. Lipid substitution on low molecular weight (0.6-2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression.

    PubMed

    Bahadur, K C Remant; Landry, Breanne; Aliabadi, Hamidreza Montazeri; Lavasanifar, Afsaneh; Uludağ, Hasan

    2011-05-01

    Cationic polymers are desirable gene carriers because of their better safety profiles than viral delivery systems. Low molecular weight (MW) polymers are particularly attractive, since they display little cytotoxicity, but they are also ineffective for gene delivery. To create effective carriers from low MW polymers palmitic acid (PA) was substituted on 0.6-2.0 kDa polyethylenimines (PEIs) and their efficiency for plasmid DNA (pDNA) delivery was evaluated. The extent of lipid substitution was dependent on the lipid/PEI feed ratio and the polymer MW. While the hydrodynamic size of the polymer/pDNA complexes (polyplexes) increased or decreased depending on the extent of lipid substitution, the ζ potential of the assembled complexes was consistently higher as a result of lipid substitution. Lipid substitution generally increased the in vitro toxicity of the PEIs, but it was significantly lower than that of the 25 kDa branched PEI. The in vitro transfection efficiency of the lipid-substituted polymers was higher than that of native PEIs, which were not at all effective. The delivery efficiency was proportional to the extent of lipid substitution as well as the polymer MW. This correlated with the increased uptake of lipid-substituted polyplexes, based on confocal microscopic investigations with FITC-labeled pDNA. The addition of chloroquine further increased the transfection efficiency of lipid-substituted PEIs, indicating that endosomal release was a limiting factor affecting the efficiency of these carriers. This study indicates that lipid substitution on low MW PEIs makes their assembly more effective, resulting in better delivery of pDNA into mammalian cells.

  19. Purification and characterization of a approximately 34 kDa antioxidant protein (beta-turmerin) from turmeric (Curcuma longa) waste grits.

    PubMed

    Smitha, S; Dhananjaya, B L; Dinesha, R; Srinivas, Leela

    2009-09-01

    Beta-turmerin from turmeric (Curcuma longa) waste grits obtained after extraction of curcumin was purified by successive gel permeation chromatography. Homogeneity of beta-turmerin was confirmed by its movement as single band both in SDS-PAGE and as well as in native (basic) PAGE. The apparent molecular mass is approximately 34 kDa by SDS-PAGE. It is more hydrophobic protein and showed sharp single peak in RP-HPLC with retention time of 62.17 min. It is a glycoprotein as it shows the presence of amino sugars up to 0.021 gm%. In three different model systems i.e., linolenic acid micelles, erythrocyte membrane systems and liposomes, beta-turmerin at 0.125 microM offered 70%, 64%, and 60% inhibition of lipid peroxidation, which is 3200 times more efficient than the standard antioxidants BHA (400 microM) and alpha-tocopherol (400 microM). beta-turmerin inhibited diene-triene and tetraene conjugation up to 54%, 72% and 47%, respectively. beta-turmerin also effectively scavenges hydroxyl radicals when compared to BHA and alpha-tocopherol. beta-turmerin (2.5 microM) further inhibited the activation of PMNL mediated by fMLP up to the extent of 75%, where as standards BHA (400 microM) and mannitol (10 microM) inhibited the same to 65% and 55%, respectively. At 0.125 microM dose beta-turmerin prevented t-BOOH induced cell death at all time intervals. In addition to the above properties, it is non-toxic to lymphocytes as it did not affect the viability of cells. The mechanism of antioxidant action of beta-turmerin could probably be by counteracting/quenching of reactive oxygen species (ROS). We report the purification and characterization of beta-turmerin ( approximately 34 kDa), a potent antioxidant protein from turmeric waste grits.

  20. Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins.

    PubMed

    Yamauchi, Yasuo; Sugimoto, Yukihiro

    2010-04-01

    Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSIIOEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSIIOEE were modified, but when only OEC33 or PSIIOEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40 degrees C but not at 25 degrees C. In spinach leaves treated at 40 degrees C under light, maximal efficiency of PSII photochemistry (F(v)/F(m) ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40 degrees C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.

  1. A low-molecular-weight (25-kDa) IGF-binding protein is increased with growth inhibition in the fasting striped bass, Morone saxatilis.

    PubMed

    Siharath, K; Kelley, K M; Bern, H A

    1996-06-01

    The effect of fasting on circulating IGFBPs in the striped bass was assessed in relation to changes in growth and metabolism. Thirty-day-fasted (30DF) and 60-day-fasted (60DF) fish, and 60DF fish refed for 14 additional days (REFED), were compared with control, fed fish. Growth and metabolic status of each animal were assessed by determining body length (BL) and body weight (BW) changes, hepatosomatic index (HSI), condition factor (CF), and serum glucose concentration, and by assaying for incorporation of [35S]sulfate (proteoglycan synthetic activity) and [3H]thymidine (mitotic activity) in ceratobranchial cartilage explants in vitro. Serum IGFBP concentrations were assessed by a Western ligand blot procedure using 125I-labeled human IGF-I tracer. Both 30DF and 60DF fish exhibited hypoglycemia and reduced HSI and CF, and their BL and BW growth rates were significantly inhibited. Strongly correlated with the inhibited body growth indices were significantly depressed levels of cartilage [35S]sulfate incorporation in both 30DF and 60DF animals. The 60DF group also exhibited reduced [3H]thymidine incorporation. Associated with this growth inhibition was a dramatic increase in the serum levels of a 25-kDa IGFBP (sbIGFBP-1). A 35-kDa IGFBP (sbIGFBP-3), on the other hand, was not significantly altered with fasting. All fasting-induced changes in growth, metabolism, and IGFBP levels were restored in the REFED group. These results demonstrate that an IGFBP of low molecular weight is increased with growth inhibition in the fasting striped bass, suggesting that a teleost fish counterpart to mammalian IGFBP-1 may exist.

  2. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression.

    PubMed

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81-106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  3. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

    PubMed Central

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D.

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  4. Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

    PubMed

    Kimata-Ariga, Yoko; Hase, Toshiharu

    2014-01-01

    Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

  5. Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    PubMed Central

    Chen, Fenfang; Lin, Xia; Xu, Pinglong; Zhang, Zhengmao; Chen, Yanzhen; Wang, Chao; Han, Jiahuai; Zhao, Bin; Xiao, Mu

    2015-01-01

    Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation. PMID:25755279

  6. Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca(2+)-calmodulin modulation.

    PubMed Central

    Chen, T Y; Illing, M; Molday, L L; Hsu, Y T; Yau, K W; Molday, R S

    1994-01-01

    The cGMP-gated cation channel mediating visual transduction in retinal rods was recently found to comprise at least two subunits, 1 and 2 (or alpha and beta). SDS gels of the purified channel show, in addition to a 63-kDa protein band (subunit 1), a 240-kDa protein band that binds Ca(2+)-calmodulin, a modulator of the channel. To examine any connection between subunit 2 and the 240-kDa protein, cGMP-gated channels formed from the expressed cloned subunits in human embryonic kidney (HEK) 293 cells were tested for Ca(2+)-calmodulin effect. Homooligomeric channels formed by subunit 1 alone showed no sensitivity to Ca(2+)-calmodulin, and neither did heterooligomeric channels formed by subunit 1 and the short alternatively spliced form of subunit 2 (2a). By contrast, the cGMP half-activation constant (K1/2) for heterooligomeric channels formed from subunit 1 and the long form of subunit 2 (2b) was increased 1.5- to 2-fold by Ca(2+)-calmodulin, similar to the increase observed for the native channel. In Western blots of rod outer segment membranes, a subunit 2-specific antibody also recognized the 240-kDa protein. Finally, amino acid sequences derived from peptide fragments of the bovine 240-kDa protein showed approximately 80% identity to regions of subunit 2b of the human channel. These results together suggest that subunit 2b of the rod channel is a component of the 240-kDa protein and that it mediates the Ca(2+)-calmodulin modulation of the channel. Images PMID:7526403

  7. The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower.

    PubMed

    Horn, R; Hustedt, J E; Horstmeyer, A; Hahnen, J; Zetsche, K; Friedt, W

    1996-02-01

    In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor. PMID:8605303

  8. Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

    PubMed Central

    Lafleur, M A; Hollenberg, M D; Atkinson, S J; Knäuper, V; Murphy, G; Edwards, D R

    2001-01-01

    Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization. PMID:11415441

  9. HISTONE DEACETYLASE 7 (HDAC7) REGULATES MYOCYTE MIGRATION AND DIFFERENTIATION

    PubMed Central

    Gao, Chengzhuo; Liu, Yu; Lam, Minh; Kao, Hung-Ying

    2010-01-01

    Summary Class IIa HDACs including HDAC7 play a role in gene expression, cell differentiation, and animal development through their association with transcription factors such as myogenic enhancer factors 2 (MEF2s). In this study, we show that endogenous HDAC7 localizes to both the nucleus and the cytoplasm of C2C12 myoblasts, but is exclusively retained in the cytoplasm of myotubes after completion of differentiation process. To elucidate the role of differential distribution of HDAC7 during myogenesis, we examined the effects of stably expressed HDAC7 mutants on myogenesis. Expression of nuclear-retained HDAC7 mutants significantly inhibits myogenesis in C2C12 cells and reduces the expression of muscle-specific myosin heavy chain (MHC) and myogenin. The inhibition in myocyte differentiation can be partially relieved by introduction of a mutation disrupting HDAC7:MEF2 interaction. Since phosphorylation of HDAC7 plays an important role in its nucleocytoplasmic shuttling, we further investigated the expression and distribution of phosphorylated HDAC7. To our surprise, the phosphorylation levels of HDAC7 at S344 and S479 were slightly decreased upon differentiation, whereas the phosphorylation of S178 was unchanged. Interestingly, a significant fraction of pS344- and/or pS479-HDAC7 localizes to plasma membrane of myotubes. In addition, Ser178-phosphorylated (pS178) HDAC7 shows a predominant actin filament-like staining prior to muscle differentiation and cytoplasmic and plasma membrane staining after differentiation. Consistent with this notion, HDAC7 partially co-localizes with actin filaments; in particular, pS178-HDAC7 largely colocalizes with actin filaments as indicated by phalloidin counter staining in myocytes. Furthermore, C2C12 cells expressing nuclear-retained HDAC7 display defects in migration. Our results provide novel insight into the mechanisms that regulate myocyte differentiation and migration by controlling the subcellular distribution of HDAC7 in

  10. Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

    PubMed Central

    Chen, Tingjin; Ning, Dan; Sun, Hengchang; Li, Ran; Shang, Mei; Li, Xuerong; Wang, Xiaoyun; Chen, Wenjun; Liang, Chi; Li, Wenfang; Mao, Qiang; Li, Ye; Deng, Chuanhuan; Wang, Lexun; Wu, Zhongdao; Huang, Yan; Xu, Jin; Yu, Xinbing

    2014-01-01

    Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors

  11. Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

    PubMed

    Gundimeda, Usha; McNeill, Thomas H; Barseghian, Barsegh A; Tzeng, William S; Rayudu, David V; Cadenas, Enrique; Gopalakrishna, Rayudu

    2015-01-01

    Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 μM) or more effectively through a steady-state generation (1-2 μM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

  12. Protection of pigs from swine dysentery by vaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae.

    PubMed

    La, Tom; Phillips, Nyree D; Reichel, Michael P; Hampson, David J

    2004-08-19

    Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0

  13. Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

    PubMed

    Gundimeda, Usha; McNeill, Thomas H; Barseghian, Barsegh A; Tzeng, William S; Rayudu, David V; Cadenas, Enrique; Gopalakrishna, Rayudu

    2015-01-01

    Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 μM) or more effectively through a steady-state generation (1-2 μM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.> PMID

  14. The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

    PubMed

    Vassall, Kenrick A; Bessonov, Kyrylo; De Avila, Miguel; Polverini, Eugenia; Harauz, George

    2013-01-01

    The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure

  15. Detection of Tuberculosis in HIV Co-infected Individuals: Use of Multiple ELISA Responses to 38kDa, Lipoarabinomannan and ESAT– 6 of M. tuberculosis

    PubMed Central

    Gutlapalli, Ravi; Sykam, Aparna; Tenali, Sandeep P; Chandran, Priscilla; Suneetha, Sujai

    2016-01-01

    Introduction There is a constant search for more sensitive and specific laboratory markers for tuberculosis (TB) infection. The early detection of TB in HIV co infected individuals is a diagnostic challenge. This is further compounded in those harbouring extrapulmonary disease. Aim To evaluate the use of multiple Enzyme Linked Immunosorbent Assays (ELISA) quantifying antibody responses to 38kDa, LAM and ESAT-6 M.tb antigens in detection of TB in patients with TB and HIV-TB co-infection. Materials and Methods This is a cross-sectional study carried out in Hyderabad, India. Patient groups included 124 HIV-TB {62 with pulmonary TB (PTB) and 62 with extrapulmonary TB (ETB)}, 39 TB, 56 HIV and 57 healthy subjects (HS). A combination of anti 38kDa and LAM ELISAs measuring IgG, IgM and IgA levels and another ELISA measuring anti ESAT-6 combined antibody levels of IgG, IgM and IgA were evaluated. One-way ANOVA was performed to compare antibody responses among groups. To assess the efficacy of multiple ELISAs in detecting TB, concomitant seropositivity of an individual for all four ELISAs were evaluated for sensitivity and specificity. Results A single ELISA carried out to detect TB in HIV patients showed a sensitivity ranging from 39% to 72%. The sensitivities of concomitant evaluation of multiple ELISAs were 92% for any single, 72% for any two, 44% for any three and 14% for any four. Based on the specificities, a simple algorithm for TB detection can be deduced. When four ELISAs are positive (specificity 100%) in a patient-confirmed TB; when three ELISAs are positive (specificity 98%) – probably TB; when two ELISAs are positive (specificity 95%) – possibly TB; and when one ELISA is positive (specificity 70%) – suspicion of TB. Conclusion The present study establishes the value of combining two or more M.tb antigen based ELISAs to enhance the sensitivity and specificity of TB detection in patients with tuberculosis as well as in those co-infected with HIV. PMID

  16. Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics.

    PubMed

    Ho, Chin Yee; Jaalouk, Diana E; Vartiainen, Maria K; Lammerding, Jan

    2013-05-23

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

  17. Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

    PubMed Central

    Ho, Chin Yee; Jaalouk, Diana E.; Vartiainen, Maria K.; Lammerding, Jan

    2013-01-01

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458

  18. Identification of a novel Drosophila melanogaster heat-shock gene, lethal(2)denticleless [l(2)dtl], coding for an 83-kDa protein.

    PubMed

    Kurzik-Dumke, U; Neubauer, M; Debes, A

    1996-06-01

    In this study, we describe the identification of a novel Drosophila melanogaster (Dm) gene, l(2)dtl, characterized by elevated expression under heat-shock (HS) conditions. It encodes a protein of 83 kDa with no homology to known members of the HSP90 family and other proteins. Gene l(2)dtl is located on the right arm of the second chromosome at locus 59F5, close to the tumor suppressor gene l(2)tid, a homolog of the dnaJ encoding a chaperone strongly conserved in evolution. In the following, we present the sequence of l(2)dtl, the putative protein it encodes, and its molecular localization in a closely interspaced gene cluster consisting of at least four nested genes spanning an approximately 10-kb genomic interval. Furthermore, we present the temporal expression of l(2)dtl in the wild type under normal and HS conditions, and describe the isolation and the phenotype of eight embryonic lethal l(2)dtl mutants.

  19. Cholera toxin partially inhibits the T-cell response to phytohaemagglutinin through the ADP-ribosylation of a 45 kDa membrane protein.

    PubMed Central

    Nel, A E; Vandenplas, M; Wooten, M M; Cooper, R; Vandenplas, S; Rheeder, A; Daniels, J

    1988-01-01

    This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA). CT suppressed lectin-induced [3H]thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP. The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen. The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover. Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP. This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane. These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT. It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C. Our study does not exclude the existence of non-CT-sensitive transducers in this capacity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2851989

  20. The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes.

    PubMed

    Hack, N; Carey, F; Crawford, N

    1984-10-01

    Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

  1. Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+.

    PubMed

    Hack, N; Authi, K S; Crawford, N

    1988-08-01

    A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca2+ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J.M. and Crawford, N. 1988, Biochem. J. 250, 355-361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20-25 micrograms/ml) commensurate with total LDH release, sequestration of Ca2+ into intracellular non-mitochondrial stores is inhibited by the antibody (approximately 50% inhibition at 20 micrograms/ml IgG). At higher saponin concentrations when intracellular binding of 125I-labelled mAb is maximum, inhibition of Ca2+ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca2+ sequestration. No effect of the antibody were observed against IP3-induced release of prestored Ca2+, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca2+ translocating channel protein associated with the intracellular membrane (Ca2+ + Mg2+) ATPase and not to Ca2+ channels responsive to IP3.

  2. Pretreatment with a 55-kDa tumor necrosis factor receptor-immunoglobulin fusion protein attenuates activation of coagulation, but not of fibrinolysis, during lethal bacteremia in baboons.

    PubMed

    van der Poll, T; Jansen, P M; Van Zee, K J; Hack, C E; Oldenburg, H A; Loetscher, H; Lesslauer, W; Lowry, S F; Moldawer, L L

    1997-07-01

    Baboons (Papio anubis) receiving a lethal intravenous infusion with live Escherichia coli were pretreated with either a 55-kDa tumor necrosis factor (TNF) receptor-IgG fusion protein (TNFR55:IgG) (n = 4, 4.6 mg/kg) or placebo (n = 4). Neutralization of TNF activity in TNFR55:IgG-treated animals was associated with a complete prevention of mortality and a strong attenuation of coagulation activation as reflected by the plasma concentrations of thrombin-antithrombin III complexes (P < .05). Activation of fibrinolysis was not influenced by TNFR55:IgG (plasma tissue-type plasminogen activator and plasmin-alpha2-antiplasmin complexes), whereas TNFR55:IgG did inhibit the release of plasminogen activator inhibitor type I (P < .05). Furthermore, TNFR55:IgG inhibited neutrophil degranulation (plasma levels of elastase-alpha1-antitrypsin complexes, P < .05) and modestly reduced release of secretory phospholipase A2. These data suggest that endogenous TNF contributes to activation of coagulation, but not to stimulation of fibrinolysis, during severe bacteremia.

  3. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

  4. Attenuated migration by green tea extract (-)-epigallocatechin gallate (EGCG): involvement of 67 kDa laminin receptor internalization in macrophagic cells.

    PubMed

    Ren, Xuezhi; Guo, Xingzhi; Chen, Li; Guo, Minxia; Peng, Ning; Li, Rui

    2014-08-01

    Excessive activation of the microglia in the brain is involved in the development of several neurodegenerative diseases. Previous studies have indicated that (-)-epigallocatechin gallate (EGCG), a major active constituent of green tea, exhibits potent suppressive effects on the activation of microglia. As the 67 kDa laminin receptor (67LR) is a key element in cellular activation and migration, we investigated the effect of EGCG on cell migration and 67LR in lipopolysaccharide (LPS)-activated macrophagic RAW264.7 cells. The presence of EGCG (1-25 μM) markedly attenuated LPS-induced cell migration in a dose-dependent manner. However, the total amount of 67LR protein in the RAW264.7 cells was unaffected by EGCG, as revealed by Western blot analysis. In addition, confocal immunofluorescence microscopy indicated that EGCG caused a marked membrane translocation of 67LR from the membrane surface towards the cytoplasm. Cell-surface biotinylation analysis confirmed that EGCG induced a significant internalization of 67LR by 24-68% in a dose-dependent manner. This study helps to explain the pharmacological action of EGCG on 67LR, suggesting its potential use in the treatment of diseases associated with macrophage/microglia activation, such as neurodegenerative diseases and cancer.

  5. Fibroblast Growth Factor-2 Isoform (Low Molecular Weight/18 kDa) Overexpression in Preosteoblast Cells Promotes Bone Regeneration in Critical Size Calvarial Defects in Male Mice

    PubMed Central

    Xiao, Liping; Ueno, Daisuke; Catros, Sylvain; Homer-Bouthiette, Collin; Charles, Lyndon; Kuhn, Liisa

    2014-01-01

    Repair of bone defects remains a significant clinical problem. Bone morphogenetic protein 2 (BMP2) is US Food and Drug Administration–approved for fracture healing but is expensive and has associated morbidity. Studies have shown that targeted overexpression of the 18-kDa low-molecular-weight fibroblast growth factor 2 isoform (LMW) by the osteoblastic lineage of transgenic mice increased bone mass. This study tested the hypotheses that overexpression of LMW would directly enhance healing of a critical size calvarial bone defect in mice and that this overexpression would have a synergistic effect with low-dose administration of BMP2 on critical size calvarial bone defect healing. Bilateral calvarial defects were created in LMW transgenic male mice and control/vector transgenic (Vector) male mice and scaffold with or without BMP2 was placed into the defects. New bone formation was assessed by VIVA-computed tomography of live animals over a 27-week period. Radiographic and computed tomography analysis revealed that at all time points, healing of the defect was enhanced in LMW mice compared with that in Vector mice. Although the very low concentration of BMP2 did not heal the defect in Vector mice, it resulted in complete healing of the defect in LMW mice. Histomorphometric and gene analysis revealed that targeted overexpression of LMW in osteoblast precursors resulted in enhanced calvarial defect healing due to increased osteoblast activity and increased canonical Wnt signaling. PMID:24424065

  6. A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin.

    PubMed

    Rothnie, Alice; Clarke, Anthony R; Kuzmic, Petr; Cameron, Angus; Smith, Corinne J

    2011-04-26

    An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

  7. Repeated administration of AC-5216, a ligand for the 18 kDa translocator protein, improves behavioral deficits in a mouse model of post-traumatic stress disorder.

    PubMed

    Qiu, Zhi-Kun; Zhang, Li-Ming; Zhao, Nan; Chen, Hong-Xia; Zhang, You-Zhi; Liu, Yan-Qin; Mi, Tian-Yue; Zhou, Wen-Wen; Li, Yang; Yang, Ri-Fang; Xu, Jiang-Ping; Li, Yun-Feng

    2013-08-01

    Post-traumatic stress disorder (PTSD) is a severely disabling anxiety disorder that may occur following exposure to a serious traumatic event. It is a psychiatric condition that can afflict anyone who has experienced a life-threatening or violent event. Previous studies have shown that changes in 18 kDa translocator protein (TSPO) expression (or function), a promising target for treating neurological disorders without benzodiazepine-like side effects, may correlate with PTSD. However, few studies have investigated the anti-PTSD effects of TSPO ligands. AC-5216, a ligand for TSPO, induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether AC-5216 ameliorates PTSD behavior in mice. Following the training session consisting of exposure to inescapable electric foot shocks, animals were administered AC-5216 daily during the behavioral assessments, i.e., situational reminders (SRs), the open field (OF) test, the elevated plus-maze (EPM) test, and the staircase test (ST). The results indicated that exposure to foot shocks induced long-term behavioral deficiencies in the mice, including freezing and anxiety-like behavior, which were significantly ameliorated by repeated treatment with AC-5216 but without any effect on spontaneous locomotor activity or body weight. In summary, this study demonstrated the anti-PTSD effects of AC-5216 treatment, suggesting that TSPO may represent a therapeutic target for anti-PTSD drug discovery and that TSPO ligands may be a promising new class of drugs for the future treatment of PTSD.

  8. A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.

    PubMed Central

    García-Cuéllar, C; Montañez, C; Tenorio, V; Reyes-Esparza, J; Durán, M J; Negrete, E; Guerrero, A; de la Garza, M

    2000-01-01

    Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 6. Figure 7. PMID:10805246

  9. Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

    PubMed Central

    Wang, Wei; Zhang, Liming; Zhang, Xiaoying; Xue, Rui; Li, Lei; Zhao, Weixing; Fu, Qiang; Mi, Weidong; Li, Yunfeng

    2016-01-01

    The 18 kDa translocator protein (TSPO) is involved in the immune/inflammatory response. However, the exact role that TSPO plays in neuroinflammation-induced cognitive impairment is still elusive. The purpose of our present study was to investigate the effects of lentiviral-mediated hippocampal overexpression of the TSPO in a mouse model of LPS-induced cognitive impairment. We established a mouse cognitive impairment model using systematic daily administration of lipopolysaccharide (LPS) (0.5 mg/kg). Microinjection of the dentate gyrus of the mouse with lentiviral vectors, which contained a cDNA targeting TSPO (Lv-TSPO), resulted in a significant increase in TSPO expression and allopregnanolone production. Mice treated with LPS showed cognitive deficits in the novel object recognition test and the Morris water maze test that could be ameliorated by TSPO overexpression. In addition, TSPO overexpression reversed LPS-induced microglial activation and accumulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Moreover, TSPO overexpression attenuated the LPS-induced impairment of hippocampal neurogenesis. Our results suggest that local overexpression of TSPO in the hippocampal dentate gyrus alleviated LPS-induced cognitive deficits, and its effects might be mediated by the attenuation of inflammatory cytokines, inhibition of microglial activation, and promotion of neurogenesis. PMID:27803668

  10. Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM).

    PubMed

    Skilton, R A; Bishop, R P; Wells, C W; Spooner, P R; Gobright, E; Nkonge, C; Musoke, A J; Macklin, M; Iams, K P

    1998-10-01

    To identify the genes encoding novel immunodominant antigens of Theileria parva a lambda gt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host-sporozoite interaction. PMID:9820853

  11. Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion.

    PubMed

    Bassan, Juliana C; Goulart, Antonio J; Nasser, Ana L M; Bezerra, Thaís M S; Garrido, Saulo S; Rustiguel, Cynthia B; Guimarães, Luis H S; Monti, Rubens

    2015-01-01

    Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

  12. Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus.

    PubMed

    Tvete, T; Haugan, K

    2008-05-01

    Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.

  13. The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

    PubMed

    Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

    2015-01-30

    Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. PMID:25543059

  14. Genetic polymorphism of 3' untranslated region of zeta-chain associated protein kinase 70 kDa in southern Taiwanese patients with rheumatoid arthritis.

    PubMed

    Chen, Shih-Yao; Liu, Ming-Fei; Wang, Chrong-Reen

    2016-03-01

    T cell activation participates in the pathogenesis of rheumatoid arthritis (RA), and the signaling molecule zeta-chain-associated protein kinase 70 kDa (ZAP-70) plays a crucial role in this process. Different mutations in the coding sequence of ZAP-70 are involved in a variety of immunological phenotypes, and recent evidence indicates that genetic variations within the 3' untranslated regions (UTR) of microRNA binding sites may affect the hybridization with target mRNAs, leading to phenotype changes with disease status. In this study, we evaluated the possible effect of ZAP-70 polymorphism as a genetic risk factor in RA by examining the single-nucleotide polymorphism in 100 patients and 100 ethnicity- and sex-matched healthy individuals from southern Taiwan. In both groups, the genotype distribution of rs2278699 in the 3' UTR was in the Hardy-Weinberg equilibrium. In RA, there were higher frequencies of the G allele (15.5 versus 8.0 %, odds ratio 2.1, P = 0.020) and significant differences in the trend of various genotypes (P = 0.024). The results suggest that genetic polymorphism in the 3' UTR of ZAP-70 is associated with RA susceptibility in southern Taiwanese.

  15. Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-04-01

    This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS. PMID:16208518

  16. Inhibitory effect of plant-originated glycoprotein (27 kDa) on expression of matrix metalloproteinase-9 in cadmium chloride-induced BNL CL.2 cells.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2011-12-01

    Cadmium is very harmful to the environment and to human beings because of its long lifetime. The toxicity of cadmium as an industrial pollutant and a food contaminant, and as one of the major components in cigarette smoke is well known. Cadmium can cause a number of lesions in many organs, such as the kidney, the lung, the liver, the brain, the blood system. However, the mechanism of toxicity of cadmium is not yet clear. Also, it has been well known as human carcinogen which is indirectly caused inflammation-mediated hepatocarcinoma. In the present study it was demonstrated that glycoprotein (27 kDa) isolated from Gardenia jasminoides Ellis (GJE) protects BNL CL.2 cells from expression of inflammation-related factors stimulated by cadmium chloride (10 μM). Intracellular ROS and intracellular Ca(2+) using fluorescence, activities of activator protein (AP)-1, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, and arachidonic acid (AA) using immunoblot analysis or radioactivity were evaluated. The results obtained from this experiment indicated that GJE glycoprotein (100 μg/mL) inhibits the production of intracellular ROS, and intracellular Ca(2+) mobilization. Also, it significantly suppressed inflammatory