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Sample records for centaurins control subcellular

  1. Torso RTK controls Capicua degradation by changing its subcellular localization.

    Science.gov (United States)

    Grimm, Oliver; Sanchez Zini, Victoria; Kim, Yoosik; Casanova, Jordi; Shvartsman, Stanislav Y; Wieschaus, Eric

    2012-11-01

    The transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts. PMID:23048183

  2. Subcellular controls of mercury trophic transfer to a marine fish

    Energy Technology Data Exchange (ETDEWEB)

    Dang Fei [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2010-09-15

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  3. Subcellular controls of mercury trophic transfer to a marine fish

    International Nuclear Information System (INIS)

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  4. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

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    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  5. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    Science.gov (United States)

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-01

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity. PMID:17105729

  6. Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

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    Meier Iris

    2005-11-01

    Full Text Available Abstract Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Results Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. Conclusion MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell.

  7. Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin [alpha]1

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Yufeng; Tempel, Wolfram; Wang, Hui; Yamada, Kaori; Shen, Limin; Senisterra, Guillermo A.; MacKenzie, Farrell; Chishti, Athar H.; Park, Hee-Won (Toronto); (UICM)

    2011-11-07

    Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin {alpha}1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P2). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

  8. A celiac cellular phenotype, with altered LPP sub-cellular distribution, is inducible in controls by the toxic gliadin peptide P31-43.

    Directory of Open Access Journals (Sweden)

    Merlin Nanayakkara

    Full Text Available Celiac disease (CD is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP gene was identified as strongly associated with CD using genome-wide association studies (GWAS. The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD and controls, without and with treatment with A-gliadin peptide P31-43. We observed a "CD cellular phenotype" in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.

  9. Recent advances in imaging subcellular processes.

    Science.gov (United States)

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  10. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

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

  11. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

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

    2016-01-01

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

  12. Neuronal Computations Made Visible with Subcellular Resolution.

    Science.gov (United States)

    Kaschula, Richard; Salecker, Iris

    2016-06-30

    Sensory information is gradually processed within dedicated neural circuits to generate specific behaviors. In this issue, Yang et al. push technology boundaries to measure both voltage and calcium signals from subcellular compartments of genetically defined interconnected neurons and shed light on local neural computations critical for motion detection. PMID:27368098

  13. Subcellular distribution of curium in beagle liver

    International Nuclear Information System (INIS)

    The subcellular distribution of curium (243244Cm) was studied in canine liver from 2 hr to 47 days after injection of 3 μCi243244Cm/kg of body weight. The pattern of distribution for Cm was similar to other trivalent actinide elements studied previously (Am, Cf). Initially (2 hr), most of the nuclide was found in the cytosol and at least 90 percent was protein bound. About 70 percent of the Cm was bound to ferritin, approximately 5 percent was associated with a protein of MW approximately 200,000, and approximately 25 percent was found in the low-molecular-weight region (approximately 5000). The decrease in the Cm content of cytosol, nuclei, and microsomes coincided with an increase in the amount associated with mitochondria and lysosomes. The concentration of the Cm in the mitochondrial fraction was higher than it was in the lysosomal fraction at each time studied. In the mitochondrial fraction approximately 30 percent of the Cm was bound to membranous or granular material, and 70 percent was found in the soluble fraction. The Cm concentration initially associated with cell nuclei was high but had diminished to 20 percent of the 2 hr concentration by 20 days post injection (PI). The subcellular distribution of Cm in the liver of a dog which had received the same dose and was terminated because of severe liver damage was studied at 384 days PI. The liver weighed 130 g and contained approximately 30 percent of the injected Cm. In contrast, a normal liver weighs 280 g and at 2 hr PI contains approximately 40 percent of the injected dose. The subcellular distribution of Cm in this severely damaged liver differed from the pattern observed at earlier times after injection. The relative concentration of Cm in the cytosol was doubled; it was higher in the nuclei-debris fraction; and it was lower in the mitochondrial and lysosomal fractions when compared to earlier times

  14. Gene ontology based transfer learning for protein subcellular localization

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    Zhou Shuigeng

    2011-02-01

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

  15. Quantitative Dose-Response Curves from Subcellular Lipid Multilayer Microarrays

    Science.gov (United States)

    Kusi-Appiah, A. E.; Lowry, T. W.; Darrow, E. M.; Wilson, K.; Chadwick, B. P.; Davidson, M. W.; Lenhert, S.

    2015-01-01

    The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate. PMID:26167949

  16. Adaptations of proteins to cellular and subcellular pH

    OpenAIRE

    Garcia-Moreno, Bertrand

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69

  17. Research and teaching with the AFTOL SBD: an informatics resource for fungal subcellular and biochemical data.

    Science.gov (United States)

    Arun Kumar, T K; Blackwell, Meredith; Letcher, Peter M; Roberson, Robert W; McLaughlin, David J

    2013-12-01

    The Structural and Biochemical Database (SBD), developed as part of the US NSF-funded Assembling the Fungal Tree of Life (AFTOL), is a multi-investigator project. It is a major resource to present and manage morphological and biochemical information on Fungi and serves as a phyloinformatics tool for the scientific community. It also is an important resource for teaching mycology. The database, available at http://aftol.umn.edu, includes new and previously published subcellular data on Fungi, supplemented with images and literature links. Datasets automatically combined in NEXUS format from the site permit independent and combined (with molecular data) phylogenetic analyses. Character lists, a major feature of the site, serve as primary reference documents of subcellular and biochemical characters that distinguish taxa across the major fungal lineages. The character lists illustrated with images and drawings are informative for evolutionary and developmental biologists as well as educators, students and the public. Fungal Subcellular Ontology (FSO), developed as part of this effort is a primary initiative to provide a controlled vocabulary describing subcellular structures unique to Fungi. FSO establishes a full complement of terms that provide an operating ontological framework for the database. Examples are provided for using the database for teaching. PMID:24563838

  18. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    OpenAIRE

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Bur...

  19. Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

    OpenAIRE

    1984-01-01

    Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determine...

  20. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    Science.gov (United States)

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  1. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

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

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. PMID:26356766

  2. A formal ontology of subcellular neuroanatomy

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    Stephen D Larson

    2007-11-01

    Full Text Available The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO, covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described.

  3. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-05-13

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

  4. Object Type Recognition for Automated Analysis of Protein Subcellular Location

    OpenAIRE

    Zhao, Ting; Velliste, Meel; Boland, Michael V.; Murphy, Robert F.

    2005-01-01

    The new field of location proteomics seeks to provide a comprehensive, objective characterization of the subcellular locations of all proteins expressed in a given cell type. Previous work has demonstrated that automated classifiers can recognize the patterns of all major subcellular organelles and structures in fluorescence microscope images with high accuracy. However, since some proteins may be present in more than one organelle, this paper addresses a more difficult task: recognizing a pa...

  5. Automated analysis of protein subcellular location in time series images

    OpenAIRE

    Hu, Yanhua; Osuna-Highley, Elvira; Hua, Juchang; Nowicki, Theodore Scott; Stolz, Robert; McKayle, Camille; Murphy, Robert F.

    2010-01-01

    Motivation: Image analysis, machine learning and statistical modeling have become well established for the automatic recognition and comparison of the subcellular locations of proteins in microscope images. By using a comprehensive set of features describing static images, major subcellular patterns can be distinguished with near perfect accuracy. We now extend this work to time series images, which contain both spatial and temporal information. The goal is to use temporal features to improve...

  6. Adaptations of proteins to cellular and subcellular pH.

    Science.gov (United States)

    Garcia-Moreno, Bertrand

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69. PMID:20017887

  7. Cholinesterase of skeletal muscle and its subcellular components.

    OpenAIRE

    Fujii,Masafumi; Namba, Tatsuji

    1982-01-01

    The cholinesterase activity of skeletal muscle and its subcellular components, including motor endplates, was compared chemically in human, mouse and rat. The total cholinesterase activity of muscle per unit protein was in the descending order of human, mouse and rat. Cholinesterase was present in all subcellular components fractionated by differential centrifugation, and was greatest in the microsome fraction followed, in descending order, by the mitochondria, myofibril, and supernatant frac...

  8. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    Science.gov (United States)

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  9. Global, quantitative and dynamic mapping of protein subcellular localization

    Science.gov (United States)

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  10. Subcellular Proteomics of Soybean under Flooding Stress

    Institute of Scientific and Technical Information of China (English)

    Setsuko Komatsu

    2012-01-01

    Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate,and causes significant reductions in the growth and yield of several crops.The application of proteomics techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops.To understand the response mechanism of soybean under flooding stress,proteomics analysis was carried out.Especially,subcellular proteomics studies have led to a better understanding of the mechanism of flooding stress tolerance in soybean.The effects of flooding stress on root plasma membrane were analyzed using an aqueous two-phase partitioning method in combination with gel-based and gel-free proteomics techniques.The results led to the following conclusions:proteins located in the cell wall were increased in the plasma membrane of flooded plants,indicating the contribution of plasma membrane to modification of the cell wall; superoxide dismutase was increased,indicating that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to flooding stress; heat shock cognate 70 kDa protein likely plays a significant role in protecting other proteins from denaturation and degradation during flooding stress; and signaling proteins might work cooperatively to regulate plasma membrane H +-ATPase and maintain ion homeostasis.Cell wall proteins were isolated from root of flooding stressed plants via sucrose gradient centrifugation and analyzed using gel-based proteomics technique.Cell wall proteins identified were related to lignification,and these results indicated that a decrease of lignification-related proteins is related to flooding decreased ROS and jasmonate biosynthesis.And also,lignin staining confirmed that lignification was suppressed in the roots of flooding stressed soybeans.Mitochondrial fractions were purified from root of flooding stressed

  11. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

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

  12. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    The mechanisms underlying heavy metal accumulation, toxicity and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent of, subcellular site of and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109CdCl2 or 112CdSO4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissue followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. Particulate materials containing other cell components were also labeled. Of the 109Cd supplied to plants, 2 to 10% was recovered in both cytosol preparations and in particulate materials. Cytosol contained proteinaceous--Cd complexes, free metal and low molecular weight Cd complexes. Labeling of protoplasts gave similar results. No evidence was obtained for the production of volatile Cd complexes in tobacco

  13. How Cells Measure Length on Subcellular Scales.

    Science.gov (United States)

    Marshall, Wallace F

    2015-12-01

    Cells are not just amorphous bags of enzymes, but precise and complex machines. With any machine, it is important that the parts be of the right size, yet our understanding of the mechanisms that control size of cellular structures remains at a rudimentary level in most cases. One problem with studying size control is that many cellular organelles have complex 3D structures that make their size hard to measure. Here we focus on linear structures within cells, for which the problem of size control reduces to the problem of length control. We compare and contrast potential mechanisms for length control to understand how cells solve simple geometry problems. PMID:26437596

  14. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement.

    Science.gov (United States)

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  15. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    Science.gov (United States)

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-01-01

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics. PMID:27078038

  16. Self-organization of the MinE ring in subcellular Min oscillations

    OpenAIRE

    Derr, Julien; Hopper, Jason T.; Sain, Anirban; Rutenberg, Andrew D.

    2009-01-01

    We model the self-organization of the MinE ring that is observed during subcellular oscillations of the proteins MinD and MinE within the rod-shaped bacterium {\\it Escherichia coli}. With a steady-state approximation, we can study the MinE-ring generically -- apart from the other details of the Min oscillation. Rebinding of MinE to depolymerizing MinD filament tips controls MinE ring formation through a scaled cell shape parameter $\\tilde{r}$. We find two types of E-ring profiles near the fil...

  17. Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

    OpenAIRE

    Harhaj, Edward W; Sun, Shao-Cong

    1999-01-01

    Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between ...

  18. Brain glycogen – new perspectives on its metabolic function and regulation at the subcellular level

    Directory of Open Access Journals (Sweden)

    Linea Frimodt Obel

    2012-03-01

    Full Text Available Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g. liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies – it is a highly dynamic molecule with versatile implications in brain function, i.e. synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on i the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP, ii alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and iii a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g. turnover is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  19. Spreading the news: subcellular and organellar reactive oxygen species production and signalling.

    Science.gov (United States)

    Mignolet-Spruyt, Lorin; Xu, Enjun; Idänheimo, Niina; Hoeberichts, Frank A; Mühlenbock, Per; Brosché, Mikael; Van Breusegem, Frank; Kangasjärvi, Jaakko

    2016-06-01

    As plants are sessile organisms that have to attune their physiology and morphology continuously to varying environmental challenges in order to survive and reproduce, they have evolved complex and integrated environment-cell, cell-cell, and cell-organelle signalling circuits that regulate and trigger the required adjustments (such as alteration of gene expression). Although reactive oxygen species (ROS) are essential components of this network, their pathways are not yet completely unravelled. In addition to the intrinsic chemical properties that define the array of interaction partners, mobility, and stability, ROS signalling specificity is obtained via the spatiotemporal control of production and scavenging at different organellar and subcellular locations (e.g. chloroplasts, mitochondria, peroxisomes, and apoplast). Furthermore, these cellular compartments may crosstalk to relay and further fine-tune the ROS message. Hence, plant cells might locally and systemically react upon environmental or developmental challenges by generating spatiotemporally controlled dosages of certain ROS types, each with specific chemical properties and interaction targets, that are influenced by interorganellar communication and by the subcellular location and distribution of the involved organelles, to trigger the suitable acclimation responses in association with other well-established cellular signalling components (e.g. reactive nitrogen species, phytohormones, and calcium ions). Further characterization of this comprehensive ROS signalling matrix may result in the identification of new targets and key regulators of ROS signalling, which might be excellent candidates for engineering or breeding stress-tolerant plants. PMID:26976816

  20. Subcellular targeting and dynamic regulation of PTEN: Implications for neuronal cells and neurological disorders

    Directory of Open Access Journals (Sweden)

    Ivo Lieberam

    2014-04-01

    Full Text Available PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum, the mitochondria or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  1. Sequential changes of serum and liver subcellular oxidants and antoxidant concentrations in silymarin treated male rats

    Directory of Open Access Journals (Sweden)

    J.A.A. Al-Sa'aidi

    2016-04-01

    Full Text Available The present study aimed to investigate the role of silymarin as an antioxidant and/or it activity in induction of the endogenous antioxidants in intact adult male rats. Seventy males were randomly devided into control and silymarin treated groups (35 each, and were drenched with drinking water and silymarin suspension (200 mg/ kg b.w daily for 40 days. Each group was allocated to 5 equal subgroups; sacrificed before treatment (0 day, and after 10, 20, 30, and 40 days of treatment. At the end of each period, males were anaesthesized, dissected and blood samples were obtained for assessment of MDA, SOD, CAT and GSH concentrations. liver samples (1 g have been removed and homogenized for assessment of liver subcellular MDA, SOD, CAT and GSH concentrations. At the end of each periods, serum and liver subcellular MAD concentrations showed no significant changes between groups, whereas SOD, CAT, and GSH concentrations significantly increased at 10, 20, 30, and 40 day periods in silymarin treated males compared with control. It can be concluded that silymarin antioxidant activity is of pharmacological value not only as an antioxidant by itself but also as an inducer of endogenous enzymatic and non-enzymatic antioxidants even in normal intact male.

  2. Imaging trace element distributions in single organelles and subcellular features

    Science.gov (United States)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  3. Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

    CERN Document Server

    Zurbuchen, Mark A; Kohan, Sirus A; Leung, Belinda; Bouchard, Louis-S

    2015-01-01

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

  4. Convolutional LSTM Networks for Subcellular Localization of Proteins

    OpenAIRE

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

    2015-01-01

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model on the other hand are designed to handle sequences. In this study we demonstrate that LSTM networks predict the subcellular location of proteins given only the protein sequence with high accuracy (...

  5. Evaluation and comparison of mammalian subcellular localization prediction methods

    Directory of Open Access Journals (Sweden)

    Fink J Lynn

    2006-12-01

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

  6. Discriminative Motif Finding for Predicting Protein Subcellular Localization

    OpenAIRE

    Lin, Tien-ho; Murphy, Robert F.; Bar-Joseph, Ziv

    2011-01-01

    Many methods have been described to predict the subcellular location of proteins from sequence information. However, most of these methods either rely on global sequence properties or use a set of known protein targeting motifs to predict protein localization. Here we develop and test a novel method that identifies potential targeting motifs using a discriminative approach based on hidden Markov models (discriminative HMMs). These models search for motifs that are present in a compartment but...

  7. Subcellular site and nature of intracellular cadmium in plants

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, George J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent of, subcellular site of and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied /sup 109/CdCl/sub 2/ or /sup 112/CdSO/sub 4/ accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissue followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. Particulate materials containing other cell components were also labeled. Of the /sup 109/Cd supplied to plants, 2 to 10% was recovered in both cytosol preparations and in particulate materials. Cytosol contained proteinaceous--Cd complexes, free metal and low molecular weight Cd complexes. Labeling of protoplasts gave similar results. No evidence was obtained for the production of volatile Cd complexes in tobacco.

  8. Comparative studies on the influence of some radioprotectors on the postirradiation hydrolytic function of subcellular fractions of pancreas

    International Nuclear Information System (INIS)

    Comparative studies on the influence of the two radioprotectors Fenchlorfos and cysteamine on the hydroproteolytic activity of pancreatic subcellular fractions of rats were carried out. The animals were irradiated with the single dose of 800 R of X-rays. Biochemical estimation of lipase, β-glucuronidase, acid phosphatase and catheptic (caseonolytic) activity were done 24 hours after exposition. The two radioprotective substances have a lot of common in their action against hydrolytic activity. They induce the decrease of some enzymes of subcellular fractions we checked both in control as well as in irradiated groups. The ambivalent influence of Trichlorfon on the lipase activity in granules and supernatant fraction seems to be of interest. (orig.)

  9. Abnormal subcellular distribution of GLUT4 protein in obese and insulin-treated diabetic female dogs

    Directory of Open Access Journals (Sweden)

    Vargas A.M.

    2004-01-01

    Full Text Available The GLUT4 transporter plays a key role in insulin-induced glucose uptake, which is impaired in insulin resistance. The objective of the present study was to investigate the tissue content and the subcellular distribution of GLUT4 protein in 4- to 12-year-old control, obese and insulin-treated diabetic mongrel female dogs (4 animals per group. The parametrial white adipose tissue was sampled and processed to obtain both plasma membrane and microsome subcellular fractions for GLUT4 analysis by Western blotting. There was no significant difference in glycemia and insulinemia between control and obese animals. Diabetic dogs showed hyperglycemia (369.9 ± 89.9 mg/dl. Compared to control, the plasma membrane GLUT4, reported per g tissue, was reduced by 55% (P < 0.01 in obese dogs, and increased by 30% (P < 0.05 in diabetic dogs, and the microsomal GLUT4 was increased by ~45% (P < 0.001 in both obese and diabetic animals. Considering the sum of GLUT4 measured in plasma membrane and microsome as total cellular GLUT4, percent GLUT4 present in plasma membrane was reduced by ~65% (P < 0.001 in obese compared to control and diabetic animals. Since insulin stimulates GLUT4 translocation to the plasma membrane, percent GLUT4 in plasma membrane was divided by the insulinemia at the time of tissue removal and was found to be reduced by 75% (P < 0.01 in obese compared to control dogs. We conclude that the insulin-stimulated translocation of GLUT4 to the cell surface is reduced in obese female dogs. This probably contributes to insulin resistance, which plays an important role in glucose homeostasis in dogs.

  10. Influence of prey type on nickel and thallium assimilation, subcellular distribution and effects in juvenile fathead minnows (Pimephales promelas).

    Science.gov (United States)

    Lapointe, Dominique; Gentès, Sophie; Ponton, Dominic E; Hare, Landis; Couture, Patrice

    2009-11-15

    Because fish take up metals from prey, it is important to measure factors controlling metal transfer between these trophic levels so as to explain metal bioaccumulation and effects in fish. To achieve this, we exposed two types of invertebrates, an oligochaete (Tubifex tubifex) and a crustacean (Daphnia magna), to environmentally relevant concentrations of two important contaminants, nickel (Ni) and thallium (Tl), and fed these prey to juvenile fathead minnows (Pimephales promelas). We then measured the assimilation efficiency (AE), subcellular distribution and effects of these metals in fish. Fish assimilated dietary Tl more efficiently from D. magna than from T. tubifex, and more efficiently than Ni, regardless of prey type. However, the proportion of metal bound to prey subcellular fractions that are likely to be trophically available (TAM) had no significant influence on the efficiency with which fish assimilated Ni or Tl. In fish, the majority of their Ni and Tl was bound to subcellular fractions that are purportedly detoxified, and prey type had a significant influence on the proportion of detoxified Ni and Tl in fish. We measured higher activities of cytochrome C oxidase and glutathione S-transferase in fish fed D. magna compared to fish fed T. tubifex, regardless of the presence or absence of Ni or Tl in prey. However, we measured decreased activities of glutathione S-transferase and nucleoside diphosphate kinase in fish fed Tl-contaminated D. magna compared to fish from the three other treatment levels. PMID:20028068

  11. Image analysis of the movement of sub-cellular structures

    Czech Academy of Sciences Publication Activity Database

    Matula, Pe.; Ondřej, Vladan; Kozubek, Stanislav

    Brno : Masarykova univerzita v Brně, 2004 - (Kozubek, S.; Kozubek, M.), s. 62-65 ISBN 80-210-3560-9. [Biophysics of the Genome. Brno (CZ), 12.10.2004-13.10.2004] R&D Projects: GA ČR GA202/04/0907; GA AV ČR IBS5004010; GA AV ČR IAA5004306 Institutional research plan: CEZ:AV0Z5004920 Keywords : sub-cellular structures * image analysis * graph theory Subject RIV: BO - Biophysics

  12. A System for Predicting Subcellular Localization of Yeast Genome Using Neural Network

    CERN Document Server

    Thampi, Sabu M

    2007-01-01

    The subcellular location of a protein can provide valuable information about its function. With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important. Many efforts have been made to predict protein subcellular localization. This paper aims to merge the artificial neural networks and bioinformatics to predict the location of protein in yeast genome. We introduce a new subcellular prediction method based on a backpropagation neural network. The results show that the prediction within an error limit of 5 to 10 percentage can be achieved with the system.

  13. Subcellular Distribution of Glutathione Precursors in Arabidopsis thafiana

    Institute of Scientific and Technical Information of China (English)

    Barbara Eva Koffier; Romana Maier; Bernd Zechmann

    2011-01-01

    Glutathione is an important antioxidant and has many important functions in plant development,growth and defense.Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors,cysteine,glutamate,glycine and y-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis.The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense.The aim of this study was to investigate the compartmentspecific importance of glutathione precursors in Arabidopsis thaliana.The subcellular distribution was compared between wild type plants (Col-0),plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant,wild type plants treated with buthionine sulfoximine),and one complemented line (OE3) with restored glutathione synthesis.Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine,glutamate and glycine in most cell compartments including plastids and the cytosol.A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments.These experiments demonstrated that the inhibition of y-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.

  14. Cholinesterase of skeletal muscle and its subcellular components.

    Directory of Open Access Journals (Sweden)

    Fujii,Masafumi

    1982-06-01

    Full Text Available The cholinesterase activity of skeletal muscle and its subcellular components, including motor endplates, was compared chemically in human, mouse and rat. The total cholinesterase activity of muscle per unit protein was in the descending order of human, mouse and rat. Cholinesterase was present in all subcellular components fractionated by differential centrifugation, and was greatest in the microsome fraction followed, in descending order, by the mitochondria, myofibril, and supernatant fractions. Each of these fractions had greater cholinesterase activity in human muscle than in mouse muscle, and in mouse muscle than in rat muscle. The ratio of the activity of the microsome fraction to the activity of muscle homogenate was 11.1 in human, 4.6 in mouse and 3.4 in rat. Because of its relatively greater proportion, the myofibril fraction seems to contribute most to the total cholinesterase activity of muscle. Muscle membrane contained high cholinesterase activity of motor endplates, and the activity was greater than the activity of the microsome fraction in rat. Cholinesterase activity per motor endplate was in the descending order of rat, human and mouse, and the variation was less than the variation in the total muscle cholinesterase activity among these species.

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Amanda Getty

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

  17. Imaging protein interactions in vivo with sub-cellular resolution

    CERN Document Server

    Raicu, Valerica; Fung, Russell; Melnichuk, Mike; Jansma, David B; Pisterzi, Luca; Fox, Michael; Wells, James W; Saldin, Dilano K

    2008-01-01

    Resonant Energy Transfer (RET) from an optically excited donor molecule (D) to a non-excited acceptor molecule (A) residing nearby is widely used to detect molecular interactions in living cells. Stoichiometric information, such as the number of proteins forming a complex, has been obtained so far for a handful of proteins, but only after exposing the sample sequentially to at least two different excitation wavelengths. During this lengthy process of measurement, the molecular makeup of a cellular region may change, and this has so far limited the applicability of RET to determination of cellular averages. Here we demonstrate a method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a spectrally-resolved two-photon microscope, a simple but competent theory, and a keen selection of fluorescent tags. This technology may eventually lead to tracking dynamics of macromolecular complex formation and dissociation with spatial resolution inside living cell...

  18. Quantification of asymmetric microtubule nucleation at sub-cellular structures

    Science.gov (United States)

    Zhu, Xiaodong; Kaverina, Irina

    2012-01-01

    Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Qian-Quan Sun

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

  1. Cellular and subcellular localization of Marlin-1 in the brain

    Directory of Open Access Journals (Sweden)

    Luján Rafael

    2009-04-01

    Full Text Available Abstract Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  2. Protein-protein interaction as a predictor of subcellular location

    Directory of Open Access Journals (Sweden)

    Davis Melissa J

    2009-02-01

    Full Text Available Abstract Background Many biological processes are mediated by dynamic interactions between and among proteins. In order to interact, two proteins must co-occur spatially and temporally. As protein-protein interactions (PPIs and subcellular location (SCL are discovered via separate empirical approaches, PPI and SCL annotations are independent and might complement each other in helping us to understand the role of individual proteins in cellular networks. We expect reliable PPI annotations to show that proteins interacting in vivo are co-located in the same cellular compartment. Our goal here is to evaluate the potential of using PPI annotation in determining SCL of proteins in human, mouse, fly and yeast, and to identify and quantify the factors that contribute to this complementarity. Results Using publicly available data, we evaluate the hypothesis that interacting proteins must be co-located within the same subcellular compartment. Based on a large, manually curated PPI dataset, we demonstrate that a substantial proportion of interacting proteins are in fact co-located. We develop an approach to predict the SCL of a protein based on the SCL of its interaction partners, given sufficient confidence in the interaction itself. The frequency of false positive PPIs can be reduced by use of six lines of supporting evidence, three based on type of recorded evidence (empirical approach, multiplicity of databases, and multiplicity of literature citations and three based on type of biological evidence (inferred biological process, domain-domain interactions, and orthology relationships, with biological evidence more-effective than recorded evidence. Our approach performs better than four existing prediction methods in identifying the SCL of membrane proteins, and as well as or better for soluble proteins. Conclusion Understanding cellular systems requires knowledge of the SCL of interacting proteins. We show how PPI data can be used more effectively to

  3. Multi-scale continuum modeling of biological processes: from molecular electro-diffusion to sub-cellular signaling transduction

    Science.gov (United States)

    Cheng, Y.; Kekenes-Huskey, P.; Hake, J. E.; Holst, M. J.; McCammon, J. A.; Michailova, A. P.

    2012-01-01

    This paper presents a brief review of multi-scale modeling at the molecular to cellular scale, with new results for heart muscle cells. A finite element-based simulation package (SMOL) was used to investigate the signaling transduction at molecular and sub-cellular scales (http://mccammon.ucsd.edu/smol/, http://FETK.org) by numerical solution of the time-dependent Smoluchowski equations and a reaction-diffusion system. At the molecular scale, SMOL has yielded experimentally validated estimates of the diffusion-limited association rates for the binding of acetylcholine to mouse acetylcholinesterase using crystallographic structural data. The predicted rate constants exhibit increasingly delayed steady-state times, with increasing ionic strength, and demonstrate the role of an enzyme's electrostatic potential in influencing ligand binding. At the sub-cellular scale, an extension of SMOL solves a nonlinear, reaction-diffusion system describing Ca2+ ligand buffering and diffusion in experimentally derived rodent ventricular myocyte geometries. Results reveal the important role of mobile and stationary Ca2+ buffers, including Ca2+ indicator dye. We found that alterations in Ca2+-binding and dissociation rates of troponin C (TnC) and total TnC concentration modulate sub-cellular Ca2+ signals. The model predicts that reduced off-rate in the whole troponin complex (TnC, TnI, TnT) versus reconstructed thin filaments (Tn, Tm, actin) alters cytosolic Ca2+ dynamics under control conditions or in disease-linked TnC mutations. The ultimate goal of these studies is to develop scalable methods and theories for the integration of molecular-scale information into simulations of cellular-scale systems.

  4. Multi-scale continuum modeling of biological processes: from molecular electro-diffusion to sub-cellular signaling transduction

    International Nuclear Information System (INIS)

    This paper presents a brief review of multi-scale modeling at the molecular to cellular scale, with new results for heart muscle cells. A finite element-based simulation package (SMOL) was used to investigate the signaling transduction at molecular and sub-cellular scales (http://mccammon.ucsd.edu/smol/, http://FETK.org) by numerical solution of the time-dependent Smoluchowski equations and a reaction-diffusion system. At the molecular scale, SMOL has yielded experimentally validated estimates of the diffusion-limited association rates for the binding of acetylcholine to mouse acetylcholinesterase using crystallographic structural data. The predicted rate constants exhibit increasingly delayed steady-state times, with increasing ionic strength, and demonstrate the role of an enzyme's electrostatic potential in influencing ligand binding. At the sub-cellular scale, an extension of SMOL solves a nonlinear, reaction-diffusion system describing Ca2+ ligand buffering and diffusion in experimentally derived rodent ventricular myocyte geometries. Results reveal the important role of mobile and stationary Ca2+ buffers, including Ca2+ indicator dye. We found that alterations in Ca2+-binding and dissociation rates of troponin C (TnC) and total TnC concentration modulate sub-cellular Ca2+ signals. The model predicts that reduced off-rate in the whole troponin complex (TnC, TnI, TnT) versus reconstructed thin filaments (Tn, Tm, actin) alters cytosolic Ca2+ dynamics under control conditions or in disease-linked TnC mutations. The ultimate goal of these studies is to develop scalable methods and theories for the integration of molecular-scale information into simulations of cellular-scale systems.

  5. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

    KAUST Repository

    Bayoumi, Maged Fouad

    2012-12-12

    To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards. © 2012 American Chemical Society.

  6. Subcellular localization of the antidepressant-sensitive norepinephrine transporter

    Directory of Open Access Journals (Sweden)

    Winder Danny G

    2009-06-01

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

  7. Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

    Institute of Scientific and Technical Information of China (English)

    Juan TAN; Kai WU; Rui CHANG; Qi-min CHEN; Yun-qi GENG; Wen-tao QIAO

    2008-01-01

    The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.

  8. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

    Science.gov (United States)

    Serag, Maged F; Braeckmans, Kevin; Habuchi, Satoshi; Kaji, Noritada; Bianco, Alberto; Baba, Yoshinobu

    2012-12-12

    To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards. PMID:23170917

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

    International Nuclear Information System (INIS)

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

  10. Determination of arsenic in subcellular fractions of rat liver by ENAA

    International Nuclear Information System (INIS)

    The concentration of arsenic in subcellular fractions of rat liver is determined by differential centrifugation combined with epithermal neutron activation analysis (ENAA). The analytical quality has been verified by analysis of some standard reference materials. The results show that the distribution of As in subcellular fractions of rat liver cells is not homogeneous. The highest concentration of As is found in microsome, while the lowest content is found in nuclei for arsenic poisoned rat. The lowest content of As is noted in mitochondria of contrast rats. The differences of As contents in subcellular fractions of lives cells between arsenic poisoned and normal rats are compared by means of t-test and exceedingly significant differences are found (P<0.001). In As poisoned rat, the As content in microsome is significantly higher than that in other subcellular fractions (P<0.05), except lysosome

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

    Directory of Open Access Journals (Sweden)

    Jarrod S Johnson

    2011-05-01

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

  12. Subcellular adaptation of the human diaphragm in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Orozco-Levi, M; Gea, J; Lloreta, J L; Félez, M; Minguella, J; Serrano, S; Broquetas, J M

    1999-02-01

    Pulmonary hyperinflation impairs the function of the diaphragm in patients with chronic obstructive pulmonary disease (COPD). However, it has been recently demonstrated that the muscle can counterbalance this deleterious effect, remodelling its structure (i.e. changing the proportion of different types of fibres). The aim of this study was to investigate whether the functional impairment present in COPD patients can be associated with structural subcellular changes of the diaphragm. Twenty individuals (60+/-9 yrs, 11 COPD patients and 9 subjects with normal spirometry) undergoing thoracotomy were included. Nutritional status and respiratory function were evaluated prior to surgery. Then, small samples of the costal diaphragm were obtained and processed for electron microscopy analysis. COPD patients showed a mean forced expiratory volume in one second (FEV1) of 60+/-9% predicted, a higher concentration of mitochondria (n(mit)) in their diaphragm than controls (0.62+/-0.16 versus 0.46+/-0.16 mitochondrial transections (mt) x microm(-2), p37%) disclosed not only a higher n(mit) (0.63+/-0.17 versus 0.43+/-0.07 mt x microm(-2), p<0.05) but shorter sarcomeres (L(sar)) than subjects without this functional abnormality (2.08+/-0.16 to 2.27+/-0.15 microm, p<0.05). Glycogen stores were similar in COPD and controls. The severity of airways obstruction (i.e. FEV1) was associated with n(mit) (r=-0.555, p=0.01), while the amount of air trapping (i.e. RV/TLC) was found to correlate with both n(mit) (r=0.631, p=0.005) and L(sar) (r=-0.526, p<0.05). Finally, maximal inspiratory pressure (PI,max) inversely correlated with n(mit) (r=-0.547, p=0.01). In conclusion, impairment in lung function occurring in patients with chronic obstructive pulmonary disease is associated with subcellular changes in their diaphragm, namely a shortening in the length of sarcomeres and an increase in the concentration of mitochondria. These changes form a part of muscle remodelling, probably contributing

  13. Predicting multiplex subcellular localization of proteins using protein-protein interaction network: a comparative study

    OpenAIRE

    Jiang Jonathan Q; Wu Maoying

    2012-01-01

    Abstract Background Proteins that interact in vivo tend to reside within the same or "adjacent" subcellular compartments. This observation provides opportunities to reveal protein subcellular localization in the context of the protein-protein interaction (PPI) network. However, so far, only a few efforts based on heuristic rules have been made in this regard. Results We systematically and quantitatively validate the hypothesis that proteins physically interacting with each other probably shar...

  14. Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

    OpenAIRE

    Greiser, Maura; Kerfant, Benoît-Gilles; Williams, George S. B.; Voigt, Niels; Harks, Erik; Dibb, Katharine M.; Giese, Anne; Meszaros, Janos; Verheule, Sander; Ravens, Ursula; Allessie, Maurits A.; Gammie, James S.; Van Der Velden, Jolanda; Lederer, W. Jonathan; Dobrev, Dobromir

    2014-01-01

    Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ wa...

  15. Multiple Features Based Two-stage Hybrid Classifier Ensembles for Subcellular Phenotype Images Classification

    OpenAIRE

    Bailing Zhang - China; Tuan D. Pham - Australia

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. As an interesting ``bio-image informatics'' application, an automatic, reliable and efficient prediction system for protein subcellular localization can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, we propose a two-stage multiple classifier system to improve classification...

  16. Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

    OpenAIRE

    Masedunskas, Andrius; Porat-Shliom, Natalie; Tora, Muhibullah; Milberg, Oleg; Weigert, Roberto

    2013-01-01

    Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most o...

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

    Science.gov (United States)

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

    2014-09-01

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

  18. Linking Subcellular Disturbance to Physiological Behavior and Toxicity Induced by Quantum Dots in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Qin; Zhou, Yanfeng; Song, Bin; Zhong, Yiling; Wu, Sicong; Cui, Rongrong; Cong, Haixia; Su, Yuanyuan; Zhang, Huimin; He, Yao

    2016-06-01

    The wide-ranging applications of fluorescent semiconductor quantum dots (QDs) have triggered increasing concerns about their biosafety. Most QD-related toxicity studies focus on the subcellular processes in cultured cells or global physiological effects on whole animals. However, it is unclear how QDs affect subcellular processes in living organisms, or how the subcellular disturbance contributes to the overall toxicity. Here the behavior and toxicity of QDs of three different sizes in Caenorhabditis elegans (C. elegans) are systematically investigated at both the systemic and the subcellular level. Specifically, clear size-dependent distribution and toxicity of the QDs in the digestive tract are observed. Short-term exposure of QDs leads to acute toxicity on C. elegans, yet incurring no lasting, irreversible damage. In contrast, chronic exposure of QDs severely inhibits development and shortens lifespan. Subcellular analysis reveals that endocytosis and nutrition storage are disrupted by QDs, which likely accounts for the severe deterioration in growth and longevity. This work reveals that QDs invasion disrupts key subcellular processes in living organisms, and may cause permanent damage to the tissues and organs over long-term retention. The findings provide invaluable information for safety evaluations of QD-based applications and offer new opportunities for design of novel nontoxic nanoprobes. PMID:27121203

  19. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu

    2006-01-01

    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  20. A sub-cellular viscoelastic model for cell population mechanics.

    Directory of Open Access Journals (Sweden)

    Yousef Jamali

    Full Text Available Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and 'in silico' (computational models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM, effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the

  1. Rhesus macaque MHC class I molecules show differential subcellular localizations.

    Science.gov (United States)

    Rosner, Cornelia; Kruse, Philip H; Lübke, Torben; Walter, Lutz

    2010-03-01

    The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding alpha1 and alpha2 domains, suggesting failure of peptide binding is responsible for retaining 'intracellular' Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes. PMID:20151120

  2. Subcellular localization and compartmentation of thiamine derivatives in rat brain.

    Science.gov (United States)

    Bettendorff, L; Wins, P; Lesourd, M

    1994-05-26

    The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate. PMID:8186256

  3. Predicting Protein Subcellular Location Using Digital Signal Processing

    Institute of Scientific and Technical Information of China (English)

    Yu-Xi PAN; Da-Wei LI; Yun DUAN; Zhi-Zhou ZHANG; Ming-Qing XU; Guo-Yin FENG; Lin HE

    2005-01-01

    The biological functions of a protein are closely related to its attributes in a cell. With the rapid accumulation of newly found protein sequence data in databanks, it is highly desirable to develop an automated method for predicting the subcellular location of proteins. The establishment of such a predictor will expedite the functional determination of newly found proteins and the process of prioritizing genes and proteins identified by genomic efforts as potential molecular targets for drug design. The traditional algorithms for predicting these attributes were based solely on amino acid composition in which no sequence order effect was taken into account. To improve the prediction quality, it is necessary to incorporate such an effect. However, the number of possible patterns in protein sequences is extremely large, posing a formidable difficulty for realizing this goal. To deal with such difficulty, a well-developed tool in digital signal processing named digital Fourier transform (DFT) [1] was introduced. After being translated to a digital signal according to the hydrophobicity of each amino acid, a protein was analyzed by DFT within the frequency domain. A set of frequency spectrum parameters, thus obtained, were regarded as the factors to represent the sequence order effect. A significant improvement in prediction quality was observed by incorporating the frequency spectrum parameters with the conventional amino acid composition. One of the crucial merits of this approach is that many existing tools in mathematics and engineering can be easily applied in the predicting process. It is anticipated that digital signal processing may serve as a useful vehicle for many other protein science areas.

  4. Movies of cellular and sub-cellular motion by digital holographic microscopy

    Directory of Open Access Journals (Sweden)

    Yu Lingfeng

    2006-03-01

    Full Text Available Abstract Background Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. Methods A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Results Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable

  5. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs;

    2015-01-01

    content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies...... for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h...... in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular...

  6. Sub-cellular modeling of platelet transport in blood flow through microchannels with constriction.

    Science.gov (United States)

    Yazdani, Alireza; Karniadakis, George Em

    2016-05-11

    Platelet transport through arterial constrictions is one of the controlling processes influencing their adhesive functions and the formation of thrombi. We perform high-fidelity mesoscopic simulations of blood flow in microchannels with constriction, resembling arterial stenoses. The wall shear rates inside the constrictions reach levels as high as ≈8000 s(-1), similar to those encountered in moderate atherosclerotic plaques. Both red blood cells and platelets are resolved at sub-cellular resolution using the Dissipative Particle Dynamics (DPD) method. We perform a systematic study on the red blood cell and platelet transport by considering different levels of constriction, blood hematocrit and flow rates. We find that higher levels of constriction and wall shear rates lead to significantly enhanced margination of platelets, which may explain the experimental observations of enhanced post-stenosis platelet aggregation. We also observe similar margination effects for stiff particles of spherical shapes such as leukocytes. To our knowledge, such numerical simulations of dense blood through complex geometries have not been performed before, and our quantitative findings could shed new light on the associated physiological processes such as ATP release, plasma skimming, and thrombus formation. PMID:27087267

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

    International Nuclear Information System (INIS)

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

  8. In situPCR on Plant Material with Sub-cellular Resolution

    DEFF Research Database (Denmark)

    Johansen, Bo

    1997-01-01

    In situPCR and reverse transcribedin situPCR have been tested on leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5% GA.In situPCR amplification of the gene coding for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was successfully performed following all three fixation protocols. As...... expected the PCR product was restricted to the plastids of all cells. Reverse transcribedin situPCR was performed onrbcL mRNA and in this case the PCR product was restricted to the plastids of the bundle sheath cells. This is the first report ofin situPCR on plant material and only the second report ofin...... situPCR with sub-cellular resolution.In situPCR onrbcL may prove to be a valuable positive control for futurein situPCR studies on plant material.Copyright 1997 Annals of Botany Company 10.1006/anbo.1997.0502...

  9. Subcellular differences in handling Cu excess in three freshwater fish species contributes greatly to their differences in sensitivity to Cu

    Energy Technology Data Exchange (ETDEWEB)

    Eyckmans, Marleen, E-mail: marleen.eyckmans@ua.ac.be [Laboratory for Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp (Belgium); Blust, Ronny; De Boeck, Gudrun [Laboratory for Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp (Belgium)

    2012-08-15

    Since changes in metal distribution among tissues and subcellular fractions can provide insights in metal toxicity and tolerance, we investigated this partitioning of Cu in gill and liver tissue of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). These fish species are known to differ in their sensitivity to Cu exposure with gibel carp being the most tolerant and rainbow trout the most sensitive. After an exposure to 50 {mu}g/l (0.79 {mu}M) Cu for 24 h, 3 days, 1 week and 1 month, gills and liver of control and exposed fish were submitted to a differential centrifugation procedure. Interestingly, there was a difference in accumulated Cu in the three fish species, even in control fishes. Where the liver of rainbow trout showed extremely high Cu concentrations under control conditions, the amount of Cu accumulated in their gills was much less than in common and gibel carp. At the subcellular level, the gills of rainbow trout appeared to distribute the additional Cu exclusively in the biologically active metal pool (BAM; contains heat-denaturable fraction and organelle fraction). A similar response could be seen in gill tissue of common carp, although the percentage of Cu in the BAM of common carp was lower compared to rainbow trout. Gill tissue of gibel carp accumulated more Cu in the biologically inactive metal pool (BIM compared to BAM; contains heat-stable fraction and metal-rich granule fraction). The liver of rainbow trout seemed much more adequate in handling the excess Cu (compared to its gills), since the storage of Cu in the BIM increased. Furthermore, the high % of Cu in the metal-rich granule fraction and heat-stable fraction in the liver of common carp and especially gibel carp together with the better Cu handling in gill tissue, pointed out the ability of the carp species to minimize the disadvantages related to Cu stress. The differences in Cu distribution at the subcellular level of gills

  10. Subcellular differences in handling Cu excess in three freshwater fish species contributes greatly to their differences in sensitivity to Cu

    International Nuclear Information System (INIS)

    Since changes in metal distribution among tissues and subcellular fractions can provide insights in metal toxicity and tolerance, we investigated this partitioning of Cu in gill and liver tissue of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). These fish species are known to differ in their sensitivity to Cu exposure with gibel carp being the most tolerant and rainbow trout the most sensitive. After an exposure to 50 μg/l (0.79 μM) Cu for 24 h, 3 days, 1 week and 1 month, gills and liver of control and exposed fish were submitted to a differential centrifugation procedure. Interestingly, there was a difference in accumulated Cu in the three fish species, even in control fishes. Where the liver of rainbow trout showed extremely high Cu concentrations under control conditions, the amount of Cu accumulated in their gills was much less than in common and gibel carp. At the subcellular level, the gills of rainbow trout appeared to distribute the additional Cu exclusively in the biologically active metal pool (BAM; contains heat-denaturable fraction and organelle fraction). A similar response could be seen in gill tissue of common carp, although the percentage of Cu in the BAM of common carp was lower compared to rainbow trout. Gill tissue of gibel carp accumulated more Cu in the biologically inactive metal pool (BIM compared to BAM; contains heat-stable fraction and metal-rich granule fraction). The liver of rainbow trout seemed much more adequate in handling the excess Cu (compared to its gills), since the storage of Cu in the BIM increased. Furthermore, the high % of Cu in the metal-rich granule fraction and heat-stable fraction in the liver of common carp and especially gibel carp together with the better Cu handling in gill tissue, pointed out the ability of the carp species to minimize the disadvantages related to Cu stress. The differences in Cu distribution at the subcellular level of gills and

  11. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    Science.gov (United States)

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states. PMID:26005782

  12. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    Science.gov (United States)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  13. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier.

    Science.gov (United States)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  14. MOLECULAR CLONING, EXPRESSION AND SUBCELLULAR LOCALIZATION OF HUMAN OCT-4 PROTEIN

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To clone, express, purify human Oct-4 and detect its subcellular localization.Methods Human Oct-4 cDNA was amplified by RT-PCR strategy. Oct-4 protein was induced and expressed in BL21 (DE3) strain. Furthermore, the protein was purified with Ni-NTA resin. Subsequently, site-directed mutagenesis of Oct-4 (aa 236 -240) was introduced. Finally, the subcellular localization of wide type Oct-4 and mutant Oct-4 was examined by immunofluorescent cytochemistry staining and confocal laser scanning microscope analysis.Results The full length cDNA of human Oct-4 was 1083bp. Human Oct-4 encoded a 55 kd protein by prokaryotic vector in E coli. Compared with pure nuclear localization of wide type Oct-4, mutant Oct-4 was mostly enriched in the cytoplasm. Conclusion The cloning, expression and investigation of subcellular localization of human Oct-4 are basis of studying its biological function.

  15. Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Beaumelle, Léa [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Gimbert, Frédéric [Laboratoire Chrono-Environnement, UMR 6249 University of Franche-Comté/CNRS Usc INRA, 16 route de Gray, 25030 Besançon Cedex (France); Hedde, Mickaël [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Guérin, Annie [INRA, US 0010 LAS Laboratoire d' analyses des sols, 273 rue de Cambrai, 62000 Arras (France); Lamy, Isabelle, E-mail: lamy@versailles.inra.fr [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France)

    2015-07-01

    Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl{sub 2}-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl{sub 2} extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm. - Highlights: • Earthworms were exposed to a wide panel of historically contaminated soils • Subcellular partitioning of Cd, Pb and Zn was investigated in earthworms • Three proxies of soil metal availability were

  16. Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils

    International Nuclear Information System (INIS)

    Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl2-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl2 extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm. - Highlights: • Earthworms were exposed to a wide panel of historically contaminated soils • Subcellular partitioning of Cd, Pb and Zn was investigated in earthworms • Three proxies of soil metal availability were assessed

  17. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    Directory of Open Access Journals (Sweden)

    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  18. Multi-label multi-kernel transfer learning for human protein subcellular localization.

    Directory of Open Access Journals (Sweden)

    Suyu Mei

    Full Text Available Recent years have witnessed much progress in computational modelling for protein subcellular localization. However, the existing sequence-based predictive models demonstrate moderate or unsatisfactory performance, and the gene ontology (GO based models may take the risk of performance overestimation for novel proteins. Furthermore, many human proteins have multiple subcellular locations, which renders the computational modelling more complicated. Up to the present, there are far few researches specialized for predicting the subcellular localization of human proteins that may reside in multiple cellular compartments. In this paper, we propose a multi-label multi-kernel transfer learning model for human protein subcellular localization (MLMK-TLM. MLMK-TLM proposes a multi-label confusion matrix, formally formulates three multi-labelling performance measures and adapts one-against-all multi-class probabilistic outputs to multi-label learning scenario, based on which to further extends our published work GO-TLM (gene ontology based transfer learning model for protein subcellular localization and MK-TLM (multi-kernel transfer learning based on Chou's PseAAC formulation for protein submitochondria localization for multiplex human protein subcellular localization. With the advantages of proper homolog knowledge transfer, comprehensive survey of model performance for novel protein and multi-labelling capability, MLMK-TLM will gain more practical applicability. The experiments on human protein benchmark dataset show that MLMK-TLM significantly outperforms the baseline model and demonstrates good multi-labelling ability for novel human proteins. Some findings (predictions are validated by the latest Swiss-Prot database. The software can be freely downloaded at http://soft.synu.edu.cn/upload/msy.rar.

  19. Arbuscular mycorrhizal colonization alters subcellular distribution and chemical forms of cadmium in Medicago sativa L. and resists cadmium toxicity.

    Directory of Open Access Journals (Sweden)

    Yuanpeng Wang

    Full Text Available Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd in alfalfa (Medicago sativa L. that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25-60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg kg(-1 Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mgkg(-1 Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2-80.5% in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation.

  20. Study of Se subcellular distribution in human tissues by neutron activation analysis

    International Nuclear Information System (INIS)

    Subcellular distribution of selenium in certain important human tissues is studied by neutron activation analysis combined with differential centrifugation method. The standard reference materials, bovine liver NIST 1577a and horse kidney IAEA H8, are used to validate the analytical quality. The experimental results show that the slenium levels in various individuals are significantly different. Body selenium is highly enriched in human kidney. Different subcellular selenium distribution patterns are also found in different tissues. The selenium levels in various organelles are sequenced as mitochondria >nuclei>cytosol>lysosome, microsome in liver; nuclei>mitochondria>cytosol>lysosome, microsome in kidney; and mitochondria, nuclei>cytosol, lysosome, microsome in heart

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

    International Nuclear Information System (INIS)

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

  2. Bioaccumulation, subcellular distribution and toxicity of sediment-associated copper in the ragworm Nereis diversicolor: The relative importance of aqueous copper, copper oxide nanoparticles and microparticles

    International Nuclear Information System (INIS)

    The sediment-dwelling ragworm, Nereis diversicolor was exposed to sediment spiked with aqueous Cu (CuAq, CuCl2), CuO nanoparticles (CuONP) or CuO microparticles (CuOMicro) at 150 μg Cu g−1 dw sediment for 10d. Exposures to CuAq and CuOMicro caused mortality (62.5 and 37.5%, respectively), whereas mean burrowing time increased during exposure to CuAq and CuONP from 0.12 h (controls) to 19.3 and 12.2 h, respectively. All Cu treatments bioaccumulated, especially CuAq (up to 4 times more than the other treatments). Cu was roughly equally distributed among the five subcellular fractions in controls and worms exposed to CuONP or CuOMicro. In contrast, ≈50% of accumulated Cu in CuAq exposed worms was found in metal rich granules and significantly more Cu was present in heat-denatured proteins and organelles than in worms exposed to CuOMicro or in controls. Our results suggest that Cu form affects its bioaccumulation and subsequent toxicity and detoxification in a polychaete like N. diversicolor. - Highlights: • CuAq exposure resulted in Cu body burden up to 4 times higher than CuONP and CuOMicro. • CuAq was the most toxic (62.5% mortality) of the treatments tested. • Subcellular Cu distribution after CuAq exposure differed significantly from control. • Cu in CuAq exposed worms was mainly found in metal rich granules (≈50%). - CuAq was more bioavailable and toxic, and showed a different subcellular distribution compared to the particulate forms tested (CuONP and CuOMicro)

  3. Oxidative stress and the subcellular localization of the telomerase reverse transcriptase (TERT) in papillary thyroid cancer.

    Science.gov (United States)

    Muzza, Marina; Colombo, Carla; Cirello, Valentina; Perrino, Michela; Vicentini, Leonardo; Fugazzola, Laura

    2016-08-15

    During hormonogenesis, thyrocytes are physiologically exposed to high levels of oxidative stress (OS) which could either be involved in the pathogenesis of thyroid cancer or exert a cytotoxic effect. We analyzed the oxidative status of papillary thyroid cancer (PTC) both directly, by measuring H2O2 generation by NADPH oxidases (NOXs), and indirectly, by evaluating the antioxidant activity of glutathione peroxidase (GPX), which neutralizes H2O2 excess, and the lipid peroxidation (LP). Moreover, we investigated the subcellular localization of telomerase reverse transcriptase (TERT), and the H2O2 levels in the mitochondria of tumor and normal tissues. The calcium-dependent and independent H2O2 generation activity was significantly higher in tumors than in normal tissues. The GPX activity was higher in PTCs than in normal tissues, and, consistently, no differences were found in LP levels. Moreover, while TERT nuclear expression was similar in tumor and normal tissues, the mitochondrial localization was significantly higher in tumors. At the mitochondrial level, no differences were found in H2O2 generation between tumor and normal tissues. In conclusion, present data demonstrate that the intracellular H2O2 generation by NOXs is significantly higher in PTCs than in normal thyroid tissues. The increased GPX activity found in tumors counteracts the potential cytotoxic effects of high OS exposure. The significantly higher mitochondrial localization of TERT in tumors is consistent with its shuttling from the nucleus upon exposure to high OS. Finally, mitochondrial OS was not significantly different in tumors and normal tissues, supporting the postulated role of mitochondrial TERT in the control of local H2O2 production. PMID:27164443

  4. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Recovery from cadmium exposure

    International Nuclear Information System (INIS)

    Studies in the recovery from metal stress and the tolerance development to metal exposure of aquatic organisms are important for the understanding of epidemic pollution. In this study, the responses of a marine diatom, Thalassiosira nordenskioeldii, following recovery from environmental cadmium (Cd) stress were investigated. The diatoms were exposed to different concentrations of Cd for 7 days, and were then allowed different periods of time to recover. The Cd sensitivity increased after recovery from Cd stress, followed by a gradual restoration. The extent of restoration depended on both the recovery time and the environmental Cd stress during the exposure period. A complete restoration of Cd tolerance proved to be impossible for cells pre-exposed to High-Cd. The Cd cellular burden and subcellular Cd concentration decreased to the control level within the first day of recovery, indicating that the elevated sensitivity may have been due to the accumulation of functional damage caused by Cd exposure instead of a result of physical Cd accumulation. The rapid change in phytochelatins (PC) to both the increase in and the withdrawal of environmental Cd stress made it a good quantitative bioindicator of environmental Cd contamination. However, the relationships between Cd distribution in the metal sensitive fraction (MSF-Cd) or intracellular Cd to thiol ratio (intra-Cd/PC-SH) and the relative change in the median inhibition [Cd2+] ([Cd2+]-based-IC50, i.e., Cd sensitivity) differed for the various exposure and recovery periods tested. Our study suggests that more attention should be given to the recovery of aquatic organisms from episodic metal exposure.

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

    Directory of Open Access Journals (Sweden)

    P.B. Lintongan

    2004-06-01

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

  6. Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    Björn, Erik; Nygren, Yvonne; Nguyen, Tam T. T. N.;

    2007-01-01

    A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines...

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

    International Nuclear Information System (INIS)

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

  8. Subcellular Localization of Galloylated Catechins in Tea Plants (Camellia sinensis (L. O. Kuntze Assessed via Immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Huanhuan eXu

    2016-05-01

    Full Text Available Galloylated catechins, as the main secondary metabolites in the tea plant, including (--epigallocatechin-3-gallate and (--epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilised for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level.

  9. High resolution intravital imaging of subcellular structures of mouse abdominal organs using a microstage device.

    Science.gov (United States)

    Cao, Liqin; Kobayakawa, Satoru; Yoshiki, Atsushi; Abe, Kuniya

    2012-01-01

    Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox. PMID:22479464

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

    Directory of Open Access Journals (Sweden)

    Kohlbacher Oliver

    2009-09-01

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

  11. Subcellular binding of 239Pu in the liver of selected species of rodents

    International Nuclear Information System (INIS)

    The subcellular distribution of 239Pu in the liver of selected rodent species was investigated as well as the relation between 239Pu and the iron metabolism. The goal of the investigation was to find out why the liver discharge of 239Pu from the liver varies so much between species. (orig.)

  12. Different subcellular locations of secretome components of Gram-positive bacteria

    NARCIS (Netherlands)

    Buist, Girbe; Ridder, Anja N. J. A.; Kok, Jan; Kuipers, Oscar P.

    2006-01-01

    Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various

  13. A multi-label predictor for identifying the subcellular locations of singleplex and multiplex eukaryotic proteins.

    Directory of Open Access Journals (Sweden)

    Xiao Wang

    Full Text Available Subcellular locations of proteins are important functional attributes. An effective and efficient subcellular localization predictor is necessary for rapidly and reliably annotating subcellular locations of proteins. Most of existing subcellular localization methods are only used to deal with single-location proteins. Actually, proteins may simultaneously exist at, or move between, two or more different subcellular locations. To better reflect characteristics of multiplex proteins, it is highly desired to develop new methods for dealing with them. In this paper, a new predictor, called Euk-ECC-mPLoc, by introducing a powerful multi-label learning approach which exploits correlations between subcellular locations and hybridizing gene ontology with dipeptide composition information, has been developed that can be used to deal with systems containing both singleplex and multiplex eukaryotic proteins. It can be utilized to identify eukaryotic proteins among the following 22 locations: (1 acrosome, (2 cell membrane, (3 cell wall, (4 centrosome, (5 chloroplast, (6 cyanelle, (7 cytoplasm, (8 cytoskeleton, (9 endoplasmic reticulum, (10 endosome, (11 extracellular, (12 Golgi apparatus, (13 hydrogenosome, (14 lysosome, (15 melanosome, (16 microsome, (17 mitochondrion, (18 nucleus, (19 peroxisome, (20 spindle pole body, (21 synapse, and (22 vacuole. Experimental results on a stringent benchmark dataset of eukaryotic proteins by jackknife cross validation test show that the average success rate and overall success rate obtained by Euk-ECC-mPLoc were 69.70% and 81.54%, respectively, indicating that our approach is quite promising. Particularly, the success rates achieved by Euk-ECC-mPLoc for small subsets were remarkably improved, indicating that it holds a high potential for simulating the development of the area. As a user-friendly web-server, Euk-ECC-mPLoc is freely accessible to the public at the website http://levis.tongji.edu.cn:8080/bioinfo

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-10-01

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

  16. Sub-cellular force microscopy in single normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  17. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  18. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Science.gov (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  19. Sub-cellular force microscopy in single normal and cancer cells

    International Nuclear Information System (INIS)

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain

  20. Predicting the subcellular location of apoptosis proteins based on recurrence quantification analysis and the Hilbert-Huang transform

    Institute of Scientific and Technical Information of China (English)

    Han Guo-Sheng; Yu Zu-Guo; Anh Vo

    2011-01-01

    Apoptosis proteins play an important role in the development and homeostasis of an organism.The elucidation of the subcellular locations and functions of these proteins is helpful for understanding the mechanism of programmed cell death.In this paper,the recurrent quantification analysis,Hilbert-Huang transform methods,the maximum relevance and minimum redundancy method and support vector machine are used to predict the subcellular location of apoptosis proteins.The validation of the jackknife test suggests that the proposed method can improve the prediction accuracy of the subcellular location of apoptosis proteins and its application may be promising in other fields.

  1. Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein

    Institute of Scientific and Technical Information of China (English)

    Wei-wei Pan; Jing Long; Jun-ji Xing; Chun-fu Zheng

    2011-01-01

    The function of the herpes simplex virus type 1(HSV-1)UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study,fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein(EYFP),the nuclear export signals(NES)of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition,the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4.Furthermore,the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1(CRM l)-dependent manner involving RanGTP hydrolysis.

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

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Ørtenblad, Niels

    2013-01-01

    and fatigability. Based on all these data, the available literature strongly indicates that the subcellular localization of glycogen has to be taken into consideration to fully understand and appreciate the role and regulation of glycogen metabolism and signaling in skeletal muscle. A full......Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electron microscopy images as roughly spherical particles (known as β-particles of 10-45 nm in diameter), which are distributed in distinct localizations within the myofibers and are physically associated...... with metabolic and scaffolding proteins. Although the subcellular localization of glycogen has been recognized for more than 40 years, the physiological role of the distinct localizations has received sparse attention. Recently, however, studies involving stereological, unbiased, quantitative methods...

  3. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    Science.gov (United States)

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  4. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin.

    Science.gov (United States)

    Upadhyay, Srijana; Xu, Xinping; Lowry, David; Jackson, Jennifer C; Roberson, Robert W; Lin, Xiaorong

    2016-03-22

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes. PMID:26972005

  5. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

    Directory of Open Access Journals (Sweden)

    Srijana Upadhyay

    2016-03-01

    Full Text Available Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  6. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    International Nuclear Information System (INIS)

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  7. Emergent cell and tissue dynamics from subcellular modeling of active biomechanical processes

    International Nuclear Information System (INIS)

    Cells and the tissues they form are not passive material bodies. Cells change their behavior in response to external biochemical and biomechanical cues. Behavioral changes, such as morphological deformation, proliferation and migration, are striking in many multicellular processes such as morphogenesis, wound healing and cancer progression. Cell-based modeling of these phenomena requires algorithms that can capture active cell behavior and their emergent tissue-level phenotypes. In this paper, we report on extensions of the subcellular element model to model active biomechanical subcellular processes. These processes lead to emergent cell and tissue level phenotypes at larger scales, including (i) adaptive shape deformations in cells responding to slow stretching, (ii) viscous flow of embryonic tissues, and (iii) streaming patterns of chemotactic cells in epithelial-like sheets. In each case, we connect our simulation results to recent experiments

  8. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Subhash Chandra

    2008-05-30

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  9. Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yun-xiang; LIU Pei; XU Zhao-shi; CHEN Ming; LI Lian-cheng; CHEN Yao-feng; XIONG Xiang-jin; MA You-zhi

    2008-01-01

    The study aims to detect the subcellular localization of ERF (ethylene-responsive element binding factor) transcription factor W17 protein, the interaction between W17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5mmol L-1) was added to induce the expression of recombinant GST/W17 for 3h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses (e. g., salt-) induced signaling pathway.

  10. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

    OpenAIRE

    Furger, AM; Neve, J; Burger, K; Patel, R.; Gullerova, M; Li, W.; Hoque, M.; Tian, B.

    2015-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we employed a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected t...

  11. Quantifying subcellular dynamics in apoptotic cells with two-dimensional Gabor filters

    OpenAIRE

    Pasternack, Robert M.; Rabin, Bryan; Zheng, Jing-Yi; Boustany, Nada N.

    2010-01-01

    We demonstrate an optical Fourier filtering method which can be used to characterize subcellular morphology during dynamic cellular function. In this paper, our Fourier filters were based on two-dimensional Gabor elementary functions, which can be tuned to sense directly object size and orientation. We utilize this method to quantify changes in mitochondrial and nuclear structure during the first three hours of apoptosis. We find that the technique is sensitive to a decrease in particle orien...

  12. Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene

    OpenAIRE

    Guirimand, Grégory; Guihur, Anthony; Phillips, Michael A.; Oudin, Audrey; Glévarec, Gaëlle; Mahroug, Samira; Melin, Céline; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Rodríguez-Concepción, Manuel; Burlat, Vincent; Courdavault, Vincent

    2012-01-01

    Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple...

  13. APSLAP: an adaptive boosting technique for predicting subcellular localization of apoptosis protein.

    Science.gov (United States)

    Saravanan, Vijayakumar; Lakshmi, P T V

    2013-12-01

    Apoptotic proteins play key roles in understanding the mechanism of programmed cell death. Knowledge about the subcellular localization of apoptotic protein is constructive in understanding the mechanism of programmed cell death, determining the functional characterization of the protein, screening candidates in drug design, and selecting protein for relevant studies. It is also proclaimed that the information required for determining the subcellular localization of protein resides in their corresponding amino acid sequence. In this work, a new biological feature, class pattern frequency of physiochemical descriptor, was effectively used in accordance with the amino acid composition, protein similarity measure, CTD (composition, translation, and distribution) of physiochemical descriptors, and sequence similarity to predict the subcellular localization of apoptosis protein. AdaBoost with the weak learner as Random-Forest was designed for the five modules and prediction is made based on the weighted voting system. Bench mark dataset of 317 apoptosis proteins were subjected to prediction by our system and the accuracy was found to be 100.0 and 92.4 %, and 90.1 % for self-consistency test, jack-knife test, and tenfold cross validation test respectively, which is 0.9 % higher than that of other existing methods. Beside this, the independent data (N151 and ZW98) set prediction resulted in the accuracy of 90.7 and 87.7 %, respectively. These results show that the protein feature represented by a combined feature vector along with AdaBoost algorithm holds well in effective prediction of subcellular localization of apoptosis proteins. The user friendly web interface "APSLAP" has been constructed, which is freely available at http://apslap.bicpu.edu.in and it is anticipated that this tool will play a significant role in determining the specific role of apoptosis proteins with reliability. PMID:23982307

  14. Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization.

    OpenAIRE

    Fleming, R.E.; Parkkila, S; Parkkila, A K; Rajaniemi, H; Waheed, A; Sly, W S

    1995-01-01

    Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-linked isozyme previously identified on the surface of renal tubular epithelium and certain populations of vascular endothelium. This report identifies the regional, cellular, and subcellular localization of CA IV in the rat gut. Northern blot and RT-PCR analyses demonstrated little CA IV expression in stomach or proximal small intestine, but abundant expression in distal small and large intestine. In contrast, CA II mRNA was abu...

  15. Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity

    OpenAIRE

    Chenette, Emily J.; Mitin, Natalia Y.; Der, Channing J.

    2006-01-01

    Cdc42 homologous protein (Chp) is a member of the Rho family of small GTPases and shares significant sequence and functional similarity with Cdc42. However, unlike classical Rho GTPases, we recently found that Chp depends on palmitoylation, rather than prenylation, for association with cellular membranes. Because palmitoylation alone is typically not sufficient to promote membrane association, we evaluated the possibility that other carboxy-terminal residues facilitate Chp subcellular associa...

  16. Cellular and subcellular localization of protein I in the peripheral nervous system.

    OpenAIRE

    Fried, G.; Nestler, E J; De Camilli, P; Stjärne, L; Olson, L; Lundberg, J M; Hökfelt, T; Ouimet, C C; Greengard, P

    1982-01-01

    The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated tha protein I is present throughout the peripheral nervous system. Denervation studies of adrenal medulla and iris suggested that the protein I contained in peripheral...

  17. Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

    OpenAIRE

    Sengeløv, H.; Kjeldsen, L; Diamond, M S; Springer, T A; Borregaard, N

    1993-01-01

    The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% ...

  18. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

    OpenAIRE

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-ichi; Ashida, Hitoshi

    2009-01-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning f...

  19. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

    OpenAIRE

    Srijana Upadhyay; Xinping Xu; David Lowry; Jennifer C. Jackson; Robert W. Roberson; Xiaorong Lin

    2016-01-01

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs, and how it is deposited as extracellular granules, remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are ...

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

    OpenAIRE

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

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles...

  1. *CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

    OpenAIRE

    Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

    2006-01-01

    Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity ...

  2. Predicting the Subcellular Localization of Human Proteins Using Machine Learning and Exploratory Data Analysis

    Institute of Scientific and Technical Information of China (English)

    George K. Acquaah-Mensah; Sonia M. Leach; Chittibabu Guda

    2006-01-01

    Identifying the subcellular localization of proteins is particularly helpful in the functional annotation of gene products. In this study, we use Machine Learning and Exploratory Data Analysis (EDA) techniques to examine and characterize amino acid sequences of human proteins localized in nine cellular compartments. A dataset of 3,749 protein sequences representing human proteins was extracted from the SWISS-PROT database. Feature vectors were created to capture specific amino acid sequence characteristics. Relative to a Support Vector Machine, a Multi-layer Perceptron, and a Naive Bayes classifier, the C4.5 Decision Tree algorithm was the most consistent performer across all nine compartments in reliably predicting the subcellular localization of proteins based on their amino acid sequences (average Precision=0.88; average Sensitivity=0.86). Furthermore, EDA graphics characterized essential features of proteins in each compartment. As examples,proteins localized to the plasma membrane had higher proportions of hydrophobic amino acids; cytoplasmic proteins had higher proportions of neutral amino acids;and mitochondrial proteins had higher proportions of neutral amino acids and lower proportions of polar amino acids. These data showed that the C4.5 classifier and EDA tools can be effective for characterizing and predicting the subcellular localization of human proteins based on their amino acid sequences.

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

    Directory of Open Access Journals (Sweden)

    Xia Li

    2016-01-01

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

  4. Colocalization of fluorescence and Raman microscopic images for the identification of subcellular compartments: a validation study.

    Science.gov (United States)

    Krauß, Sascha D; Petersen, Dennis; Niedieker, Daniel; Fricke, Inka; Freier, Erik; El-Mashtoly, Samir F; Gerwert, Klaus; Mosig, Axel

    2015-04-01

    A major promise of Raman microscopy is the label-free detailed recognition of cellular and subcellular structures. To this end, identifying colocalization patterns between Raman spectral images and fluorescence microscopic images is a key step to annotate subcellular components in Raman spectroscopic images. While existing approaches to resolve subcellular structures are based on fluorescence labeling, we propose a combination of a colocalization scheme with subsequent training of a supervised classifier that allows label-free resolution of cellular compartments. Our colocalization scheme unveils statistically significant overlapping regions by identifying correlation between the fluorescence color channels and clusters from unsupervised machine learning methods like hierarchical cluster analysis. The colocalization scheme is used as a pre-selection to gather appropriate spectra as training data. These spectra are used in the second part as training data to establish a supervised random forest classifier to automatically identify lipid droplets and nucleus. We validate our approach by examining Raman spectral images overlaid with fluorescence labelings of different cellular compartments, indicating that specific components may indeed be identified label-free in the spectral image. A Matlab implementation of our colocalization software is available at . PMID:25679809

  5. Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro

    International Nuclear Information System (INIS)

    A bovine kidney cell culture system was used to assess what relationship cadmium (Cd) uptake and subcellular distribution had to cadmium chloride induced cytotoxicity. Twenty-four hour incubation with 0.1-10 μM Cd elicited 0-90% cytotoxicity. Fifty percent cytotoxicity was estimated to result from 0.8 μM Cd. A concentration-related Cd accumulation paralleled the cytotoxicity profile. The time-course for Cd accumulation was linear for the first 6 h of exposure and plateaued by 18 h post-exposure. When the degree of cytotoxicity was compared with the cellular Cd burden at 24 h post-treatment a least-squares linear regression analysis (r=0.93) indicated a direct relationship. Subcellular distribution studies indicated greater than 90% Cd recovery from the soluble supernatant (105,000 g) at all levels of cytotoxicity studied. Metallothionein sequestered less than 25% of the cellular Cd. As a result of the correlation of the degree of cytotoxicity with the cellular Cd burden and the independence of subcellular distribution from cytotoxicity, a cumulative mechanism of toxicity for Cd in MDBK cells was suggested. (author)

  6. Tissue and subcellular distribution of zinc-65 in normal and zinc sulphate administered rats

    International Nuclear Information System (INIS)

    Uptake of 65Zn by various organs of normal and ZnSO4 administered rats has been studied. Except duodenum and blood all the organs show low 65Zn incorporation in treated rats. The lower 65Zn incorporation in the tissues of ZnSO4 treated rats is due to the active turnover of zinc in these tissues. The maximum activity of 65Zn is exhibited by skin, followed in decreasing order by liver, intestine, spleen, rectum, kidney, prostate, urinary bladder, stomach, pancreas, adrenals, trachea, lungs, heart, thymus, seminal vesicles, testis, cauda epididymis, caput epididymis, brain, and muscle. Since ZnSO4 administration enhanced the levels of inert zinc in liver and intestine it is likely that accumulated inert zinc in the tissues may inhibit the further incorporation of 65Zn in these tissues. At subcellular level, the cytosol fraction (11000 g supernatant fraction) exhibits the higher 65Zn activity, followed in decreasing order by 300 g fraction (cell membranes and cell debris), 900 g fraction (nuclei), 6000 g fraction (heavy mitochondria) and 1000 g fraction (light mitochondria). The 65Zn activity/mgprotein in 11000 g fraction (light mitochondria) is more than the 900 g fraction (nuclei) and 6000 g fraction (heavy mitochondria). Subcellular distribution pattern of 65Zn in liver, intestine, kidney and testis is similar. ZnSO4 administration does not change the tissue and subcellular distribution pattern of 65Zn. (author). 18 refs., 3 tables

  7. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    Science.gov (United States)

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  8. A workflow for mathematical modeling of subcellular metabolic pathways in leaf metabolism of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eNägele

    2013-12-01

    Full Text Available During the last decade genome sequencing has experienced a rapid technological development resulting in numerous sequencing projects and applications in life science. In plant molecular biology, the availability of sequence data on whole genomes has enabled the reconstruction of metabolic networks. Enzymatic reactions are predicted by the sequence information. Pathways arise due to the participation of chemical compounds as substrates and products in these reactions. Although several of these comprehensive networks have been reconstructed for the genetic model plant Arabidopsis thaliana, the integration of experimental data is still challenging. Particularly the analysis of subcellular organization of plant cells limits the understanding of regulatory instances in these metabolic networks in vivo. In this study, we develop an approach for the functional integration of experimental high-throughput data into such large-scale networks. We present a subcellular metabolic network model comprising 524 metabolic intermediates and 548 metabolic interactions derived from a total of 2769 reactions. We demonstrate how to link the metabolite covariance matrix of different Arabidopsis thaliana accessions with the subcellular metabolic network model for the inverse calculation of the biochemical Jacobian, finally resulting in the calculation of a matrix which satisfies a Lyaponov equation involving a covariance matrix. In this way, differential strategies of metabolite compartmentation and involved reactions were identified in the accessions when exposed to low temperature.

  9. KCC2-dependent subcellular ECl difference of ON-OFF retinal ganglion cells in larval zebrafish

    Directory of Open Access Journals (Sweden)

    Rongwei eZhang

    2013-05-01

    Full Text Available Subcellular difference in the reversal potential of Cl- (ECl has been found in many types of neurons. As local ECl largely determines the action of nearby GABAergic/glycinergic synapses, subcellular ECl difference can effectively regulate neuronal computation. The ON-OFF retinal ganglion cell (RGC processes both ON and OFF visual signals via its ON and OFF dendrites, respectively. It is thus interesting to investigate whether the ON and OFF dendrites of single RGCs exhibit different local ECl. Here, using in vivo gramicidin-perforated patch recording in larval zebrafish ON-OFF RGCs, we examine local ECl at the ON and OFF dendrites, and soma through measuring light-evoked ON and OFF inhibitory responses, and GABA-induced response at the soma, respectively. We find there are subcellular ECl differences between the soma and dendrite, as well as between the ON and OFF dendrites of single RGCs. These somato-dendritic and inter-dendritic ECl differences are dependent on the Cl- extruder, K+/Cl- co-transporter (KCC2, because they are largely diminished by down-regulating kcc2 expression with morpholino oligonucleotides or by blocking KCC2 function with furosemide. Thus, our findings indicate that there exists KCC2-dependent ECl difference between the ON and OFF dendrites of individual ON-OFF RGCs that may differentially affect visual processing in the ON and OFF pathways.

  10. Parasites modify sub-cellular partitioning of metals in the gut of fish

    Energy Technology Data Exchange (ETDEWEB)

    Oyoo-Okoth, Elijah, E-mail: elijaoyoo2009@gmail.com [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Admiraal, Wim [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Osano, Odipo [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Kraak, Michiel H.S. [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Gichuki, John; Ogwai, Caleb [Kenya Marine and Fisheries Research Institute, P.O. Box 1881, Kisumu (Kenya)

    2012-01-15

    Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the subcellular fractions of the gut in presence of an endoparasite (Ligula intestinalis). The fish were sampled along four sites in Lake Victoria, Kenya differing in metal contamination. Accumulation of Cd, Cr and Zn was higher in the whole body and in the gut of parasitized fish compared to non-parasitized fish, while Cu was depleted in parasitized fish. Generally, for both non-parasitized and parasitized fish, Cd, Cr and Zn partitioned in the cytosolic fractions and Cu in the particulate fraction. Metal concentrations in organelles within the particulate fractions of the non-parasitized fish were statistically similar except for Cd in the lysosome, while in the parasitized fish, Cd, Cr and Zn were accumulated more by the lysosome and microsomes. In the cytosolic fractions, the non-parasitized fish accumulated Cd, Cr and Zn in the heat stable proteins (HSP), while in the parasitized fish the metals were accumulated in the heat denatured proteins (HDP). On the contrary, Cu accumulated in the HSP in parasitized fish. The present study revealed specific binding of metals to potentially sensitive sub-cellular fractions in fish in the presence of parasites, suggesting interference with metal detoxification, and potentially affecting the health status of fish hosts in Lake Victoria.

  11. Parasites modify sub-cellular partitioning of metals in the gut of fish

    International Nuclear Information System (INIS)

    Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the subcellular fractions of the gut in presence of an endoparasite (Ligula intestinalis). The fish were sampled along four sites in Lake Victoria, Kenya differing in metal contamination. Accumulation of Cd, Cr and Zn was higher in the whole body and in the gut of parasitized fish compared to non-parasitized fish, while Cu was depleted in parasitized fish. Generally, for both non-parasitized and parasitized fish, Cd, Cr and Zn partitioned in the cytosolic fractions and Cu in the particulate fraction. Metal concentrations in organelles within the particulate fractions of the non-parasitized fish were statistically similar except for Cd in the lysosome, while in the parasitized fish, Cd, Cr and Zn were accumulated more by the lysosome and microsomes. In the cytosolic fractions, the non-parasitized fish accumulated Cd, Cr and Zn in the heat stable proteins (HSP), while in the parasitized fish the metals were accumulated in the heat denatured proteins (HDP). On the contrary, Cu accumulated in the HSP in parasitized fish. The present study revealed specific binding of metals to potentially sensitive sub-cellular fractions in fish in the presence of parasites, suggesting interference with metal detoxification, and potentially affecting the health status of fish hosts in Lake Victoria.

  12. Uptake pathways and subcellular fractionation of Cd in the polychaete Nereis diversicolor.

    Science.gov (United States)

    Li, Lianzhen; Liu, Xiaoli; You, Liping; Zhang, Linbao; Zhao, Jianmin; Wu, Huifeng

    2012-01-01

    Polychaetes have often been utilized as indicator species to investigate the impacts of pollutants, such as heavy metals. The uptake of Cd by the polychaete Nereis diversicolor was determined at varying Ca concentrations and with pre-exposure to Ca ion channel blockers and metabolic inhibitors in simulated sea water over 1 week period. The supply of Ca in simulated sea water inhibited Cd uptake and increased Ca concentration in N. diversicolor after 10 μM Cd exposure. Pre-exposure to a Ca-channel blocker (Lanthanum) significantly inhibited Cd uptake, suggesting that the uptake of Cd was exerted at a Ca channel. N-ethylmaleimide, which specifically binds to sulfhydryl groups, inhibited Cd uptake at 10 μM, implying that the transport of Cd is carrier-mediated by proteins or other SH-containing compounds. Subcellular Cd distribution analysis showed that more than 60% of the total Cd associated with the cytosolic fraction. The presence of higher concentration of Ca in simulated sea water did not impact the proportional subcellular distribution of Cd in N. diversicolor. Nevertheless, the supply of Ca could significantly lower Cd concentration in cytosol and cellular debris. The present study provides evidence that Cd transport by N. diversicolor was mediated mainly through lanthanum- sensitive Ca ion channels and accumulated by SH-containing compounds. These results help to understand the uptake mechanism and subcellular distribution of Cd in polychaetes. PMID:21858512

  13. The roles of nucleolin subcellular localization in cancer.

    Science.gov (United States)

    Berger, Caroline Madeleine; Gaume, Xavier; Bouvet, Philippe

    2015-06-01

    Nucleolin (NCL) is one of the most abundant non ribosomal protein of the nucleolus where it plays a central role in polymerase I transcription. NCL is also found outside of the nucleolus, in the nucleoplasm, cytoplasm as well as on the cell membrane. It acts in all cell compartments to control cellular homeostasis and therefore each cellular pool of NCL can play a different role in cancer development. NCL overexpression and its increased localization at the cell membrane is a common feature of several tumor cells. In cancer cells, NCL overexpression influences cell survival, proliferation and invasion through its action on different cellular pathways. In this review, we describe how the multiple functions of NCL that are associated to its multiple cellular localization can participate to the development of cancer. PMID:25866190

  14. PathLocdb: a comprehensive database for the subcellular localization of metabolic pathways and its application to multiple localization analysis

    OpenAIRE

    Zhao Min; Qu Hong

    2010-01-01

    Abstract Background In eukaryotes, the cell is divided into several compartments enclosed by unitary membranes. Such compartmentalization is critical for cells to restrict different pathways to be carried out in different subcellular regions. The summary and classification of subcellular localizations of metabolic pathways are the first steps towards understanding their roles in spatial differentiation and the specialization of metabolic pathways in different organisms. Results Integrating th...

  15. Subcellular location of the enzymes of purine breakdown in the yeast Candida famata grown on uric acid

    OpenAIRE

    Large, Peter J.; Waterham, Hans R.; Veenhuis, Marten

    1990-01-01

    The subcellular location of the enzymes of purine breakdown in the yeast Candida famata, which grows on uric acid as sole carbon and nitrogen source, has been examined by subcellular fractionation methods. Uricase was confirmed as being peroxisomal, but the other three enzymes, allantoinase, allantoicase and ureidoglycollate lyase were shown to be cytosolic. In addition the peroxisomes harboured catalase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase.

  16. Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells: FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION*

    OpenAIRE

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

    2011-01-01

    NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribo...

  17. Bioaccumulation, subcellular distribution and toxicity of sediment-associated copper in the ragworm Nereis diversicolor: The relative importance of aqueous copper, copper oxide nanoparticles and microparticles

    DEFF Research Database (Denmark)

    Thit, Amalie; Banta, Gary Thomas; Selck, Henriette

    2015-01-01

    and worms exposed to CuONP or CuOMicro. In contrast, ≈50% of accumulated Cu in CuAq exposed worms was found in metal rich granules and significantly more Cu was present in heat-denatured proteins and organelles than in worms exposed to CuOMicro or in controls. Our results suggest that Cu form affects...... mean burrowing time increased during exposure to CuAq and CuONP from 0.12 h (controls) to 19.3 and 12.2 h, respectively. All Cu treatments bioaccumulated, especially CuAq (up to 4 times more than the other treatments). Cu was roughly equally distributed among the five subcellular fractions in controls...

  18. Influence of the route of exposure on the accumulation and subcellular distribution of nickel and thallium in juvenile fathead minnows (Pimephales promelas).

    Science.gov (United States)

    Lapointe, Dominique; Couture, Patrice

    2009-10-01

    In this study, we examine the relative contribution of water and live prey (Tubifex tubifex) as sources of nickel (Ni) and thallium (Tl) in juvenile fathead minnows (Pimephales promelas). Overall, both water and prey were important sources of metals for our fish, although only approximately 35% of the metal estimated available for trophic transfer in the prey was assimilated. We also investigated the influence of exposure route on the subcellular distribution of these two metals. Once assimilated, most of the Ni was found in the granules, debris, and heat-stable protein (HSP), regardless of the route of exposure. Thallium was also mostly located in granules, debris, and HSP, and fish exposed from both water and prey had a higher proportion of Tl bound to the HSP compartment compared to control fish. Our results, obtained using environmentally relevant concentrations, suggest the presence of regulation mechanisms for both metals. Nevertheless, we measured increased metal concentrations in potentially metal-sensitive subcellular fractions when fish were exposed from water and diet simultaneously compared to a single route of exposure, suggesting that exposure to Ni and Tl from both routes could represent a risk of toxicity. PMID:19253010

  19. IRF-2 regulates NF-κB activity by modulating the subcellular localization of NF-κB

    International Nuclear Information System (INIS)

    Nuclear Factor-kappa B (NF-κB) is a transcription factor essential to the control of cell proliferation, survival, differentiation, immune response, and inflammation. Constitutive NF-κB activation has been observed in a broad variety of solid tumors and hematological malignancies, which suggests that NF-κB signaling may perform a critical role in the development of human cancers. Interferon regulatory factor-2 (IRF-2), an antagonistic transcriptional repressor of IRF-1, evidences oncogenic potential, but little is currently known regarding the mechanism underlying the oncogenic activities of IRF-2. In this study, we report that IRF-2 recruits RelA/p65 transcription factors into the nucleus via physical interaction. While the nuclear recruitment of RelA by IRF-2 augments TNFα-induced NF-κB dependent transcription, the N-terminal truncated mutant form of IRF-2 inhibits the nuclear localization of RelA, and thus interferes with NF-κB activation. Furthermore, the knockdown of IRF-2 by IRF-2 siRNA attenuates TNFα-induced NF-κB dependent transcription by inhibiting the nuclear localization of RelA. Thus, these results show that IRF-2 regulates NF-κB activity via the modulation of NF-κB subcellular localization

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

    International Nuclear Information System (INIS)

    Delta-9-tetrahydrocannabinol (Δ9-THC), the primary psychoactive component of marihuana, and its active metabolite 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC) have been reported to produce a direct cardiac depressant effect. Studies in isolated perfused rat hearts have indicated a decreased force of contraction (inotropic response) when Δ9-THC or 11-OH-Δ9-THC was administered in microgram amounts. The mechanism and site of action have not been explained or correlated with associated metabolic pathways. The purpose of this study was to investigate the effects of cannabinoids on major myocardial energy producing pathways, GP and KC, and a non-energy producing pathway, HMS. Cardiac ventricular tissue from male Sprague-Dawley rats (250-300 g) was excised and homogenized for subcellular fractionation. KC, GP and HMS activity was assayed in the appropriate fractions by measuring 14CO2 generation from 14C-2-pyruvate, 14C-6-glucose and 14C-1-glucose respectively. Duplicate assays (n=8) were performed on tissue exposed to saline (control), empty liposomes (vehicle) and four doses each of Δ9-THC and 11-OH-Δ9-THC. Changes in metabolic activity and decreases in cardiac contractile performance may be associated

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    , unpredictable failures or mutations in individual cells can lead to serious downstream conditions across the whole organism. The traditional tools of biochemistry struggle to observe such processes: the vast majority are based upon ensemble approaches analysing the properties of bulk populations, which means...

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

    Directory of Open Access Journals (Sweden)

    Feng eChen

    2013-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Sahin Aysegul

    2009-04-01

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

  4. Clinical significance of subcellular localization of KL-6 mucin in primary colorectal adenocarcinoma and metastatic tissues

    Institute of Scientific and Technical Information of China (English)

    Qian Guo; Masatoshi Makuuchi; Wei Tang; Yoshinori Inagaki; Yutaka Midorikawa; Norihiro Kokudo; Yasuhiko Sugawara; Munehiro Nakata; Toshiro Konishi; Hirokazu Nagawa

    2006-01-01

    AIM: To assess subcellular localization of KL-6 mucin and its clinicopathological significance in colorectal carcinoma as well as metastatic lymph node and liver tissues.METHODS: Colorectal carcinoma tissues as well as metastatic lymph node and liver tissues were collected from 82 patients who underwent colorectomy or hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody.RESULTS: Of the 82 colorectal carcinoma patients, 6showed no staining, 29 showed positive staining only in the apical membrane, and 47 showed positive staining in the circumferential membrane and/or cytoplasm.Positive staining was not observed in non-cancerous colorectal epithelial cells surrounding the tumor tissues.The five-year survival rate was significantly lower in cases showing positive staining in the circumferential membrane and/or cytoplasm (63.0%) than those showing positive staining only in the apical membrane (85.7%) and those showing no staining (100%).Statistical analysis between clinicopathological factors and subcellular localization of KL-6 mucin showed that KL-6 localization in the circumferential membrane and/or cytoplasm was significantly associated with the presence of venous invasion (P=0.0003), lymphatic invasion (P<0.0001), lymph node metastasis (P<0.0001),liver metastasis (P=0.058), and advanced histological stage (P<0.0001). Positive staining was observed in all metastatic lesions tested as well as in the primary colorectal carcinoma tissues.CONCLUSION: The subcellular staining pattern of KL-6 in colorectal adenocarcinoma may be an important indicator for unfavorable behaviors such as lymph node and liver metastasis, as well as for the prognosis of patients.

  5. Multiple Features Based Two-stage Hybrid Classifier Ensembles for Subcellular Phenotype Images Classification

    Directory of Open Access Journals (Sweden)

    Bailing Zhang - China

    2010-12-01

    Full Text Available Subcellular localization is a key functional characteristic of proteins. As an interesting ``bio-image informatics'' application, an automatic, reliable and efficient prediction system for protein subcellular localization can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, we propose a two-stage multiple classifier system to improve classification reliability by introducing rejection option. The system is built as a cascade of two classifier ensembles. The first ensemble consists of set of binary SVMs which generalizes to learn a general classification rule and the second ensemble, which also include three distinct classifiers, focus on the exceptions rejected by the rule. A new way to induce diversity for the classifier ensembles is proposed by designing classifiers that are based on descriptions of different feature patterns. In addition to the Subcellular Location Features (SLF generally adopted in earlier researches, three well-known texture feature descriptions have been applied to cell phenotype images, which are the local binary patterns (LBP, Gabor filtering and Gray Level Coocurrence Matrix (GLCM. The different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. Using the public benchmark 2D HeLa cell images, a high classification accuracy 96% is obtained with rejection rate $21\\%$ from the proposed system by taking advantages of the complementary strengths of feature construction and majority-voting based classifiers' decision fusions.

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

    Directory of Open Access Journals (Sweden)

    Wei Gao

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

  7. Subcellular Accumulation of Cadmium in Corn and Wheat Plants at Different Levels of Phosphorus

    Institute of Scientific and Technical Information of China (English)

    YANG; ZHENGSHAOJIAN; 等

    1999-01-01

    Corn and wheat plants were grown in a nutrient culture solution at four levels of phosphorus (0,0.12,0.60 and 3.0mmol L-1) and two levels of cadmium(0 and 4.0umol L-1) in greenhouse for a 18-day period.The concentrations of phosphorus and cadmium in cell wall,cytoplasm and vacuoles of roots and leaves were examined by cell fractionation techniques.With increasing phosphorus in medium,the contents of P in cell wall,cytoplasm and vacuoles of corn and wheat roots and leaves increased.The highest content of P was observed in cell wall,next in vacuoles,and the lowest in cytoplasm.The wheat subcellular fractions in both roots and leaves hab higher concentrations of phosphorus than those of corn.Increasing phosphorus in medium significantly inhibited the intracellular Cd accumulation in both species,However,at P concentration up to 3.0mmol L-1,the Cd content in cell wall was increased.Increasing phosphorus resulted in reduction of the subcellular Cd content in cell wall was increased.Increasing phosphorus resulted in reduction of the subcellualr Cd content in corn and wheat leaves.Compared with corn,the wheat roots had a higher Cd content in the cell wall and vacuoles and a lower in cytoplasm,while in leaf subcellular fractions the wheat cell had a higher Cd content in its vacuoles and a lower one in its cytoplasm,The results indicate that phosphorus may be involved in sequestration of Cd ionic activity in both cell wall and vaculoes by forming insoluble Cd phosphate.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  9. Subcellular localization of SUN2 is regulated by lamin a and Rab5

    OpenAIRE

    Liang, Ying; Chiu, Peng Hang; Yip, Kit Yan; Chan, Siu Yuen

    2011-01-01

    SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the n...

  10. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    Directory of Open Access Journals (Sweden)

    Chen Ji-Yao

    2009-01-01

    Full Text Available Abstract Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  11. Hematoporphyrin derivative induced photodamage to brain tumor cells: Alterations in subcellular membranes

    Science.gov (United States)

    Sreenivasan, Rajesh; Joshi, Preeti G.; Joshi, Nanda B.

    1997-01-01

    Photoinduced structural and functional changes were studied in the subcellular membranes isolated from HpD treated cells. Changes in the limiting anisotropy of lipid specific probes 1,6,Diphenyl-1,3,5,hexatriene (DPH) and 1-(4-Trimethyl ammonium 1,6 diphenyl)-1,3,5,hexatriene toulene sulphonate (TMA-DPH) incorporated into the membrane were used to assess the structural alterations while changes in the activity of the marker enzymes were used to assess the functional alterations. Our results suggest that damage to the endoplasmic reticulum may play an important role in the photosensitization of brain tumor cells.

  12. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate; Breinholt Bekker-Jensen, Dorte; Secher, Anna; Skovgaard, Tine; Kelstrup, Christian; Dmytriyev, Anatoliy; Choudhary, Chuna Ram; Lundby, Carsten; Olsen, Jesper Velgaard

    2012-01-01

    Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4...... subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle...

  13. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    Science.gov (United States)

    Zhang, Yu; Mi, Lan; Xiong, Rongling; Wang, Pei-Nan; Chen, Ji-Yao; Yang, Wuli; Wang, Changchun; Peng, Qian

    2009-07-01

    Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  14. A signal separation technique for sub-cellular imaging using dynamic optical coherence tomography

    CERN Document Server

    Ammari, Habib; Shi, Cong

    2016-01-01

    This paper aims at imaging the dynamics of metabolic activity of cells. Using dynamic optical coherence tomography, we introduce a new multi-particle dynamical model to simulate the movements of the collagen and the cell metabolic activity and develop an efficient signal separation technique for sub-cellular imaging. We perform a singular-value decomposition of the dynamic optical images to isolate the intensity of the metabolic activity. We prove that the largest eigenvalue of the associated Casorati matrix corresponds to the collagen. We present several numerical simulations to illustrate and validate our approach.

  15. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    OpenAIRE

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintain...

  16. An improved procedure for subcellular spatial alignment during live-cell CLEM.

    Directory of Open Access Journals (Sweden)

    Benjamin S Padman

    Full Text Available Live-cell correlative light and electron microscopy (CLEM offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

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

    International Nuclear Information System (INIS)

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

  18. Classification of Subcellular Phenotype Images by Decision Templates for Classifier Ensemble

    Science.gov (United States)

    Zhang, Bailing

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. An automatic, reliable and efficient prediction system for protein subcellular localization is needed for large-scale genome analysis. The automated cell phenotype image classification problem is an interesting "bioimage informatics" application. It can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, three well-known texture feature extraction methods including local binary patterns (LBP), Gabor filtering and Gray Level Coocurrence Matrix (GLCM) have been applied to cell phenotype images and the multiple layer perceptron (MLP) method has been used to classify cell phenotype image. After classification of the extracted features, decision-templates ensemble algorithm (DT) is used to combine base classifiers built on the different feature sets. Different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. For the HeLa cells, the human classification error rate on this task is of 17% as reported in previous publications. We obtain with our method an error rate of 4.8%.

  19. Radioimmunoassay of steroids in homogenates and subcellular fractions of testicular tissue

    International Nuclear Information System (INIS)

    Radioimmunoassays for testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha, 17beta-diol (DIOL) in homogenates of whole testis, interstitial tissue and seminiferous tubules as well as subcellular fractions of the latter were developed. Steroids were extracted with acetone, submitted to several solvent partitions and isolated by a celite: propylene glycol: ethylene glycol column chromatography. Anit-T serum was used for the assay of T and DTH, and a specific anti-Diol serum for DIOL. Subcellular fractions were separated by differential centrifugation. The nuclear fraction was purified by centrifugation in a dense sucrose buffer followed by several washings. Losses were corrected according to recovery of DNA. Optimal conditions for purification of acetone extracts at minimal losses were established. Validation of the method was studied testing linear regression of logit-log transformations of standard curves and parallelism with unknowns. T was the steroid present in higher concentrations in all samples studied. It is concluded that the present method for determination of endogenous androgen concentrations in testicular tissue is valid and might be useful in studing testicular function. (orig.)

  20. Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.

    Directory of Open Access Journals (Sweden)

    Zhang Yi

    2010-09-01

    Full Text Available Abstract Background Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency. Results Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake ( Conclusions Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.

  1. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    Science.gov (United States)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  2. Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

    Science.gov (United States)

    Greiser, Maura; Kerfant, Benoît-Gilles; Williams, George S.B.; Voigt, Niels; Harks, Erik; Dibb, Katharine M.; Giese, Anne; Meszaros, Janos; Verheule, Sander; Ravens, Ursula; Allessie, Maurits A.; Gammie, James S.; van der Velden, Jolanda; Lederer, W. Jonathan; Dobrev, Dobromir; Schotten, Ulrich

    2014-01-01

    Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia. PMID:25329692

  3. Analysis of potato virus X replicase and TGBp3 subcellular locations

    International Nuclear Information System (INIS)

    Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

  4. Mutational analyses of the signals involved in the subcellular location of DSCR1

    Directory of Open Access Journals (Sweden)

    Henrique-Silva Flávio

    2002-09-01

    Full Text Available Abstract Background Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR, to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human. Results The protein's location was preferentially nuclear, independently of the isoform, cell line and insertion in the GFP's N- or C-terminal. A segment in the C-terminal, which is important in the location of the protein, was identified by deletion. On the other hand, site-directed mutational analyses have indicated the involvement of some serine and threonine residues in this event. Conclusion In this paper, we discuss the identification of amino acids which can be important for subcellular location of DSCR1. The involvement of residues that are prone to phosphorylation suggests that the location and function of DSCR1 may be regulated by kinases and/or phosphatases.

  5. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L

    International Nuclear Information System (INIS)

    Phytolacca americana L. (pokeweed) is a promising species for Cd phytoextraction with large biomass and fast growth rate. To further understand the mechanisms involved in Cd tolerance and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in pokeweed. Subcellular fractionation of Cd-containing tissues indicated that both in root and leaves, the majority of the element was located in soluble fraction and cell walls. Meanwhile, Cd taken up by pokeweed existed in different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 80% ethanol in roots, followed by 1 M NaCl, d-H2O and 2% HAc, while in leaves and stems, most of the Cd was extracted by 1 M NaCl, and the subdominant amount of Cd was extracted by 80% ethanol. It could be suggested that Cd compartmentation with organo-ligands in vacuole or integrated with pectates and proteins in cell wall might be responsible for the adaptation of pokeweed to Cd stress.

  6. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    Science.gov (United States)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  7. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

    Science.gov (United States)

    Tanz, Sandra K; Castleden, Ian; Hooper, Cornelia M; Small, Ian; Millar, A Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1-Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  8. Subcellular Distribution of Gallium-67 and other Auger-emitting Radionuclides in Human and Animal Tissues

    International Nuclear Information System (INIS)

    The subcellular distributions of the Auger-emitting radiopharmaceuticals 67Ga citrate, 111In-bleomycin, 63Zn-bleomycin, 123/125/I-Conray and 123/125I-Biligram in human and animal tumours and normal tissues were studied using differential centrifugation. For 67Ga, at 19 to 54 h after injection, the observed subcellular distribution patterns were similar in all the tissues studied with 2-5% of the total tissue radioactivity being associated with the cell nuclei. For 63Zn and 111In administered as the bleomycin complex or as chloride, as well as for the X ray contrast media 135/125I-Conray and 123/125I-Biligram the fraction associated with the nuclei was only 1-2%. The studies indicate that, for the 67Ga, 111In, 63Zn complexes or compounds studied, only γ1 to 4% of the Auger emissions are likely to arise in the cell nucleus. (author)

  9. β-amylase in developing apple fruits: activities, amounts and subcellular localization

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Dapeng; (张大鹏); WANG; Yongzhang(王永章)

    2002-01-01

    Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β-amylase is considered one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. The present experiment showed that β-amylase activity was progressively increasing concomitantly with decreasing starch concentrations during apple (Malus domestica Borkh cv. Starkrimson) fruit development. The apparent amount of β-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The subcellular-localization studies via immunogold electron-microscopy technique showed that β-amylase visualized by gold particles was predominantly located in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments. These data proved for the first time that the enzyme is compartmented in its functional sites in plant living cells. The predominantly plastid-distributed pattern of β-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (β-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that β-amylase is involved in starch hydrolysis in plastids of the fruit cells.

  10. Transport and subcellular distribution characteristics of human erythrocyte glucose transporter fused in adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Inho.

    1990-01-01

    Purified human erythrocyte glucose transporters (HEGT) were incorporated into the rat epididymal adipocytes by polyethylene glycol (PEG)-induced fusion. The incorporation of HEGT was found to be dependent on both the molecular weight and the concentration of PEG. Optimal incorporation of HEGT occurred when 10% PEG 8000 was used. This incorporation was found to be proportional to the increasing amounts of HEGT used. Morphology tests showed very little surface adsorption of HEGT on adipocytes after fusion. Transport activity of fused HEGT was studied by measuring equilibrium exchange of 3-O-methylglucose. In adipocytes employed this fusion protocols, the transport rate increased significantly compared with cells treated under non-fusion conditions. This fold increase was directly proportional to the amount of HEGT recovered in the plasma membrane (PM). Furthermore, calculated turnover number of HEGT in fused adipocytes was as high as that of the native adipocyte glucose transporter (AGT). Subcellular distribution of HEGT in fused adipocytes was assessed using ({sup 3}H)cytochalasin B-labeled HEGT vesicles. HEGT was distributed in each subcellular organelle at specific ratios. This relative distribution was almost constant regardless of the amount of HEGT used. The distributions of lipid-labeled HEGT, fluid-phase endocytosed HEGT and free HEGT mixed to adipocyte homogenate were shown to be completely different from that of protein-labeled HEGT fusion.

  11. Transport and subcellular distribution characteristics of human erythrocyte glucose transporter fused in adipocytes

    International Nuclear Information System (INIS)

    Purified human erythrocyte glucose transporters (HEGT) were incorporated into the rat epididymal adipocytes by polyethylene glycol (PEG)-induced fusion. The incorporation of HEGT was found to be dependent on both the molecular weight and the concentration of PEG. Optimal incorporation of HEGT occurred when 10% PEG 8000 was used. This incorporation was found to be proportional to the increasing amounts of HEGT used. Morphology tests showed very little surface adsorption of HEGT on adipocytes after fusion. Transport activity of fused HEGT was studied by measuring equilibrium exchange of 3-O-methylglucose. In adipocytes employed this fusion protocols, the transport rate increased significantly compared with cells treated under non-fusion conditions. This fold increase was directly proportional to the amount of HEGT recovered in the plasma membrane (PM). Furthermore, calculated turnover number of HEGT in fused adipocytes was as high as that of the native adipocyte glucose transporter (AGT). Subcellular distribution of HEGT in fused adipocytes was assessed using [3H]cytochalasin B-labeled HEGT vesicles. HEGT was distributed in each subcellular organelle at specific ratios. This relative distribution was almost constant regardless of the amount of HEGT used. The distributions of lipid-labeled HEGT, fluid-phase endocytosed HEGT and free HEGT mixed to adipocyte homogenate were shown to be completely different from that of protein-labeled HEGT fusion

  12. Zn Accumulation and Subcellular Distribution in the Zn Hyperaccumulator Sedum alfredii Hance

    Institute of Scientific and Technical Information of China (English)

    LI Ting-Qiang; YANG Xiao-E; YANG Jin-Yan; HE Zhen-Li

    2006-01-01

    Zn accumulation and subcellular distribution in leaves of the hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum alfredii Hance were studied using radiotracer and gradient centrifugation techniques. Leaf Zn accumulation in the HE of S. alfredii was 18.5-26.7 times greater than that in the NHE when the plants were grown at 1-500μmol Zn L-1. Leaf section uptake of 65Zn was highly dependent on external Zn levels. Greater 65Zn uptake in HE was noted only at external Zn levels ≥ 100μmol L-1. Zinc subcellular distribution in the leaves of the two ecotypes of S. alfredii was: cell wall > soluble fraction > cell organelle. However, more Zn was distributed to the leaf cell wall and soluble fractions for HE than for NHE. In the leaf of HE, 91%-94% of the Zn was found in the cell walls and the soluble fraction and only 6%-9% Zn was distributed in the cell organelle fraction. For NHE, about 20%-26% Zn was recovered in the cell organelle fraction. In stems, Zn distribution to the cell wall fraction was approximately two fold greater in the HE than that in the NHE. For the hyperaccumulating ecotype of S. alfredii, the cell wall and the vacuole played a very important role in Zn tolerance and hyperaccumulation.

  13. Subcellular localization of several heavy metals of Hg,Cd and Pb in human liver

    Institute of Scientific and Technical Information of China (English)

    CHEN Chunying; ZHANG Peiqun; CHAI Zhifang

    2005-01-01

    Liver, as an important metabolic and detoxicological organ of human body, can be used as a good bioindicator for evaluating body burden of environmental pollutants. Its elemental contents and their chemical forms are closely related to the status of human health and disease. In this paper, the liver samples collected from normal subjects were separated to different subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol by differential centrifugation. Then their concentrations of heavy metals of As, Pb, Cd, and Hg were determined by atomic absorption and atomic fluorescent spectroscopy. Our results show no significant difference with literature ones when comparing their gross concentrations. In the case of their subcellular distribution, the Hg concentrations are higher in mitochondrial, microsomal and cytosolic fractions; the Cd concentrations are higher in cytosolic and mitochondrial fractions, while As highest in nuclear fraction. The highest concentration of Pb is found in microsomal fraction with similarity to Fe. Mercury in liver is mainly in the form of inorganic, and methylmercury ranged from 9% to 50% with the average value of 20.9%(13.3%. These results indicate that the cellular distribution and the accumulated target organelles are quite different among these heavy metals, which suggest their various pathways and toxic mechanism in vivo.

  14. Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis

    Directory of Open Access Journals (Sweden)

    Cheevadhanarak Supapon

    2009-09-01

    Full Text Available Abstract The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

  15. Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Steen Redeker, E. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium)]. E-mail: erik.steenredeker@ua.ac.be; Campenhout, K. van [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Bervoets, L. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Reijnders, H. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Blust, R. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium)

    2007-07-15

    We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed. - The internal metal speciation in prey items is not clearly linked to the actual assimilation in predators.

  16. Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity

    International Nuclear Information System (INIS)

    We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed. - The internal metal speciation in prey items is not clearly linked to the actual assimilation in predators

  17. Laminar stream of detergents for subcellular neurite damage in a microfluidic device: a simple tool for the study of neuroregeneration

    Science.gov (United States)

    Lee, Chang Young; Romanova, Elena V.; Sweedler, Jonathan V.

    2013-06-01

    Objective. The regeneration and repair of damaged neuronal networks is a difficult process to study in vivo, leading to the development of multiple in vitro models and techniques for studying nerve injury. Here we describe an approach for generating a well-defined subcellular neurite injury in a microfluidic device. Approach. A defined laminar stream of sodium dodecyl sulfate (SDS) was used to damage selected portions of neurites of individual neurons. The somata and neurites unaffected by the SDS stream remained viable, thereby enabling the study of neuronal regeneration. Main results. By using well-characterized neurons from Aplysia californica cultured in vitro, we demonstrate that our approach is useful in creating neurite damage, investigating neurotrophic factors, and monitoring somata migration during regeneration. Supplementing the culture medium with acetylcholinesterase (AChE) or Aplysia hemolymph facilitated the regeneration of the peptidergic Aplysia neurons within 72 h, with longer (p < 0.05) and more branched (p < 0.05) neurites than in the control medium. After the neurons were transected, their somata migrated; intriguingly, for the control cultures, the migration direction was always away from the injury site (7/7). In the supplemented cultures, the number decreased to 6/8 in AChE and 4/8 in hemolymph, with reduced migration distances in both cases. Significance. The SDS transection approach is simple and inexpensive, yet provides flexibility in studying neuroregeneration, particularly when it is important to make sure there are no retrograde signals from the distal segments affecting regeneration. Neurons are known to not only be under tension but also balanced in terms of force, and the balance is obviously disrupted by transection. Our experimental platform, verified with Aplysia, can be extended to mammalian systems, and help us gain insight into the role that neurotrophic factors and mechanical tension play during neuronal regeneration.

  18. Subcellular distribution of [3H]-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    International Nuclear Information System (INIS)

    The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively

  19. An ensemble classifier for eukaryotic protein subcellular location prediction using gene ontology categories and amino acid hydrophobicity.

    Directory of Open Access Journals (Sweden)

    Liqi Li

    Full Text Available With the rapid increase of protein sequences in the post-genomic age, it is challenging to develop accurate and automated methods for reliably and quickly predicting their subcellular localizations. Till now, many efforts have been tried, but most of which used only a single algorithm. In this paper, we proposed an ensemble classifier of KNN (k-nearest neighbor and SVM (support vector machine algorithms to predict the subcellular localization of eukaryotic proteins based on a voting system. The overall prediction accuracies by the one-versus-one strategy are 78.17%, 89.94% and 75.55% for three benchmark datasets of eukaryotic proteins. The improved prediction accuracies reveal that GO annotations and hydrophobicity of amino acids help to predict subcellular locations of eukaryotic proteins.

  20. Bioaccumulation and subcellular partitioning of zinc in rainbow trout (Oncorhynchus mykiss): Cross-talk between waterborne and dietary uptake

    International Nuclear Information System (INIS)

    Zinc homeostasis was studied at the tissue and gill subcellular levels in rainbow trout (Oncorhynchus mykiss) following waterborne and dietary exposures, singly and in combination. Juvenile rainbow trout were exposed to 150 or 600 μg l-1 waterborne Zn, 1500 or 4500 μg g-1 dietary Zn, and a combination of 150 μg l-1 waterborne and 1500 μg g-1 dietary Zn for 40 days. Accumulation of Zn in tissues and gill subcellular fractions was measured. At the tissue level, the carcass acted as the main Zn depot containing 84-90% of whole body Zn burden whereas the gill held 4-6%. At the subcellular level, the majority of gill Zn was bioavailable with the estimated metabolically active pool being 81-90%. Interestingly, the nuclei-cellular debris fraction bound the highest amount (40%) of the gill Zn burden. There was low partitioning of Zn into the detoxified pool (10-19%) suggesting that sequestration and chelation are not major mechanisms of cellular Zn homeostasis in rainbow trout. Further, the subcellular partitioning of Zn did not conform to the spill-over model of metal toxicity because Zn binding was indiscriminate irrespective of exposure concentration and duration. The contribution of the branchial and gastrointestinal uptake pathways to Zn accumulation depended on the tissue. Specifically, in plasma, blood cells, and gill, uptake from water was dominant whereas both pathways appeared to contribute equally to Zn accumulation in the carcass. Subcellularly, additive uptake from the two pathways was observed in the heat-stable proteins (HSP) fraction. Toxicologically, Zn exposure caused minimal adverse effects manifested by a transitory inhibition of protein synthesis in gills in the waterborne exposure. Overall, subcellular fractionation appears to have value in the quest for a better understanding of Zn homeostasis and interactions between branchial and gastrointestinal uptake pathways

  1. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    Science.gov (United States)

    Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

  2. Early subcellular partitioning of cadmium in gill and liver of rainbow trout (Oncorhynchus mykiss) following low-to-near-lethal waterborne cadmium exposure

    International Nuclear Information System (INIS)

    Non-essential metals such as cadmium (Cd) accumulated in animal cells are envisaged to partition into potentially metal-sensitive compartments when detoxification capacity is exceeded. An understanding of intracellular metal partitioning is therefore important in delineation of the toxicologically relevant metal fraction for accurate tissue residue-based assessment of toxicity. In the present study, the early intracellular Cd accumulation was studied to test the prediction that it conforms to the spillover model of metal toxicity. Juvenile rainbow trout (10-15 g) were exposed for 96 h to three doses of cadmium (5, 25 and 50 μg/l) and a control (nominal 0 μg/l Cd) in hard water followed by measurement of the changes in intracellular Cd concentrations in the gill and liver, and carcass calcium (Ca) levels. There were dose-dependent increases in Cd concentration in both organs but the accumulation pattern over time was linear in the liver and biphasic in the gill. The Cd accumulation was associated with carcass Ca loss after 48 h. Comparatively, the gill accumulated 2-4x more Cd than the liver and generally the subcellular compartments reflected the organ-level patterns of accumulation. For the gill the rank of Cd accumulation in subcellular fractions was: heat-stable proteins (HSP) > heat-labile proteins (HLP) > nuclei > microsomes-lysosomes (ML) ≥ mitochondria > resistant fraction while for the liver it was HSP > HLP > ML > mitochondria > nuclei > resistant fraction. Contrary to the spillover hypothesis there was no exposure concentration or internal accumulation at which Cd was not found in potentially metal-sensitive compartments. The proportion of Cd bound to the metabolically active pool (MAP) increased while that bound to the metabolically detoxified pool (MDP) decreased in gills of Cd-exposed fish but remained unchanged in the liver. Because the Cd concentration increased in all subcellular compartments while their contribution to the total increased

  3. Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

    Science.gov (United States)

    Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

    1995-08-01

    Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles

  4. Subcellular localization of KL-6 mucin in intraductal papillary mucinous neoplasm of the pancreas.

    Science.gov (United States)

    Inagaki, Yoshinori; Seyama, Yasuji; Hasegawa, Kiyoshi; Tang, Wei; Kokudo, Norihiro

    2014-08-01

    This study aimed to clarify the expression profile of KL-6 mucin in intraductal papillary mucinous neoplasm (IPMN) and its relation to tumor malignancy. Expression of KL-6 mucin in 38 IPMNs (intraductal papillary mucinous adenoma (IPMA), 24 cases; minimally invasive intraductal papillary mucinous carcinoma (MI-IPMC), 8 cases; invasive carcinoma originating from IPMC (IC-IPMC), 6 cases) and 66 pancreatic ductal adenocarcinomas (PDACs) was evaluated immunohistochemically. IC-IPMCs and MI-IPMCs had positive staining of KL-6 mucin whereas 58% of IPMAs tested negative. Subcellular localization of KL-6 mucin varied among IPMNs whereas all of the PDAC had positive expression in the circumferential membrane and cytoplasm of cancer cells. IC-IPMCs and MI-IPMCs had a higher frequency of circumferential membrane and cytoplasmic localization of KL-6 mucin than did IPMAs. These results suggest that localization of KL-6 mucin could be used to predict the malignancy of IPMN. PMID:25047009

  5. Subcellular localization of PS1 based on PS1/GFP fusing protein

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer's disease (EOFAD). The fusion genes, GFP-PS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT Ⅱ (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution.

  6. MECHANISMS OF DAMAGING EFFECT OF MANGENESE IN TOXIC CONCENTRATIONS ON CELLULAR AND SUBCELLULAR LEVELS

    Directory of Open Access Journals (Sweden)

    Goncharenko A. V.

    2012-11-01

    Full Text Available Influence of subtoxic concentration of manganese chloride in dose equal to LD 50 on condition of plasmatic membranes (model: erythrocytes and functional activity of cell power (model: the isolated liver mitochondrion of rats was studied. It was established that manganese chloride in fixed concentration caused authentic augmentation of sorption capacity of erythrocytes towards alcian blue, influenced increasing of their spontaneous haemolysis and activation of peroxide oxidation of lipids. In experiment on the isolated mitochondrion it was proved that manganese chloride caused dissociation of an oxidizing phosphorusling and complete inhibition of respiration in concentrations of 3 and 4,5mM. These dependences testify that subtoxic concentration of manganese can damage the cell energy. Thus, this pilot research indicated damaging effect of manganese on cellular (erythrocytes and subcellular (mitochondrion levels which are realized through external functioning of membrane structures and deprived them from restoration.

  7. Expression and subcellular localization of mechano-growth factor in osteoblasts under mechanical stretch

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Mechano-growth factor (MGF) is a stretch sensitive factor in myocytes, and it might also be produced by other mechanocytes under mechanical stimulation. In this study, both the mRNA and protein expression of MGF were detected in stretched osteoblasts. Quantitative analysis showed that a cyclic stretching stimulation caused a quick and sharp increase of MGF mRNA and protein expression from a low basal level under no stretch; the mRNA and protein levels respectively peaked in 6 and 12 h to 5 and 5.2 fold over the basal level and returned to normal by 24 h. The subcellular distribution of MGF protein was revealed by immunofluorescence analysis to be restricted to the nucleus. We concluded that cyclic stretching stimulation could induce MGF expression in osteoblasts in a pulsing fashion; and the nuclear distribution of MGF suggested that MGF might act in mechanocytes as an autocrine growth factor.

  8. Taurine effects on 45Ca2+ transport in retinal subcellular fractions

    International Nuclear Information System (INIS)

    The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1-5 min in a Krebs-bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role of taurine in the regulation of calcium fluxes in the retina. (Auth.)

  9. Spatial and temporal changes in Bax subcellular localization during NPe6-PDT-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Xing, Da; Chen, Wei R.; Wan, Qingling; Zhou, Feifan

    2008-02-01

    Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Bax, a member of the Bcl-2 family of proteins, is an essential regulator of apoptosis. Bax is normally found in the cytosol of healthy cells, and translocates to mitochondria in response to many apoptotic stimuli. In this study, using real-time single-cell analysis, we have investigated the kinetics of Bax distribution during NPe6-induced apoptosis in ASTC-a-1 cells. In order to monitor Bax subcellular distribution, cells were stained with GFP-Bax and Mito Tracker Red. The results show that Bax redistribution occurred at about 170 min after treated with NPe6-PDT, and then sequestered into clusters associated with the mitochondira within 30 min. Our data clearly showed the spatial and temporal changes in Bax distribution in living cells during NPe6-induced apoptosis.

  10. Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus1

    Science.gov (United States)

    De Luca, Vincenzo; Cutler, Adrian J.

    1987-01-01

    The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline. PMID:16665811

  11. Measurement of subcellular texture by optical Gabor-like filtering with a digital micromirror device

    Science.gov (United States)

    Pasternack, Robert M.; Qian, Zhen; Zheng, Jing-Yi; Metaxas, Dimitris N.; White, Eileen; Boustany, Nada N.

    2010-01-01

    We demonstrate an optical Fourier processing method to quantify object texture arising from subcellular feature orientation within unstained living cells. Using a digital micromirror device as a Fourier spatial filter, we measured cellular responses to two-dimensional optical Gabor-like filters optimized to sense orientation of nonspherical particles, such as mitochondria, with a width around 0.45 μm. Our method showed significantly rounder structures within apoptosis-defective cells lacking the proapoptotic mitochondrial effectors Bax and Bak, when compared with Bax/Bak expressing cells functional for apoptosis, consistent with reported differences in mitochondrial shape in these cells. By decoupling spatial frequency resolution from image resolution, this method enables rapid analysis of nonspherical submicrometer scatterers in an under-sampled large field of view and yields spatially localized morphometric parameters that improve the quantitative assessment of biological function. PMID:18830354

  12. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.

    Science.gov (United States)

    Giesen, Charlotte; Wang, Hao A O; Schapiro, Denis; Zivanovic, Nevena; Jacobs, Andrea; Hattendorf, Bodo; Schüffler, Peter J; Grolimund, Daniel; Buhmann, Joachim M; Brandt, Simone; Varga, Zsuzsanna; Wild, Peter J; Günther, Detlef; Bodenmiller, Bernd

    2014-04-01

    Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies. PMID:24584193

  13. Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Vrieling, E.G.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous c

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

    Directory of Open Access Journals (Sweden)

    Lewis Victoria

    2012-04-01

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

  15. Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

    Science.gov (United States)

    Zhong, Zhenhui; Norvienyeku, Justice; Yu, Jie; Chen, Meilian; Cai, Renli; Hong, Yonghe; Chen, Liqiong; Zhang, Dongmei; Wang, Baohua; Zhou, Jie; Lu, Guodong; Chen, Xiaofeng; Wang, Zonghua

    2015-10-01

    The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue. PMID:26318870

  16. Cadmium in three marine phytoplankton: Accumulation, subcellular fate and thiol induction

    International Nuclear Information System (INIS)

    We explored the possible mechanisms leading to differential Cd sensitivity in three marine phytoplankton (the diatom Thalassiosira pseudonana, the dinoflagellate Prorocentrum minimum and the green alga Chlorella autotrophica) based on their Cd accumulation, Cd subcellular distribution, and phytochelatin (PC) synthesis. The most sensitive species, T. pseudonana, generally exhibited the highest Cd body burden and organelle (org)-Cd concentration. C. autotrophica, the most tolerant species to Cd, had the smallest org-Cd accumulation, as well as a much higher percentage of cellular debris-Cd, which may play an important role in Cd detoxification. The dinoflagellate P. minimum, with a sensitivity between the diatoms and green algae, had a comparable Cd body burden but higher percentage of org-Cd than C. autotrophica. Although the induction of PCs was dependent on the species, the intracellular (intra)-Cd/PC-SH ratio showed a strong linear log-log relationship with [Cd2+], suggesting that this ratio could possibly be a biomarker for environmental [Cd2+] stress. With the increases of the intra-Cd/PC-SH ratio, these three species of phytoplankton exhibited clearly different patterns of growth inhibition, implying that the effectiveness of PCs as a detoxification pathway is dependent on the species. The lowest intra-Cd/PC-SH toxicity threshold for T. pseudonana implied its low PC-Cd capacity. Furthermore, the sudden slowdown of growth inhibition when the intra-Cd/PC-SH ratio reached 33 implied the launch of other detoxification pathway in C. autotrophica in order to alleviate Cd toxicity. Our study demonstrated that accumulation and subcellular distribution of Cd and PC synthesis can account for the inter-species differences in Cd sensitivity in marine phytoplankton.

  17. Subcellular trafficking of the amyloid precursor protein gene family and its pathogenic role in Alzheimer's disease.

    Science.gov (United States)

    Kins, Stefan; Lauther, Nadine; Szodorai, Anita; Beyreuther, Konrad

    2006-01-01

    Changes in the intracellular transport of amyloid precursor protein (APP) affect the extent to which APP is exposed to alpha- or beta-secretase in a common subcellular compartment and therefore directly influence the degree to which APP undergoes the amyloidogenic pathway leading to generation of beta-amyloid. As the presynaptic regions of neurons are thought to be the main source of beta-amyloid in the brain, attention has been focused on axonal APP trafficking. APP is transported along axons by a fast, kinesin-dependent anterograde transport mechanism. Despite the wealth of in vivo and in vitro data that have accumulated regarding the connection of APP to kinesin transport, it is not yet clear if APP is coupled to its specific motor protein via an intracellular interaction partner, such as the c-Jun N-terminal kinase-interacting protein, or by yet another unknown molecular mechanism. The cargo proteins that form a functional complex with APP are also unknown. Due to the long lifespan, and vast extent, of neurons, in particular axons, neurons are highly sensitive to changes in subcellular transport. Recent in vitro and in vivo studies have shown that variations in APP or tau affect mitochondrial and synaptic vesicle transport. Further, it was shown that this axonal dysfunction might lead to impaired synaptic plasticity, which is crucial for neuronal viability and function. Thus, changes in APP and tau expression may cause perturbed axonal transport and changes in APP processing, contributing to cognitive decline and neurodegeneration in Alzheimer's disease. PMID:17047360

  18. Prolactin kinase activity in bovine anterior pituitary sub-cellular fractions.

    Science.gov (United States)

    Wicks, J R; Brooks, C L

    1999-01-25

    Bovine anterior pituitary cells phosphorylate prolactin (PRL). We describe the phosphorylation of endogenous and exogenous bPRL in highly enriched subcellular fractions of bovine anterior pituitary using [gamma-32P]-ATP. 32P-labeling of endogenous and exogenous bPRL occurred in all subcellular membrane fractions, but most significantly in the fraction enriched for secretory granules. Zn2+ (0.8 mM), Cu2+ (0.8 mM), and Mn2+ (9.8 mM) increased bPRL phosphorylation by 268, 214, and 154%, respectively, relative to basal phosphorylation with no added cations. Neither Mg2+ (10 mM) nor Ca2+ (0.9 mM) increased bPRL phosphorylation above basal levels. Phosphorylation was dependent on the concentration of Zn2+ with an apparent Km of 570 microM. bPRL phosphorylation occurred over a wide pH range of 5.9-8.3, with the greatest activity at pH of 6.7 or greater. Phosphorylation of bPRL was time-dependent. The apparent Kms of the bPRL kinase for exogenous bPRL and ATP were 15.3 and 267 microM, respectively. bPRL incorporation of 32P was unaffected by the presence of calcium and calmodulin, cAMP, phosphotidylserine and diolein, or spermine. From these results we conclude that in vitro phosphorylation of bPRL occurs under physiological conditions that would be found in pituitary cells. PMID:10195699

  19. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    Science.gov (United States)

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  20. Subcellular localisation of radionuclides by transmission electronic microscopy in aquatic and terrestrial organisms

    International Nuclear Information System (INIS)

    The global framework of this study is to go further in the understanding of the involved mechanisms of uranium and selenium internalisation at the subcellular level and of their toxicity towards several aquatic and terrestrial organisms. In this context, the applications and performances of a Scanning Transmission Electron Microscope (TEM/STEM) equipped with CCD camera and Energy-Dispersive- X-Ray (EDAX) analysis are reported. The principal merit of this equipment is the clear expression of element distribution with nanometer resolution. The sample for TEM analysis were prepared in ultrathin sections of 70-140 nm (thickness) and those for EDAX in sections of 200-500 nm. This method offers the possibility of a direct correlation between histological image and distribution map of trace elements. For each sample, following TEM analysis, EDAX spectra or EDAX mapping were also recorded to confirm the identity of the electron dense material in the scanned sections. Demonstration of the usefulness of this method to understand the bioaccumulation mechanisms and to study the effect of the pollutant uptake at the subcellular level was performed for target organs of a metal (U) and a metalloid (Se) in various biological models: a higher rooted plant (Phaseolus vulgaris)) and a freshwater invertebrate (Orconectes Limosus) and a unicellular green alga (Chlamydomonas reinhardtii)). TEM-EDAX analysis revealed the presence of U-deposits in gills and digestive gland in crayfish, and in vacuoles or in the cytoplasm of different rooted cells bean. In the alga, the accumulation of Se was found in electron-dense granules within cytoplasm associated with ultrastructural changes and starch accumulation. (author)

  1. Determination of ABA-binding proteins contents in subcellular fractions isolated from cotton seedlings using radioimmunoanalysis

    International Nuclear Information System (INIS)

    Full text: Knowledge of plants' hormone receptor sites is essential to understanding of the principles of phytohormone action in cells and tissues. The hormone abscisic acid (ABA) takes part in many important physiological processes of plants, including water balance and resistance to salt stress. The detection of salt tolerance in the early stages of ontogenesis is desirable for effective cultivation of cotton. Usually such characteristics are determined visually after genetic analysis of hybrids over several generations. This classic method of genetics requires a long time to grow several generations of cotton plants. In this connection we study ABA-binding protein contents in subcellular fractions isolated from seedlings of several kinds of cotton with different tolerance to salt stress. The contents of ABA-binding protein in nuclei and chloroplasts fractions isolated from cotton seedlings were determined using radioimmunoanalysis. The subcellular fractions were prepared by ultracentrifugation in 0,25 - 2,2 M sucrose gradient. ABA-binding protein was isolated from cotton seedlings by affinity chromatography. The antibodies against ABA-binding protein of cotton were developed in rabbits according standard protocols. Than the antibodies were labelled by radioisotope J125 according Greenwood et al. It was shown, that the nuclei and chloroplasts fractions isolated from cotton with high tolerance to salt stress contain ABA-binding protein up to 1,5-1,8 times more, than the same fractions from cotton with low tolerance to salt stress. So, the ABA-binding protein contents in cotton seedlings may be considered as a marker for screening of cotton kinds, which may potentially have high tolerance to salt stress

  2. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells.

    Science.gov (United States)

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  3. Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

    Science.gov (United States)

    Schilling, Stephan; Lindner, Christiane; Koch, Birgit; Wermann, Michael; Rahfeld, Jens-Ulrich; von Bohlen, Alex; Rudolph, Thomas; Reuter, Gunter; Demuth, Hans-Ulrich

    2007-09-25

    Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes. PMID:17722885

  4. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Arieh Riskin

    2015-10-01

    Full Text Available Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC in culture. Methods: Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC. To study GLUT1 targeting and recycling in living mouse MEC (MMEC in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM, or exposed to secretion medium (SM, containing prolactin. Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

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

    Science.gov (United States)

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

    2011-01-01

    HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus. PMID:21054678

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

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2008-11-01

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

  7. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

    Directory of Open Access Journals (Sweden)

    Kunichika Tsumoto

    Full Text Available Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na+ channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na+ channels in the arrhythmogenicity under ischemia remain unclear.Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na+ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.We found in the myofiber model that the subcellular redistribution of the Na+ channels under myocardial ischemia, decreasing in Na+ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism did not reproduce the conduction slowing caused by the subcellular Na+ channel redistribution. Furthermore, Na+ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na+ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.Proarrhythmia of Na+ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na+ channel redistribution.

  8. Bioaccumulation, subcellular distribution and toxicity of sediment-associated copper in the ragworm Nereis diversicolor: The relative importance of aqueous copper, copper oxide nanoparticles and microparticles.

    Science.gov (United States)

    Thit, Amalie; Banta, Gary T; Selck, Henriette

    2015-07-01

    The sediment-dwelling ragworm, Nereis diversicolor was exposed to sediment spiked with aqueous Cu (CuAq, CuCl2), CuO nanoparticles (CuONP) or CuO microparticles (CuOMicro) at 150 μg Cu g(-1) dw sediment for 10d. Exposures to CuAq and CuOMicro caused mortality (62.5 and 37.5%, respectively), whereas mean burrowing time increased during exposure to CuAq and CuONP from 0.12 h (controls) to 19.3 and 12.2 h, respectively. All Cu treatments bioaccumulated, especially CuAq (up to 4 times more than the other treatments). Cu was roughly equally distributed among the five subcellular fractions in controls and worms exposed to CuONP or CuOMicro. In contrast, ≈50% of accumulated Cu in CuAq exposed worms was found in metal rich granules and significantly more Cu was present in heat-denatured proteins and organelles than in worms exposed to CuOMicro or in controls. Our results suggest that Cu form affects its bioaccumulation and subsequent toxicity and detoxification in a polychaete like N. diversicolor. PMID:25800937

  9. Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient

    Energy Technology Data Exchange (ETDEWEB)

    Seebaugh, David R. [Department of Biology, Graduate School and University Center, City University of New York, 365 Fifth Avenue, New York, NY 10016 (United States); Wallace, William G., E-mail: wallace@mail.csi.cuny.edu [Department of Biology, Graduate School and University Center, City University of New York, 365 Fifth Avenue, New York, NY 10016 (United States); Department of Biology, College of Staten Island, 6S-310, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States)

    2009-06-28

    Chronic exposure to polluted field conditions can impact metal bioavailability in prey and may influence metal transfer to predators. The present study investigated the assimilation of Cd, Hg and organic carbon by grass shrimp Palaemonetes pugio, collected along an impact gradient within the New York/New Jersey Harbor Estuary. Adult shrimp were collected from five Staten Island, New York study sites, fed {sup 109}Cd- or {sup 203}Hg-labeled amphipods or {sup 14}C-labeled meals and analyzed for assimilation efficiencies (AE). Subsamples of amphipods and shrimp were subjected to subcellular fractionation to isolate metal associated with a compartment presumed to contain trophically available metal (TAM) (metal associated with heat-stable proteins [HSP - e.g., metallothionein-like proteins], heat-denatured proteins [HDP - e.g., enzymes] and organelles [ORG]). TAM-{sup 109}Cd% and TAM-{sup 203}Hg% in radiolabeled amphipods were {approx}64% and {approx}73%, respectively. Gradients in AE-{sup 109}Cd% ({approx}54% to {approx}75%) and AE-{sup 203}Hg% ({approx}61% to {approx}78%) were observed for grass shrimp, with the highest values exhibited by shrimp collected from sites within the heavily polluted Arthur Kill complex. Population differences in AE-{sup 14}C% were not observed. Assimilated {sup 109}Cd% partitioned to the TAM compartment in grass shrimp varied between {approx}67% and {approx}75%. {sup 109}Cd bound to HSP in shrimp varied between {approx}15% and {approx}47%, while {sup 109}Cd associated with metal-sensitive HDP was {approx}17% to {approx}44%. Percentages of assimilated {sup 109}Cd bound to ORG were constant at {approx}10%. Assimilated {sup 203}Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated {sup 203}Hg bound to HDP ({approx}47%) and ORG ({approx}11%) did not vary among populations and partitioning of {sup 203}Hg to HSP was not observed. Using a simplified biokinetic model of metal accumulation from

  10. Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient

    International Nuclear Information System (INIS)

    Chronic exposure to polluted field conditions can impact metal bioavailability in prey and may influence metal transfer to predators. The present study investigated the assimilation of Cd, Hg and organic carbon by grass shrimp Palaemonetes pugio, collected along an impact gradient within the New York/New Jersey Harbor Estuary. Adult shrimp were collected from five Staten Island, New York study sites, fed 109Cd- or 203Hg-labeled amphipods or 14C-labeled meals and analyzed for assimilation efficiencies (AE). Subsamples of amphipods and shrimp were subjected to subcellular fractionation to isolate metal associated with a compartment presumed to contain trophically available metal (TAM) (metal associated with heat-stable proteins [HSP - e.g., metallothionein-like proteins], heat-denatured proteins [HDP - e.g., enzymes] and organelles [ORG]). TAM-109Cd% and TAM-203Hg% in radiolabeled amphipods were ∼64% and ∼73%, respectively. Gradients in AE-109Cd% (∼54% to ∼75%) and AE-203Hg% (∼61% to ∼78%) were observed for grass shrimp, with the highest values exhibited by shrimp collected from sites within the heavily polluted Arthur Kill complex. Population differences in AE-14C% were not observed. Assimilated 109Cd% partitioned to the TAM compartment in grass shrimp varied between ∼67% and ∼75%. 109Cd bound to HSP in shrimp varied between ∼15% and ∼47%, while 109Cd associated with metal-sensitive HDP was ∼17% to ∼44%. Percentages of assimilated 109Cd bound to ORG were constant at ∼10%. Assimilated 203Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated 203Hg bound to HDP (∼47%) and ORG (∼11%) did not vary among populations and partitioning of 203Hg to HSP was not observed. Using a simplified biokinetic model of metal accumulation from the diet, it is estimated that site-specific variability in Cd AE by shrimp and tissue Cd burdens in field-collected prey (polychaetes Nereis spp.) could potentially

  11. Ecological nanotoxicology: integrating nanomaterial hazard considerations across the subcellular, population, community, and ecosystems levels.

    Science.gov (United States)

    Holden, Patricia A; Nisbet, Roger M; Lenihan, Hunter S; Miller, Robert J; Cherr, Gary N; Schimel, Joshua P; Gardea-Torresdey, Jorge L

    2013-03-19

    Research into the health and environmental safety of nanotechnology has seriously lagged behind its emergence in industry. While humans have often adopted synthetic chemicals without considering ancillary consequences, the lessons learned from worldwide pollution should motivate making nanotechnology compatible with environmental concerns. Researchers and policymakers need to understand exposure and harm of engineered nanomaterials (ENMs), currently nanotechnology's main products, to influence the ENM industry toward sustainable growth. Yet, how should research proceed? Standard toxicity testing anchored in single-organism, dose-response characterizations does not adequately represent real-world exposure and receptor scenarios and their complexities. Our approach is different: it derives from ecology, the study of organisms' interactions with each other and their environments. Our approach involves the characterization of ENMs and the mechanistic assessment of their property-based effects. Using high throughput/content screening (HTS/HCS) with cells or environmentally-relevant organisms, we measure the effects of ENMs on a subcellular or population level. We then relate those effects to mechanisms within dynamic energy budget (DEB) models of growth and reproduction. We reconcile DEB model predictions with experimental data on organism and population responses. Finally, we use microcosm studies to measure the potential for community- or ecosystem-level effects by ENMs that are likely to be produced in large quantities and for which either HTS/HCS or DEB modeling suggest their potential to harm populations and ecosystems. Our approach accounts for ecological interactions across scales, from within organisms to whole ecosystems. Organismal ENM effects, if propagated through populations, can alter communities comprising multiple populations (e.g., plant, fish, bacteria) within food webs. Altered communities can change ecosystem services: processes that cycle carbon

  12. Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

    Science.gov (United States)

    Pratt, Evan P S; Owens, Jake L; Hockerman, Gregory H; Hu, Chang-Deng

    2016-01-01

    Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software. PMID:27515079

  13. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    International Nuclear Information System (INIS)

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the location of Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments

  14. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types

    International Nuclear Information System (INIS)

    Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells. - Selenium assimilation by a predatory aquatic insect depends on Se availability in the cells of its prey

  15. Protein subcellular localization in human and hamster cell lines: employing local ternary patterns of fluorescence microscopy images.

    Science.gov (United States)

    Tahir, Muhammad; Khan, Asifullah; Kaya, Hüseyin

    2014-01-01

    Discriminative feature extraction technique is always required for the development of accurate and efficient prediction systems for protein subcellular localization so that effective drugs can be developed. In this work, we showed that Local Ternary Patterns (LTPs) effectively exploit small variations in pixel intensities; present in fluorescence microscopy based protein images of human and hamster cell lines. Further, Synthetic Minority Oversampling Technique is applied to balance the feature space for the classification stage. We observed that LTPs coupled with data balancing technique could enable a classifier, in this case support vector machine, to yield good performance. The proposed ensemble based prediction system, using 10-fold cross-validation, has yielded better performance compared to existing techniques in predicting various subcellular compartments for both 2D HeLa and CHO datasets. The proposed predictor is available online at: http://111.68.99.218/Protein_SubLoc/, which is freely accessible to the public. PMID:23988793

  16. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types.

    Science.gov (United States)

    Dubois, Maïtée; Hare, Landis

    2009-03-01

    Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells. PMID:19110352

  17. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types

    Energy Technology Data Exchange (ETDEWEB)

    Dubois, Maitee [Institut national de la recherche scientifique - Eau, Terre et Environnement, Universite du Quebec, Quebec City, Quebec, G1K 9A9 (Canada); Hare, Landis [Institut national de la recherche scientifique - Eau, Terre et Environnement, Universite du Quebec, Quebec City, Quebec, G1K 9A9 (Canada)], E-mail: landis@ete.inrs.ca

    2009-03-15

    Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells. - Selenium assimilation by a predatory aquatic insect depends on Se availability in the cells of its prey.

  18. A radiotracer study on the chronic effects and subcellular partitioning of metals in the Manila clam Ruditapes Philippinarum

    International Nuclear Information System (INIS)

    A laboratory microcosm study was employed to evaluate chronic physiological and histopathological effects of wate-rborne Ag to the Manila clam Ruditapes philippinarum. Additionally, radiotracers of gamma emitting isotopes of 109Cd, 51Cr, and 65Zn were used to study the metal bioaccumulation mechanisms and subcellular partitioning in the clam Ruditapes philippinarum. Bioaccumulation of Ag in the clam increased linearly with Ag concentration in seawater and exposure time. All the physiological parameters (e.g., filtration rate, scope for growth) and histopathological data had negative relationships with tissue Agconcentrations in the clams. The uptake rate of 3 metals increased in the order of Zn > Cd > Cr and had inverse relationship with salinity and with individual clam size (dry weight). The result suggest that subcellular partitioning of metals in the clams into different binding pools could be important factors determining Biologically Detoxified Metal (BDM), Trophically Available Metal (TAM) and Trophically Available Metal (TAM)

  19. Subcellular clearance and accumulation of Huntington disease protein: A mini-review

    Directory of Open Access Journals (Sweden)

    Ting eZhao

    2016-04-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ tract in the N-terminal region of mutant huntingtin (mHtt. As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies of examining mHtt clearance and accumulation in different subcellular regions

  20. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  1. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Bindesh Shrestha

    Full Text Available Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI, an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen, were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  2. Subcellular object quantification with Squassh3C and SquasshAnalyst.

    Science.gov (United States)

    Rizk, Aurélien; Mansouri, Maysam; Ballmer-Hofer, Kurt; Berger, Philipp

    2015-11-01

    Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst. Squassh3C extends the functionality of Squassh to three fluorescence channels and live-cell movie analysis. SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. It provides segmentation image overview and data exploration, figure generation, object and image filtering, and a statistical significance test in an easy-to-use interface. The overall procedure combines the Squassh3C plug-in for the free biological image processing program ImageJ and a web application working in conjunction with the free statistical environment R, and it is compatible with Linux, MacOS X, or Microsoft Windows. Squassh3C and SquasshAnalyst are available for download at www.psi.ch/lbr/SquasshAnalystEN/SquasshAnalyst.zip. PMID:26554508

  3. Photoaffinity labelling of tobacco subcellular fractions with [32P]-azido-UDP-glucose

    International Nuclear Information System (INIS)

    Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, [32P]-N3 UDPG. Two stromal polypeptides (Mr = 42 and 21 kD) were the major photolabelled chloroplast polypeptides. UDPG protected the 42 but not the 21 kD polypeptide against photoincorporation of [32P]-N3 UDPG. In a cytosol-enriched fraction, the major photolabelling polypeptides had Mr of 92, 50, 42, 30 and 17 kD. Photolabelling of the 42, 30, and 17 kD polypeptides was unaffected by UDPG, but UDPG blocked incorporation into the 92 and 50 kD polypeptides. In addition to photolabelled polypeptides, a polypeptide identified as phosphoglucomutase (PGM) was labelled in both the chloroplast and cytosol fraction. 32P-labelling of PGM was independent of UV irradiation, occurring via phosphoryl transfer from contaminating [32P]G-I-P. The plastid and cytosolic PGM isozymes had Mr of 69 and 62.8 kD, respectively, and both were labelled in a leaf extract

  4. Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

    International Nuclear Information System (INIS)

    Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species

  5. Microscopy with spatial filtering for sorting particles and monitoring subcellular morphology

    Science.gov (United States)

    Zheng, Jing-Yi; Qian, Zhen; Pasternack, Robert M.; Boustany, Nada N.

    2009-02-01

    Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

  6. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

    Science.gov (United States)

    Baghirova, Sabina; Hughes, Bryan G.; Hendzel, Michael J.; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C PMID:26740924

  7. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase

    International Nuclear Information System (INIS)

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the nonspecificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination

  8. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    Science.gov (United States)

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications. PMID:25451863

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

    International Nuclear Information System (INIS)

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

  10. Protein Subcellular Localization Prediction and Genomic Polymorphism Analysis of the SARS Coronavirus

    Institute of Scientific and Technical Information of China (English)

    季星来; 柳树群; 李岭; 孙之荣

    2004-01-01

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus (CoV).Several sequences of the complete genome of SARS-CoV have been determined.The subcellular localization (SubLocation) of annotated open-reading frames of the SARS-CoV genome was predicted using a support vector machine.Several gene products were predicted to locate in the Golgi body and cell nucleus.The SubLocation information was combined with predicted transmembrane information to develop a model of the viral life cycle.The results show that this information can be used to predict the functions of genes and even the virus pathogenesis.In addition,the entire SARS viral genome sequences currently available in GenBank were compared to identify the sequence variations among different isolates.Some variations in the Hong Kong strains may be related to the special clinical manifestations and provide clues for understanding the relationship between gene functions and evolution.These variations reflect the evolution of the SARS virus in human populations and may help development of a vaccine.

  11. Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

    Institute of Scientific and Technical Information of China (English)

    QIU; Quansheng; (邱全胜); WANG; Zezhou(王泽宙); CAI; Qigui(蔡起贵); JIANG; Rongxi(姜荣锡)

    2002-01-01

    The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by microprojectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclohexylcarbodiimid, indicating that the cytoplasmic DHN1 was newly synthesized under osmotic stress. Furthermore, ABA promoted the presence of green fluorescence in the cytoplasm, and the GFP fluorescence was visualized within a shorter time under a higher osmotic potential.

  12. Subcellular Clearance and Accumulation of Huntington Disease Protein: A Mini-Review

    Science.gov (United States)

    Zhao, Ting; Hong, Yan; Li, Xiao-Jiang; Li, Shi-Hua

    2016-01-01

    Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ) tract in the N-terminal region of mutant huntingtin (mHtt). As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system (UPS) and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies examining mHtt clearance and accumulation in different subcellular regions. PMID:27147961

  13. Monte-Carlo study of energy deposition by heavy charged particles in sub-cellular volumes

    International Nuclear Information System (INIS)

    Detailed-history Monte-Carlo code is used to study the energy deposition from proton and alpha particle tracks at the sub-cellular level. Inelastic cross sections for both the vapour and liquid phases of water have been implemented into the code in order to explore the influence of non-linear density effects associated with the condensed-phase cellular environment. Results of energy deposition and its straggling for 0.5 to 5 MeV amu-1 protons and alpha particles traversing or passing near spherical volumes of 2-200 nm in diameter relevant to DNA- and chromosome-size targets are presented. It is shown that the explicit account of δ-ray transport reduces the dose by as much as 10-60%, whereas stochastic fluctuations lead to a relative uncertainty ranging from 20% to more than 100%. Protons and alpha particles of the same velocity exhibit a similar δ-ray effect, whereas the relative uncertainty of the alphas is almost half that of protons. The effect of the phase is noticeable (10-15%) mainly through differences on the transport of δ-rays, which in liquid water have higher penetration distances. It is expected that the implementation of such results into multi-scale biophysical models of radiation effects will lead to a more realistic predictions on the efficacy of new radiotherapeutic modalities that employ either external proton beam irradiation or internal alpha-emitting radionuclides. (authors)

  14. Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer.

    Science.gov (United States)

    Friedrich, Teresa; Söhn, Michaela; Gutting, Tobias; Janssen, Klaus-Peter; Behrens, Hans-Michael; Röcken, Christoph; Ebert, Matthias P A; Burgermeister, Elke

    2016-06-01

    Full-length (FL) docking protein-1 (DOK1) is an adapter protein which inhibits growth factor and immune response pathways in normal tissues, but is frequently lost in human cancers. Small DOK1 variants remain in cells of solid tumors and leukemias, albeit, their functions are elusive. To assess the so far unknown role of DOK1 in colorectal cancer (CRC), we generated DOK1 mutants which mimic the domain structure and subcellular distribution of DOK1 protein variants in leukemia patients. We found that cytoplasmic DOK1 activated peroxisome-proliferator-activated-receptor-gamma (PPARγ) resulting in inhibition of the c-FOS promoter and cell proliferation, whereas nuclear DOK1 was inactive. PPARγ-agonist increased expression of endogenous DOK1 and interaction with PPARγ. Forward translation of this cell-based signaling model predicted compartmentalization of DOK1 in patients. In a large series of CRC patients, loss of DOK1 protein was associated with poor prognosis at early tumor stages (*p=0.001; n=1492). In tumors with cytoplasmic expression of DOK1, survival was improved, whereas nuclear localization of DOK1 correlated with poor outcome, indicating that compartmentalization of DOK1 is critical for CRC progression. Thus, DOK1 was identified as a prognostic factor for non-metastatic CRC, and, via its drugability by PPARγ-agonist, may constitute a potential target for future cancer treatments. PMID:27428427

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

    Science.gov (United States)

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

    2016-03-01

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

  16. Incorporation of 15N into subcellular fractions and soluble proteins in rice seedlings

    International Nuclear Information System (INIS)

    15N incorporation into the subcellular and protein fractions of rice seedlings was investigated, when (15NH4)2SO4 was fed through culture solution, and the following was found. (1) Optical emission spectroscopic method is very useful for the 15N analysis of small amount of protein as much as separated by Sephadex G-200. (2) In the developed leaves, where nitrogen was balanced by continuous influx and efflux, 6 to 9 percent of total nitrogen was exchanged in a day by newly absorbed nitrogen, and the absorbed nitrogen was incorporated into chloroplast, 30,000 x g precipitate and soluble protein fractions by almost equal rates. (3) In the developing leaf, where the amount of nitrogen was increasing, newly absorbed and redistributed nitrogen almost equally contributed to the increase of total and protein nitrogen. (4) The speed of 15N incorporation into Fraction 1 Protein was the same in the developing leaf and slow in the developed leaves in comparison with other soluble proteins. (author)

  17. A General Procedure to Study Subcellular Models of Transsynaptic Signaling at Inhibitory Synapses.

    Science.gov (United States)

    Lupascu, Carmen A; Morabito, Annunziato; Merenda, Elisabetta; Marinelli, Silvia; Marchetti, Cristina; Migliore, Rosanna; Cherubini, Enrico; Migliore, Michele

    2016-01-01

    Computational modeling of brain circuits requires the definition of many parameters that are difficult to determine from experimental findings. One way to help interpret these data is to fit them using a particular kinetic model. In this paper, we propose a general procedure to fit individual synaptic events recorded from voltage clamp experiments. Starting from any given model description (mod file) in the NEURON simulation environment, the procedure exploits user-defined constraints, dependencies, and rules for the parameters of the model to fit the time course of individual spontaneous synaptic events that are recorded experimentally. The procedure, implemented in NEURON, is currently available in ModelDB. A Python version is installed, and will be soon available for public use, as a standalone task in the Collaboratory Portal of the Human Brain Project. To illustrate the potential application of the procedure, we tested its use with various sets of experimental data on GABAergic synapses; gephyrin and gephyrin-dependent pathways were chosen as a suitable example of a kinetic model of synaptic transmission. For individual spontaneous inhibitory events in hippocampal pyramidal CA1 neurons, we found that gephyrin-dependent subcellular pathways may shape synaptic events at different levels, and can be correlated with cell- or event-specific activity history and/or pathological conditions. PMID:27445784

  18. Subcellular distribution of Al, Cu, Mg, Mn and other elements in the human liver

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.; Zhang Peiqun; Lu Xiangli; Hou Xiaolin; Chai Zhifang [Institute of High Energy Physics and Laboratory of Nuclear Analysis Techniques, Chinese Academy of Sciences, Beijing (China)

    1999-03-01

    The elemental concentrations and chemical species of Al, Br, Cl, Cu, K, Mg, Mn, Na and I in human liver and its subcellular fractions were studied by several biochemical techniques combined with neutron activation analysis. The highest concentrations of Al, Mg, and I were found in the nuclei, whereas those of Br, Cu, Cl, K, Mn and Na in the cytosol. About 20% of Br, half of Al and most of Cu (78.8%), Mg (65.9%) and Mn (80.6%) remained in the cellulose bags after dialysis of liver homogenate, which were suggested to be bound to macromolecules. K (100%) and more than 95% of Cl and Na were found to be in the dialyzates. Similar results were found in the fractions of nuclei, mitochondria, lysosome and microsome, respectively, after the same treatment. Further study was carried out to elucidate the elemental distribution in the cytosol by ethanol precipitation and by ammonium sulfate fractionation. The results suggested that several kinds of Cu-, Mn- and Mg-bound proteins existed in the cytosol of human liver cells. (orig.) With 2 figs., 6 tabs., 14 refs.

  19. ELECTRON MICROSCOPIC AUTORADIOGRAPHIC STUDY ON SUBCELLULAR LOCALIZATION OF FISSION PRODUCT 147Pm IN TISSUE CELLS

    Institute of Scientific and Technical Information of China (English)

    朱寿彭; 汪源长

    1994-01-01

    The early risk of internal contaminated accumualtion of 147Pm is in blood cells and endothelial cells,especially in red blood cells.Then 147Pm is selectively deposited in ultrastructure of liver cells,such as in nucleus,nucleolus,rough endoplasmic reticulum,mitochondria and microbodies,Dense tracks also appear in mitochondria and lysosome of pedal cells within renal corpuscle,and so dose in nucleus as well as in mitochondria and microbodies of epicyte of kidney near-convoluted tubule.With the prolongation of observing time,147Pm is selectively and steadily depostied in subcellular level of organic ocmponent for bone.Substantial amount of 147Pm is taken up into the nuclear fraction of osteoclasts and osteoblasts.Particularly,in organelles 147Pm is mainly accumulated in rough endoplasmic reticulum and in mitochondria.Autoradiographic tracks especially localize in combined point between Golgi complex and transitive vesicle of rough endoplasmic reticulum.In addition,numerous 147Pm deposited in collagenous fibre within interstitial of bone cells is hardly excreted.

  20. Subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

    International Nuclear Information System (INIS)

    Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes

  1. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  2. Technical advance: Inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments.

    Science.gov (United States)

    Paschke, Stephan; Weidner, Astrid Franziska; Paust, Tobias; Marti, Othmar; Beil, Michael; Ben-Chetrit, Eldad

    2013-11-01

    Colchicine is an efficient drug for the management of inflammatory diseases, such as gouty arthritis and familial Mediterranean fever. It affects neutrophil activity by interfering with the formation of microtubules. To test the hypothesis that therapeutic concentrations of colchicine modulate the mechanical properties of these cells, we applied a combination of biophysical techniques (optical stretching and microrheology) to analyze cellular deformability. The contribution of the subcellular compartments to the regulation of cell mechanics was determined by fitting a multicomponent model of cellular viscoelasticity to time-dependent deformation curves. Neutrophils were found to be less deformable in response to 10 ng/ml colchicine. The model-based analysis of cellular deformation revealed a decrease in cytoplasmatic elasticity and a substantial increase in both elasticity and viscosity of the cell membrane compartment in response to colchicine. These results correlate with a reduced number of cytoplasmatic microtubules and an increase in subcortical actin filaments. The latter finding was confirmed by microrheology and fluorescence microscopy. Neutrophil migration through small pores requiring substantial cellular deformations, but not through large pores, was significantly impaired by colchicine. These data demonstrate that colchicine determines mechanics of neutrophils and, thereby, motility in confined spaces, which is crucial during extravasation of neutrophils in response to inflammatory stimuli. PMID:23901122

  3. Localization and subcellular distribution of N-copine in mouse brain.

    Science.gov (United States)

    Nakayama, T; Yaoi, T; Kuwajima, G

    1999-01-01

    N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+-dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+-dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of "double C2 domain-containing proteins," including synaptotagmin I, in presynaptic events. PMID:9886090

  4. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation

    Science.gov (United States)

    Rodríguez-Escudero, María; Cid, Víctor J.; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors

  5. Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

    OpenAIRE

    Reddick, L. Evan; Alto, Neal M.

    2012-01-01

    The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our under...

  6. Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products

    OpenAIRE

    Cummins, Timothy D.; Higdon, Ashlee N; Kramer, Philip A.; Chacko, Balu K; Riggs, Daniel W.; Salabei, Joshua K.; Dell’Italia, Louis J.; Zhang, Jianhua; Darley-Usmar, Victor M.; Hill, Bradford G.

    2012-01-01

    Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled...

  7. Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator

    International Nuclear Information System (INIS)

    A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites

  8. Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

    OpenAIRE

    Wang, Yuanpeng; Huang, Jing; Gao, Yanzheng

    2012-01-01

    Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were g...

  9. Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    OpenAIRE

    Kathryn R Bowles; Brooks, Simon P.; Dunnett, Stephen B.; Lesley Jones

    2015-01-01

    Huntington’s disease is a neurodegenerative disorder characterised primarily by motor abnormalities, and is caused by an expanded polyglutamine repeat in the huntingtin protein. Huntingtin dynamically shuttles between subcellular compartments, and the mutant huntingtin protein is mislocalised to cell nuclei, where it may interfere with nuclear functions, such as transcription. However, the mechanism by which mislocalisation of mutant huntingtin occurs is currently unknown. An immortalised emb...

  10. Multi-Scale Continuum Modeling of Biological Processes: From Molecular Electro-Diffusion to Sub-Cellular Signaling Transduction

    OpenAIRE

    Cheng, Y; Kekenes-Huskey, P; Hake, JE; Holst, MJ; McCammon, JA; Michailova, AP

    2012-01-01

    This article provides a brief review of multi-scale modeling at the molecular to cellular scale, with new results for heart muscle cells. A finite element-based simulation package (SMOL) was used to investigate the signaling transduction at molecular and sub-cellular scales (http://mccammon.ucsd.edu/smol/, http://FETK.org) by numerical solution of time-dependent Smoluchowski equations and a reaction-diffusion system. At the molecular scale, SMOL has yielded experimentally-validated estimates ...

  11. MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria.

    Science.gov (United States)

    Magnus, Marcin; Pawlowski, Marcin; Bujnicki, Janusz M

    2012-12-01

    Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction. PMID:22705560

  12. Effects of carbohydrate supplements on exercise-induced menstrual dysfunction and ovarian subcellular structural changes in rats

    Directory of Open Access Journals (Sweden)

    Can Zhao

    2014-09-01

    Conclusion: Female adult rats with 9-week continuous exercise can cause menstrual dysregulation as a model for EAMD. Post-EAMD intervention with glucose and oligosaccharide intake can normalize the menstrual cycle, restore the follicular subcellular structure, and reverse the exercise-induced reduction of ovary sex hormones. It suggests a positive feedback of hypothalamus–pituitary–ovary axis might be involved in the molecular mechanisms of energy intake in treating EAMD.

  13. mGOASVM: Multi-label protein subcellular localization based on gene ontology and support vector machines

    OpenAIRE

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

    2012-01-01

    Abstract Background Although many computational methods have been developed to predict protein subcellular localization, most of the methods are limited to the prediction of single-location proteins. Multi-location proteins are either not considered or assumed not existing. However, proteins with multiple locations are particularly interesting because they may have special biological functions, which are essential to both basic research and drug discovery. Results This paper proposes an effic...

  14. PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

    Institute of Scientific and Technical Information of China (English)

    Evangelia I. Petsalaki; Pantelis G. Bagos; Zoi I. Litou; Stavros J. Hamodrakas

    2006-01-01

    The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function.We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast,thylakoid, mitochondrion, secretory pathway, and "other". When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:http://bioinformatics.biol.uoa.gr/PredSL/.

  15. Precision automation of cell type classification and sub-cellular fluorescence quantification from laser scanning confocal images

    Directory of Open Access Journals (Sweden)

    Hardy Craig Hall

    2016-02-01

    Full Text Available While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to 1 segment radial plant organs into individual cells, 2 classify cells into cell type categories based upon random forest classification, 3 divide each cell into sub-regions, and 4 quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types.

  16. Subcellular distribution of trace elements and liver histology of landlocked Arctic char (Salvelinus alpinus) sampled along a mercury contamination gradient.

    Science.gov (United States)

    Barst, Benjamin D; Rosabal, Maikel; Campbell, Peter G C; Muir, Derek G C; Wang, Xioawa; Köck, Günter; Drevnick, Paul E

    2016-05-01

    We sampled landlocked Arctic char (Salvelinus alpinus) from four lakes (Small, 9-Mile, North, Amituk) in the Canadian High Arctic that span a gradient of mercury contamination. Metals (Hg, Se, Tl, and Fe) were measured in char tissues to determine their relationships with health indices (relative condition factor and hepatosomatic index), stable nitrogen isotope ratios, and liver histology. A subcellular partitioning procedure was employed to determine how metals were distributed between potentially sensitive and detoxified compartments of Arctic char livers from a low- and high-mercury lake (Small Lake and Amituk Lake, respectively). Differences in health indices and metal concentrations among char populations were likely related to differences in feeding ecology. Concentrations of Hg, Se, and Tl were highest in the livers of Amituk char, whereas concentrations of Fe were highest in Small and 9-Mile char. At the subcellular level we found that although Amituk char had higher concentrations of Tl in whole liver than Small Lake char, they maintained a greater proportion of this metal in detoxified fractions, suggesting an attempt at detoxification. Mercury was found mainly in potentially sensitive fractions of both Small and Amituk Lake char, indicating that Arctic char are not effectively detoxifying this metal. Histological changes in char livers, mainly in the form of melano-macrophage aggregates and hepatic fibrosis, could be linked to the concentrations and subcellular distributions of essential or non-essential metals. PMID:26986088

  17. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    Science.gov (United States)

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi. PMID:20053634

  18. Content of peroxide compounds and 14C-leucine incorporation in subcellular fractions of new embryos obtained from poultry fed with antioxidants

    International Nuclear Information System (INIS)

    The formation of peroxide compounds in feed mixtures and the poultry organism was investigated. Two groups of broilers were fed with whole fodder mixture. The controls received a basic diet, while the experimental group got in addition 125 ppm each of the ethoxyquin antioxidant. 14C leucine was applied to the 14-day embryos in the eggs air space in a 20 microcurie dose per embryo. The incorporation of the labelled precursor was determined on the 3rd hour after introduction of the isotope. The radiometric analysis was made by the filter method with a liquid scintillation counter. The relative distribution of labelled leucine in the liver subcellular fractions of the control embryos revealed a pattern characteristic of their protein synthesizing capacity. The relative distribution of the labelled precursor in the experimental group showed the same pattern. The greater precursor incorporation in the nuclei of the embryos in the experimental group, however, suggests that ethoxyquin might stimulate nuclear protein synthesis or the passage of more protein from the cytoplasm, which is then utilized by the nucleolus for the development of the ribosomal apparatus

  19. The subcellular distribution of 239Pu, 241Am and 59Fe in the liver of rat and Chinese hamster as dependent on time

    International Nuclear Information System (INIS)

    The subcellular distribution of monomeric 239Pu, 241Am and iron in rat and Chinese hamster liver has been investigated by sucrose-, metrizamide- and Percoll-density gradients. In rat liver, the transuranium elements become and remain bound to typical lysosomes primary storage organelle in Chinese hamster liver. However, their apparent density in sucrose decreases with time, which possible indicates transition into telolysomes. The transuranium nuclides show a subcellular distribution which is quite different from that of iron. (orig.)

  20. Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

    OpenAIRE

    Pufeng Du; Lusheng Wang

    2014-01-01

    One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as w...

  1. The sub-cellular fate of mercury in the liver of wild mullets (Liza aurata) – Contribution to the understanding of metal-induced cellular toxicity

    International Nuclear Information System (INIS)

    Highlights: • This study is one of the first reports of Hg sub-cellular partitioning in wild fish. • Hg partition in Liza aurata liver differed between reference and contaminated sites. • Levels of Hg in sub-cellular fractions were in line with environmental contamination. • Lysosomes plus microsomes was the main fraction bounding Hg at the contaminated site. • Sub-cellular partitioning of Hg revealed to be a promising indicator of cellular toxicity. - Abstract: Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas – contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys + Mic). At LAR, Lys + Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions

  2. Combined phase and X-Ray fluorescence imaging at the sub-cellular level

    International Nuclear Information System (INIS)

    This work presents some recent developments in the field of hard X-ray imaging applied to biomedical research. As the discipline is evolving quickly, new questions appear and the list of needs becomes bigger. Some of them are dealt with in this manuscript. It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimental setup to investigate diverse aspects of cellular research. Together with its high spatial resolution, high flux and high energy range the experimental setup provides bigger field of view, is less sensitive to radiation damages (while taking phase contrast images) and suits well chemical analysis with emphasis on endogenous metals (Zn, Fe, Mn) but also with a possibility for exogenous one's like these found in nanoparticles (Au, Pt, Ag) study. Two synchrotron-based imaging techniques, fluorescence and phase contrast imaging were used in this research project. They were correlated with each other on a number of biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA, red blood cells). The explorations made in the chapter 5 allowed preparation of more established and detailed analysis, described in the next chapter where both techniques, X-ray fluorescence and phase contrast imaging, were exploited in order to access absolute metal projected mass fraction in a whole cell. The final image presents for the first time true quantitative information at the sub-cellular level, not biased by the cell thickness. Thus for the first time a fluorescence map serves as a complete quantitative image of a cell without any risk of misinterpretation. Once both maps are divided by each other pixel by pixel (fluorescence map divided by the phase map) they present a complete and final result of the metal (Zn in this work) projected mass fraction in ppm of dry weight. For the purpose of this calculation the analysis was extended to calibration (non-biological) samples. Polystyrene spheres of a known diameter and known

  3. Radioisotopic studies of the action of anticancer compounds in subcellular level

    International Nuclear Information System (INIS)

    Full text: Determination of mechanisms of biological action alkylating compounds with the purpose of an establishment of interrelation between structure and physiological action is one of the major problems of our time. Synthesis of new compounds and studying of corresponding processes in nucleus of cells is considered a priority of chemists and biologists. N-(β-chlorethyl)-salsoline has been earlier studied on antineoplastic activity in system in vitro, with the help marked thymidine. Thus it has been shown that this compound inhibits biosynthesis of nucleinic acids and proteins. In the present work influence of two antineoplastic preparations N-(β-chlorethyl)-salsoline and N-(β-chlorethyl)-hexamethylenimine on protein synthetic ability (PSA) at not sharing cells of animals of different age with use marked lysine has been investigated. Activity of these two preparations checked in identical concentration of 150 mkg/ml. Results of research have shown that N-(β-chlorethyl)-salsoline in a doze of 150 mkg/ml suppresses PSA nucleus of not sharing cells of a brain: on 11 % at 5-days animals; on 26 % at 25-days animals; on 14 % at 35 and 90-days animals. N-(β-chlorethyl)-hexamethylenimine in the same dozes on the contrary insignificantly stimulates this process. It is possible, that non-ribosomal alkylating does not occur in not sharing cells by these preparations (in normal cells). The action probably depends on structure of preparations. It is possible, that these preparations influence through immune system tumoral cells. Thus, antineoplastic compounds N-(β-chlorethyl)-salsoline and N-(β-chlorethyl)-hexamethylenimine do not operate on PSA at a subcellular level. Work is executed at financial support of the grant 'Research of natural connections with cancerolitic activity and creation on their basis of antineoplastic means'. This work was supported by the Center of Science and Technology RU (4F - 4.19)

  4. Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions.

    Science.gov (United States)

    Valente, Valeria; Maia, Rafaela Martins; Vianna, Murilo Carlos Bizam; Paçó-Larson, Maria Luisa

    2010-11-01

    Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family. PMID:20977671

  5. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    Science.gov (United States)

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis. PMID:26733305

  6. Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

    Science.gov (United States)

    Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

    2016-08-01

    Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed. PMID:26820914

  7. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    Directory of Open Access Journals (Sweden)

    Meredith J. Layton

    2014-04-01

    Full Text Available Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α to its PM (plasma membrane localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v FBS. Following stimulation of RTKs (receptor tyrosine kinases, microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.

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

    Directory of Open Access Journals (Sweden)

    Lucchetti-Miganeh Céline

    2010-03-01

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

  9. Identification of two subcellular sites for γ-glutamyltranspeptidase propeptide cleavage

    International Nuclear Information System (INIS)

    Renal intracellular and brush border membranes were purified from rats injected with [35S]methionine. Solubilized transpeptidase (γGT) was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophresis (PAGE). The initial precipitates contain 3 bands corresponding to the core glycosylated propeptide (75K) and the mature heterodimer subunits (50K and 30K). The propeptide represents 75% of the radioactivity in γGT from 5 to 45 min postinjection consistent with cotranslational cleavage of 25% of the propeptide to subunits. By 20 min postinjection all three bands are more diffuse and endoglycosidase H-resistant. Between 20 and 30 min postinjection, propeptide and heterodimer coincidentally reach the brush border membrane. Propeptide then rapidly disappears (t/sub 1/2/ < 1 h) whereas heterodimer accumulates for 4 h then disappears with a t/sub 1/2/ of 2.5 d. The basis for these two distinct subcellular sites of γGT propeptide cleavage is unknown. Both purified γGT heterodimer and immunoprecipitates of labeled γGT occasionally exhibit high M/sub r/ bands (85K and 100K) on SDS-PAGE. Individual subunits and high M/sub r/ species were eluted from SDS gel slices, subjected to SDS-PAGE and analyzed on immunoblots with IgG affinity-purified against individual subunits. The cumulative data show that the subunits can form both stable homodimers (60K and 100K) and heterodimers (85K) during SDS-PAGE. Thus, these high M/sub r/ species do not represent biosynthetic intermediates of γGT

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

    International Nuclear Information System (INIS)

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

  11. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    Science.gov (United States)

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A. PMID:12514140

  12. Subcellular metabolic contrast in living tissue using dynamic full field OCT (D-FFOCT) (Conference Presentation)

    Science.gov (United States)

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

    2016-03-01

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

  13. Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress.

    Science.gov (United States)

    Maitra, Dhiman; Elenbaas, Jared S; Whitesall, Steven E; Basrur, Venkatesha; D'Alecy, Louis G; Omary, M Bishr

    2015-09-25

    Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a

  14. Subcellular distribution of uranium in the roots of Spirodela punctata and surface interactions

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Xiaoqin, E-mail: xiaoqin_nie@163.com [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Key Laboratory of Radiation Physics and Technology (Sichuan University), Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu 610064 (China); Dong, Faqin, E-mail: fqdong2004@163.com [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Liu, Ning [Key Laboratory of Radiation Physics and Technology (Sichuan University), Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu 610064 (China); Liu, Mingxue [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Zhang, Dong; Kang, Wu [Institute of Nuclear Physics and Chemistry,China Academy of Engineering Physics, Mianyang 621900 (China); Sun, Shiyong; Zhang, Wei; Yang, Jie [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China)

    2015-08-30

    Graphical abstract: - Highlights: • The proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. • The particles including 35% Fe (wt%) released from the cells after 100 mg/L U treatment 48 h. • Most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal. • FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed. - Abstract: The subcellular distribution of uranium in roots of Spirodela punctata (duckweed) and the process of surface interaction were studied upon exposure to U (0, 5–200 mg/L) at pH 5. The concentration of uranium in each subcelluar fraction increased significantly with increasing solution U level, after 200 mg/L uranium solution treatment 120 h, the proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. OM SEM and EDS showed after 5–200 mg/L U treatment 4–24 h, some intracellular fluid released from the root cells, after 100 mg/L U treatment 48 h, the particles including 35% Fe (wt%) and other organic matters such as EPS released from the cells, most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal, similar crystal has been found in the cell wall and organelle fractions after 50 mg/L U treatment 120 h. FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed.

  15. Arabinogalactan glycosyltransferases target to a unique subcellular compartment that may function in unconventional secretion in plants.

    Science.gov (United States)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Mouille, Grégory; Burow, Meike; Geshi, Naomi

    2014-11-01

    We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A [Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan (O-glycan) biosynthesis rather than N-glycan processing. The dual localization of AtGALT31A was also observed for fluorescently tagged AtGALT31A stably expressed in an Arabidopsis atgalt31a mutant background. Further, site-directed mutagenesis of a phosphorylation site of AtGALT29A (Y144) increased the frequency of the protein being targeted to the AtGALT31A-localized small compartments, suggesting a role of Y144 in subcellular targeting. The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes. However, 41% colocalized with EXO70E2 (Arabidopsis thaliana exocyst protein Exo70 homolog 2), a marker for exocyst-positive organelles, and least affected by Brefeldin A and Wortmannin. Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants. PMID:25074762

  16. Erratum to: rhesus macaque MHC class I molecules show differential subcellular localizations.

    Science.gov (United States)

    Rosner, Cornelia; Kruse, Philip H; Lübke, Torben; Walter, Lutz

    2010-06-01

    The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding alpha1 and alpha2 domains, suggesting failure of peptide binding is responsible for retaining 'intracellular' Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes. PMID:20445972

  17. Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods.

    Science.gov (United States)

    Gimbert, Frédéric; Geffard, Alain; Guédron, Stéphane; Dominik, Janusz; Ferrari, Benoit J D

    2016-02-01

    Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates. PMID:26688328

  18. Controlling

    OpenAIRE

    Lohnická, Jitka

    2009-01-01

    This thesis deals with the description and analysis of controlling methods in one unnamed company, which is the subsidiary of multinational corporation. Controlling, its function, objectives and controlling methods are theoretically defined in the thesis. The practical part of the thesis is focused on methods of planning in the company, its system of calculations, responsibility centres and reporting.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-05

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

  20. Hepatocyte nuclear structure and subcellular distribution of copper in zebrafish Brachydanio rerio and roach Rutilus rutilus (Teleostei, Cyprinidae) exposed to copper sulphate

    International Nuclear Information System (INIS)

    Copper is a trace element essential to life, but also a heavy metal with toxic effect clearly demonstrated. Cu induced perturbations in fish liver are well documented but the variability of the reported results is large. In this study two cyprinids, zebrafish and roach, were exposed to copper. Reported histocytological changes are either adaptative or degenerative depending on fish species, concentration of metal, and duration of exposure. Hepatic subcellular distribution of copper was determined by X-ray microanalysis in control and Cu-exposed roach and zebrafish. Sublethal copper sulphate contamination induced the development of a particular nucleolar alteration forming a network or honeycomb like structure in liver. This perturbation is observable in almost all the hepatocytes of zebrafish and roach exposed to copper for a minimum of 4 days of exposure. It seemed to concern more precisely the pars fibrosa. X-ray microanalysis showed that the appearance of network nucleolus was in relation to a Cu accumulation. Cu deposit was well located in the network as pars granulosa and nucloplasm showed very lower metal concentrations. The origin and consequence of network structure in nucleolus was discussed

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

    Directory of Open Access Journals (Sweden)

    Xiuzhi Jia

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

  2. Effect of subcellular distribution on nC₆₀ uptake and transfer efficiency from Scenedesmus obliquus to Daphnia magna.

    Science.gov (United States)

    Chen, Qiqing; Hu, Xialin; Yin, Daqiang; Wang, Rui

    2016-06-01

    The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension (nC60). The nC60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the nC60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The nC60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. nC60 uptake amounts in D. magna were found to be mainly relative to the NPs' distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the nC60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the nC60 uptake from CW fed group. The CO fed groups acquired high TE values for nC60, while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs' dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator. PMID:26946286

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

    Directory of Open Access Journals (Sweden)

    Shibiao Wan

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

  4. iLoc-Euk: a multi-label classifier for predicting the subcellular localization of singleplex and multiplex eukaryotic proteins.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Predicting protein subcellular localization is an important and difficult problem, particularly when query proteins may have the multiplex character, i.e., simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular location predictor can only be used to deal with the single-location or "singleplex" proteins. Actually, multiple-location or "multiplex" proteins should not be ignored because they usually posses some unique biological functions worthy of our special notice. By introducing the "multi-labeled learning" and "accumulation-layer scale", a new predictor, called iLoc-Euk, has been developed that can be used to deal with the systems containing both singleplex and multiplex proteins. As a demonstration, the jackknife cross-validation was performed with iLoc-Euk on a benchmark dataset of eukaryotic proteins classified into the following 22 location sites: (1 acrosome, (2 cell membrane, (3 cell wall, (4 centriole, (5 chloroplast, (6 cyanelle, (7 cytoplasm, (8 cytoskeleton, (9 endoplasmic reticulum, (10 endosome, (11 extracellular, (12 Golgi apparatus, (13 hydrogenosome, (14 lysosome, (15 melanosome, (16 microsome (17 mitochondrion, (18 nucleus, (19 peroxisome, (20 spindle pole body, (21 synapse, and (22 vacuole, where none of proteins included has ≥25% pairwise sequence identity to any other in a same subset. The overall success rate thus obtained by iLoc-Euk was 79%, which is significantly higher than that by any of the existing predictors that also have the capacity to deal with such a complicated and stringent system. As a user-friendly web-server, iLoc-Euk is freely accessible to the public at the web-site http://icpr.jci.edu.cn/bioinfo/iLoc-Euk. It is anticipated that iLoc-Euk may become a useful bioinformatics tool for Molecular Cell Biology, Proteomics, System Biology, and Drug Development Also, its novel approach will further stimulate the development of

  5. Subcellular compartmentation of sugar signalling: Links among carbon cellular status, route of sucrolysis, sink-source allocation, and metabolic partitioning

    Directory of Open Access Journals (Sweden)

    Axel eTiessen

    2013-01-01

    Full Text Available Recent findings suggest that both subcellular compartmentation and route of sucrolysis are important for plant development, growth, and yield. Signalling effects are dependent on the tissue, cell type and stage of development. Downstream effects also depend on the amount and localisation of hexoses and disaccharides. All enzymes of sucrose metabolism (e.g. invertase, hexokinase, fructokinase, sucrose synthase, and sucrose 6-phosphate synthase are not produced from single genes, but from paralogue families in plant genomes. Each paralogue has unique expression across plant organs and developmental stages. Multiple isoforms can be targeted to different cellular compartments (e.g. plastids, mitochondria, nuclei, and cytosol. Many of the key enzymes are regulated by post-transcriptional modifications and associate in multimeric protein complexes. Some isoforms have regulatory functions, either in addition to or in replacement of their catalytic activity. This explains why some isozymes are not redundant, but also complicates elucidation of their specific involvement in sugar signalling. The subcellular compartmentation of sucrose metabolism forces refinement of some of the paradigms of sugar signalling during physiological processes. For example, the catalytic and signalling functions of diverse paralogues needs to be more carefully analysed in the context of post-genomic biology. It is important to note that it is the differential localization of both the sugars themselves as well as the sugar-metabolizing enzymes that ultimately led to sugar signalling. We conclude that a combination of subcellular complexity and gene duplication/subfunctionalization gave rise to sugar signalling as a regulatory mechanism in plant cells.

  6. CE-PLoc: an ensemble classifier for predicting protein subcellular locations by fusing different modes of pseudo amino acid composition.

    Science.gov (United States)

    Khan, Asifullah; Majid, Abdul; Hayat, Maqsood

    2011-08-10

    Precise information about protein locations in a cell facilitates in the understanding of the function of a protein and its interaction in the cellular environment. This information further helps in the study of the specific metabolic pathways and other biological processes. We propose an ensemble approach called "CE-PLoc" for predicting subcellular locations based on fusion of individual classifiers. The proposed approach utilizes features obtained from both dipeptide composition (DC) and amphiphilic pseudo amino acid composition (PseAAC) based feature extraction strategies. Different feature spaces are obtained by varying the dimensionality using PseAAC for a selected base learner. The performance of the individual learning mechanisms such as support vector machine, nearest neighbor, probabilistic neural network, covariant discriminant, which are trained using PseAAC based features is first analyzed. Classifiers are developed using same learning mechanism but trained on PseAAC based feature spaces of varying dimensions. These classifiers are combined through voting strategy and an improvement in prediction performance is achieved. Prediction performance is further enhanced by developing CE-PLoc through the combination of different learning mechanisms trained on both DC based feature space and PseAAC based feature spaces of varying dimensions. The predictive performance of proposed CE-PLoc is evaluated for two benchmark datasets of protein subcellular locations using accuracy, MCC, and Q-statistics. Using the jackknife test, prediction accuracies of 81.47 and 83.99% are obtained for 12 and 14 subcellular locations datasets, respectively. In case of independent dataset test, prediction accuracies are 87.04 and 87.33% for 12 and 14 class datasets, respectively. PMID:21864791

  7. Nitric Oxide Reduces Hydrogen Peroxide Accumulation Involved in Water Stress-induced Subcellular Anti-oxidant Defense in Maize Plants

    Institute of Scientific and Technical Information of China (English)

    Jianrong Sang; Mingyi Jiang; Fan Lin; Shucheng Xu; Aying Zhang; Mingpu Tan

    2008-01-01

    Nitric oxide (NO) Is a bioactive molecule involved in many biological events, and has been reported as pro-oxidant as well as anti-oxidant in plants. In the present study, the sources of NO production under water stress, the role of NO in water stress-induced hydrogen peroxide (H2O2) accumulation and subcellular activities of anti-oxidant enzymes in leaves of maize (Zea mays L.) plants were investigated. Water stress Induced defense increases in the generation of NO In maize mesphyll cells and the activity of nitric oxide synthase (NOS) in the cytosolic and microsomal fractions of maize leaves. Water stress-induced defense increases in the production of NO were blocked by pretreatments with Inhibitors of NOS and nitrate reductase (NR), suggesting that NO is produced from NOS and NR in leaves of maize plants exposed to water stress. Water stress also induced increases in the activities of the chloroplastic and cytosolic anti-oxidant enzymes superoxide dismutase (SOD), ascorbate peroxidass (APX), and glutathione reductase (GR), and the increases in the activities of anti-oxidant enzymes were reduced by pretreatments with inhibitors of NOS and NR. Exogenous NO increases the activities of water stress-induced subcellular anti-oxidant enzymes, which decreases accumulation of H2O2. Our results suggest that NOS and NR are involved in water strese-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is, at least in part, due to the induction of a subcellular anti-oxidant defense.

  8. Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

    Science.gov (United States)

    Hoffman, Robert M.

    2006-02-01

    We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

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

    Directory of Open Access Journals (Sweden)

    Kriti Bahl

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

  10. Characterization of subcellular localization and stability of a splice variant of G alphai2

    Directory of Open Access Journals (Sweden)

    Wedegaertner Philip B

    2002-05-01

    Full Text Available Abstract Background Alternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sαi2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas αi2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal. Results This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sαi2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to αi2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and αi2(1–331, in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes. Finally, sαi2 displays a propensity to localize to potential aggresome-like structures. Conclusions Thus, instead of the novel C-terminus of sαi2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2.

  11. Effects of ivermectin on Danio rerio: a multiple endpoint approach: behaviour, weight and subcellular markers.

    Science.gov (United States)

    Domingues, I; Oliveira, R; Soares, A M V M; Amorim, M J B

    2016-04-01

    Ivermectin (IVM) is a broad acting antihelmintic used in various veterinary pharmaceuticals. It has been shown that IVM enters the aquatic compartment and adversely affects organisms including fish. This study is based on the hypothesis that long term exposure to IVM affects fish and thus, the main objective was to assess the chronic effects of 0.25 and 25 µg IVM/L to zebrafish using multiple endpoints representative of several levels of biological organization: weight, behaviour (swimming and feeding) and subcellular markers including biomarkers for oestrogenicity (vitellogenin-VTG), oxidative stress (catalase-CAT and glutathione-S-transferase-GST) and neurotransmission (cholinesterase-ChE). Concentrations as low as 0.25 µg IVM/L disrupted the swimming behaviour, causing fish to spend more time at the bottom of aquaria. Such reduction of the swimming performance affected the feeding ability which is likely responsible for the weight loss. The effects on weight were gender differentiated, being more pronounced in males (0.25 µg IVM/L) than in females (25 µg IVM/L). Fish exposed to 25 µg/L exhibited darker coloration and mild curvature of the spine. No effects on VTG and AChE were observed, but a reduction on CAT and GST levels was observed in fish exposed to 25 µg IVM/L, although these alterations probably only reflect the general condition of the fish which was significantly compromised at this concentration. Despite that predicted environmental concentrations of IVM are below 0.25 µg/L, the behavioural effects may be translated into important ecological impacts, e.g. at predator-prey interactions where fish competitive advantage can be decreased. Future work should address the link between behaviour disruption and population fitness. The current study was based on a one experiment and multiple endpoint (anchored) approach, allowing the results to be integrated and linked towards a mechanistic understanding. PMID:26769347

  12. LocateP: Genome-scale subcellular-location predictor for bacterial proteins

    Directory of Open Access Journals (Sweden)

    Zhou Miaomiao

    2008-03-01

    Full Text Available Abstract Background In the past decades, various protein subcellular-location (SCL predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP. Results LocateP combines many of the existing high-precision SCL identifiers with our own newly developed identifiers for specific SCLs. The LocateP pipeline was designed such that it mimics protein targeting and secretion processes. It distinguishes 7 different SCLs within Gram-positive bacteria: intracellular, multi-transmembrane, N-terminally membrane anchored, C-terminally membrane anchored, lipid-anchored, LPxTG-type cell-wall anchored, and secreted/released proteins. Moreover, it distinguishes pathways for Sec- or Tat-dependent secretion and alternative secretion of bacteriocin-like proteins. The pipeline was tested on data sets extracted from literature, including experimental proteomics studies. The tests showed that LocateP performs as well as, or even slightly better than other SCL predictors for some locations and outperforms

  13. Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

    International Nuclear Information System (INIS)

    The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate-14C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  14. Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

    Science.gov (United States)

    Montamat, E E; Vermouth, N T; Blanco, A

    1988-11-01

    Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria. PMID:3214422

  15. Dynamic full field optical coherence tomography: subcellular metabolic contrast revealed in tissues by temporal analysis of interferometric signals

    CERN Document Server

    Apelian, Clement; Thouvenin, Olivier; Boccara, A Claude

    2016-01-01

    We developed a new endogenous approach to reveal subcellular metabolic contrast in fresh ex vivo tissues taking advantage of the time dependence of the full field optical coherence tomography interferometric signals. This method reveals signals linked with local activity of the endogenous scattering elements which can reveal cells where other imaging techniques fail or need exogenous contrast agents. We benefit from the micrometric transverse resolution of full field OCT to image intracellular features. We used this time dependence to identify different dynamics at the millisecond scale on a wide range of organs in normal or pathological conditions.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    -a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter....... Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular...

  17. Subcellular localization of L-selectin ligand in the endometrium implies a novel function for pinopodes in endometrial receptivity

    Directory of Open Access Journals (Sweden)

    Nejatbakhsh Reza

    2012-06-01

    Full Text Available Abstract Background Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. The L-selectin adhesion system has been strongly proposed to play an important role in the initial steps of trophoblast adhesion and promotion of integrin-dependent processes, ultimately culminating in the establishment of the embryo-maternal interface. On the basis of these facts, we hypothesized a novel role for pinopodes as the first embryo-fetal contact sites to contain the highest subcellular expression of L-selectin ligand suggesting its role in early adhesion as predicted. Thus, the objective of this study was therefore to determine the subcellular pattern of distribution of the L-selectin ligand (MECA-79 in human endometrial apical membrane region during the window of implantation. Methods Endometrial biopsies of secretory phases from fertile females ranging in age between 25 and 42years were studied using several approaches, including scanning electron microscopy (SEM, immunostaining for light microscopy and transmission electron microscopy (TEM, and immunoblotting as well as statistical analysis of the area-related numerical densities of immunoreactive MECA-79-bound nanogolds to detect the expression pattern and the subcellular distribution pattern of L-selectin ligand (MECA-79 in human endometrium during the window of implantation. Results The endometrial biopsies were scored according the dating criteria of Noyes et al. by an experienced histologist. The SEM images of the midluteal phase specimens revealed that fully developed pinopodes were abundant in our samples. HRP-immunostaining and immunofluorescent staining as well as immunoblotting revealed that MECA-79 was expressed in the midluteal phase specimens. The results of immunogold TEM illustrated the expression of MECA-79 in human pinopodes in the midluteal phase and a higher area

  18. Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin.

    OpenAIRE

    El-Gedaily, A; Paesold, G; Krause, M.

    1997-01-01

    The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quan...

  19. 3H-DOPA: Distribution in the subcellular fractions of melanomas and its perspective use in radiotherapy

    International Nuclear Information System (INIS)

    The incorporation of 3H-DOPA (740 MBq per mouse) into the subcellular fractions of Harding-Passey's melanoma was studied simultaenously with tyrosinase activity determination up to 2 days. The kinetics of 3H-DOPA was checked by means of electron histoautoradiography 30 min, 1, 3 and 24 hours, respectively, after injection. Maximum incorporation of 3H-DOPA was found in mitochondria and melanosomes as well as high tyrosinase activity. DOPA is supposed to be used for internal radiotherapy of melanomas. (author)

  20. Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay.

    Science.gov (United States)

    Wang, Zaosheng; Feng, Chenglian; Ye, Chun; Wang, Youshao; Yan, Changzhou; Li, Rui; Yan, Yijun; Chi, Qiaoqiao

    2016-07-01

    In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from "spillover" was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically

  1. Alterations in subcellular expression of acid-sensing ion channels in the rat forebrain following chronic amphetamine administration

    OpenAIRE

    Suman, Ajay; Mehta, Bhavi; Guo, Ming-Lei; Chu, Xiang-Ping; Fibuch, Eugene E.; Mao, Li-Min; WANG, John Q.

    2010-01-01

    Acid-sensing ion channels (ASICs) are densely expressed in broad areas of mammalian brains and actively modulate synaptic transmission and a variety of neuronal activities. To explore whether ASICs are linked to addictive properties of drugs of abuse, we investigated the effect of the psychostimulant amphetamine on subcellular ASIC expression in the rat forebrain in vivo. Repeated administration of amphetamine (once daily for 7 days, 1.25 mg/kg for days 1/7, 4 mg/kg for days 2–6) induced typi...

  2. Hepatic oxidative stress and metal subcellular partitioning are affected by selenium exposure in wild yellow perch (Perca flavescens).

    Science.gov (United States)

    Ponton, Dominic E; Caron, Antoine; Hare, Landis; Campbell, Peter G C

    2016-07-01

    Yellow perch (Perca flavescens) collected from 11 lakes in the Canadian mining regions of Sudbury (Ontario) and Rouyn-Noranda (Quebec) display wide ranges in the concentrations of cadmium (Cd), nickel (Ni), selenium (Se), and thallium (Tl) in their livers. To determine if these trace elements, as well as copper (Cu) and zinc (Zn), are causing oxidative stress in these fish, we measured three biochemical indicators (glutathione (GSH), glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS)) in their livers. We observed that 44% of the yellow perch that we collected were at risk of cellular oxidative stress and lipid peroxidation. Considering all fish from all lakes, higher liver Se concentrations were coincident with both lower proportions of GSSG compared to GSH and lower concentrations of TBARS, suggesting that the essential trace-element Se acts as an antioxidant. Furthermore, fish suffering oxidative stress had higher proportions of Cd, Cu and Zn in potentially sensitive subcellular fractions (organelles and heat-denatured proteins) than did fish not suffering from stress. This result suggests that reactive oxygen species may oxidize metal-binding proteins and thereby reduce the capacity of fish to safely bind trace metals. High Cd concentrations in metal-sensitive subcellular fractions likely further exacerbate the negative effects of lower Se exposure. PMID:27131821

  3. Effects of extended growth periods on subcellular distribution, chemical forms, and the translocation of cadmium in Impatiens walleriana.

    Science.gov (United States)

    Lai, Hung-Yu; Cai, Ming-Cyuan

    2016-01-01

    Impatiens walleriana plants accumulate sufficiently high concentrations of cadmium (Cd) for this species to be considered a potential Cd hyperaccumulator. Rooted cuttings were grown hydroponically for 25 and 50 days in solutions spiked with various Cd concentrations. The subcellular distribution and chemical forms of Cd in different organs were analyzed, and its upward translocation was also assessed. The plants accumulated large amounts of Cd; the Cd concentration in the roots and shoots reached 120-1900 and 60-1600 mg/kg, respectively. Regardless of the growth period, the Cd accumulated in the roots was primarily compartmentalized in the soluble fraction or ethanol and deionized water extractable chemical forms with high migration abilities. Translocation to the shoots was followed by an association of Cd mainly in the cell wall or with pectate and protein. The roots' Cd showed a high migration capacity for predicting the shoots' Cd concentrations. Different exposure periods significantly affected the subcellular distribution of Cd in the stems, and thus the upward translocation. PMID:26247535

  4. Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    Science.gov (United States)

    Bowles, Kathryn R.; Brooks, Simon P.; Dunnett, Stephen B.; Jones, Lesley

    2015-01-01

    Huntington’s disease is a neurodegenerative disorder characterised primarily by motor abnormalities, and is caused by an expanded polyglutamine repeat in the huntingtin protein. Huntingtin dynamically shuttles between subcellular compartments, and the mutant huntingtin protein is mislocalised to cell nuclei, where it may interfere with nuclear functions, such as transcription. However, the mechanism by which mislocalisation of mutant huntingtin occurs is currently unknown. An immortalised embryonic striatal cell model of HD (StHdhQ111) was stimulated with epidermal growth factor in order to determine whether the subcellular localisation of huntingtin is dependent on kinase signalling pathway activation. Aberrant phosphorylation of AKT and MEK signalling pathways was identified in cells carrying mutant huntingtin. Activity within these pathways was found to contribute to the regulation of huntingtin and mutant huntingtin localisation, as well as to the expression of immediate-early genes. We propose that altered kinase signalling is a phenotype of Huntington’s disease that occurs prior to cell death; specifically, that altered kinase signalling may influence huntingtin localisation, which in turn may impact upon nuclear processes such as transcriptional regulation. Aiming to restore the balance of activity between kinase signalling networks may therefore prove to be an effective approach to delaying Huntington’s disease symptom development and progression. PMID:26660732

  5. Bioaccumulation, subcellular, and molecular localization and damage to physiology and ultrastructure in Nymphoides peltata (Gmel.) O. Kuntze exposed to yttrium.

    Science.gov (United States)

    Fu, Yongyang; Li, Feifei; Xu, Ting; Cai, Sanjuan; Chu, Weiyue; Qiu, Han; Sha, Sha; Cheng, Guangyu; Xu, Qinsong

    2014-02-01

    Bioaccumulation, subcellular distribution, and acute toxicity of yttrium (Y) were evaluated in Nymphoides peltata. The effects of Y concentrations of 1-5 mg L(-1) applied for 4 days were assessed by measuring changes in photosynthetic pigments, nutrient contents, enzymatic and non-enzymatic antioxidants, and ultrastructure. The accumulation of Y in subcellular fractions decreased in the order of cell wall > organelle > soluble fraction. Much more Y was located in cellulose and pectin than in other biomacromolecules. The content of some mineral elements (Mg, Ca, Fe, Mn, and Mo) increased in N. peltata, but there was an opposite effect for P and K. Meanwhile, ascorbate, and catalase activity decreased significantly for all Y concentrations. In contrast, peroxidase activity was induced, while initial rises in superoxide dismutase activity and glutathione content were followed by subsequent declines. Morphological symptoms of senescence, such as chlorosis and damage to chloroplasts and mitochondria, were observed even at the lowest Y concentration. Pigment content decreased as the Y concentration rose and the calculated EC50 and MPC of Y for N. peltata were 2 and 0.2 mg L(-1) after 4 days of exposure, respectively. The results showed that exogenous Y was highly available in water and that its high concentration in water bodies might produce harmful effects on aquatic organisms. N. peltata is proposed as a biomonitor for the assessment of metal pollution in aquatic ecosystems. PMID:24170501

  6. Prediction of protein subcellular localization by support vector machines using multi-scale energy and pseudo amino acid composition.

    Science.gov (United States)

    Shi, J-Y; Zhang, S-W; Pan, Q; Cheng, Y-M; Xie, J

    2007-07-01

    As more and more genomes have been discovered in recent years, there is an urgent need to develop a reliable method to predict the subcellular localization for the explosion of newly found proteins. However, many well-known prediction methods based on amino acid composition have problems utilizing the sequence-order information. Here, based on the concept of Chou's pseudo amino acid composition (PseAA), a new feature extraction method, the multi-scale energy (MSE) approach, is introduced to incorporate the sequence-order information. First, a protein sequence was mapped to a digital signal using the amino acid index. Then, by wavelet transform, the mapped signal was broken down into several scales in which the energy factors were calculated and further formed into an MSE feature vector. Following this, combining this MSE feature vector with amino acid composition (AA), we constructed a series of MSEPseAA feature vectors to represent the protein subcellular localization sequences. Finally, according to a new kind of normalization approach, the MSEPseAA feature vectors were normalized to form the improved MSEPseAA vectors, named as IEPseAA. Using the technique of IEPseAA, C-support vector machine (C-SVM) and three multi-class SVMs strategies, quite promising results were obtained, indicating that MSE is quite effective in reflecting the sequence-order effects and might become a useful tool for predicting the other attributes of proteins as well. PMID:17235454

  7. Subcellular integrities in Chroococcidiopsis sp. CCMEE 029 survivors after prolonged desiccation revealed by molecular probes and genome stability assays.

    Science.gov (United States)

    Billi, Daniela

    2009-01-01

    Desiccation-tolerant cells must either protect their cellular components from desiccation-induced damage and/or repair it upon rewetting. Subcellular damage to the anhydrobiotic cyanobacterium Chroococcidiopsis sp. CCMEE 029 stored in the desiccated state for 4 years was evaluated at the single-cell level using fluorescent DNA strand breakage labelling, membrane integrity and potential related molecular probes, oxidant-sensing fluorochrome and redox dye. Covalent modifications of dried genomes were assessed by testing their suitability as PCR template. Results suggest that desiccation survivors avoid/and or limit genome fragmentation and genome covalent modifications, preserve intact plasma membranes and phycobiliprotein autofluorescence, exhibit spatially-reduced ROS accumulation and dehydrogenase activity upon rewetting. Damaged cells undergo genome fragmentation, loss of plasma membrane potential and integrity, phycobiliprotein bleaching, whole-cell ROS accumulation and lack respiratory activity upon rewetting. The co-occurrence of live and dead cells within dried aggregates of Chroococcidiopsis confirms that desiccation resistance is not a simple process and that subtle modifications to the cellular milieu are required to dry without dying. It rises also intriguing questions about the triggers of dead cells in response to drying. The capability of desiccation survivors to avoid and/or reduce subcellular damage, shows that protection mechanisms are relevant in the desiccation tolerance of this cyanobacterium. PMID:18931823

  8. Fine and distributed subcellular retinotopy of excitatory inputs to the dendritic tree of a collision-detecting neuron.

    Science.gov (United States)

    Zhu, Ying; Gabbiani, Fabrizio

    2016-06-01

    Individual neurons in several sensory systems receive synaptic inputs organized according to subcellular topographic maps, yet the fine structure of this topographic organization and its relation to dendritic morphology have not been studied in detail. Subcellular topography is expected to play a role in dendritic integration, particularly when dendrites are extended and active. The lobula giant movement detector (LGMD) neuron in the locust visual system is known to receive topographic excitatory inputs on part of its dendritic tree. The LGMD responds preferentially to objects approaching on a collision course and is thought to implement several interesting dendritic computations. To study the fine retinotopic mapping of visual inputs onto the excitatory dendrites of the LGMD, we designed a custom microscope allowing visual stimulation at the native sampling resolution of the locust compound eye while simultaneously performing two-photon calcium imaging on excitatory dendrites. We show that the LGMD receives a distributed, fine retinotopic projection from the eye facets and that adjacent facets activate overlapping portions of the same dendritic branches. We also demonstrate that adjacent retinal inputs most likely make independent synapses on the excitatory dendrites of the LGMD. Finally, we show that the fine topographic mapping can be studied using dynamic visual stimuli. Our results reveal the detailed structure of the dendritic input originating from individual facets on the eye and their relation to that of adjacent facets. The mapping of visual space onto the LGMD's dendrites is expected to have implications for dendritic computation. PMID:27009157

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

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

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

  10. Changes in subcellular localization of visfatin in human colorectal HCT-116 carcinoma cell line after cytochalasin B treatment

    Directory of Open Access Journals (Sweden)

    R.J. Bułdak

    2014-09-01

    Full Text Available The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.

  11. Differential uptake and oxidative stress response in zebrafish fed a single dose of the principal copper and zinc enriched sub-cellular fractions of Gammarus pulex

    International Nuclear Information System (INIS)

    The sub-cellular compartmentalisation of trace metals and its effect on trophic transfer and toxicity in the aquatic food chain has been a subject of growing interest. In the present study, the crustacean Gammarus pulex was exposed to either 11 μg Cu l-1, added solely as the enriched stable isotope 65Cu, or 660 μg Zn l-1, radiolabeled with 2MBq 65Zn, for 16 days. Post-exposure the heat stable cytosol containing metallothionein-like proteins (MTLP) and a combined granular and exoskeletal (MRG + exo) fractions were isolated by differential centrifugation, incorporated into gelatin and fed to zebrafish as a single meal. Assimilation efficiency (AE) and intestinal lipid peroxidation, as malondialdehyde (MDA) were measured. There was a significant difference (p 65Cu, although the results pointed towards greater bioavailability of the MTLP fraction compared to MRG + exo during the slow elimination phase (24-72 h) these results were not significant (p = 0.155). Neither zinc feed provoked a lipid peroxidation response in the intestinal tissue of zebrafish compared to control fish (gelatin fed), but both 65Cu labeled feeds did. The greater effect was exerted by the MRG + exo (2.96 ± 0.29 nmol MDA mg protein-1) feed which three-fold greater than control (p -1, p 109Cd labeled G. pulex fractions were fed to zebrafish. Thus it appears that when a metal (Cu or Cd) has the potential to cause cytotoxicity via lipid peroxidation, a feed consisting of a largely unavailable fraction (MRG + exo) causes a greater intestinal stress response than the more bioavailable (MTLP) feed.

  12. Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

    OpenAIRE

    Meier Iris; Stahlberg Eric A; Schraegle Shannon J; Rose Annkatrin

    2005-01-01

    Abstract Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previousl...

  13. The subcellular distribution of 239Pu, 241Am and 59Fe in the liver of rat and Chinese hamster as dependent on time

    International Nuclear Information System (INIS)

    The subcellular distribution of monomeric 239Pu, 241Am and iron in rat and Chinese hamster liver has been investigated by sucrose-, metrizamide- and Percoll-density gradients. In rat liver, the transuranium elements become and remain bound to typical lysosomes up to several months after incorporation. Lysosomes are also the primary storage organelle in Chinese hamster liver. However, their apparent density in sucrose decreases with time, which possibly indicates transition into telolysomes. The transuranium nuclides show a subcellular distribution which is quite different from that of iron. (orig.)

  14. Epidermal Growth Factor Receptor mediated cellular and subcellular targeted delivery of Iron oxide core-Titanium dioxide shell nanoparticles

    Science.gov (United States)

    Yuan, Ye

    TiO2 nanomaterials can carry a multitude of therapeutic and diagnostic agents and the semiconductor properties of TiO2 allow for the production of cytotoxic reactive oxygen species following photoactivation. However, the delivery of these nanomaterials to specific cancer cells and specific subcellular organelles within these cells can have a substantial impact on the efficacy and safety of TiO2 nanoparticle therapeutics. Targeting cell surface receptors that are overexpressed by cancer cells is one strategy to improve the specificity of nanoparticle delivery. Therefore we decided to target the Epidermal Growth Factor Receptor (EGFR) because ligand- binding induces rapid receptor endocytosis and ligand-bound EGFR can translocate to the nucleus of cancer cells. To create NPs that can bind EGFR, we identified a peptide derived from the B-loop of Epidermal Growth Factor (EGF) that has been shown to bind and activate EGFR and conjugated it to the surface of Fe3O4 core-TiO2 shell NPs to produce B-loop NCs. We then devised a pulldown assay to show that B-loop NCs, but not bare NPs or NCs carrying a scrambled B-loop peptide, can bind and extract EGFR from HeLa cell protein extracts. Interestingly, B-loop NCs can also pulldown importin-beta, a protein that can transport EGFR to the nucleus. Furthermore, we used flow cytometry and fluorescently labeled NPs to show that B-loop peptides can significantly improve the internalization of NPs by EGFR-expressing HeLa cells. We determined that B-loop NCs can bind EGFR on the membrane of HeLa cells and that these NCs can be transported to the nucleus, by using a combination of confocal microscopy and X-ray Fluorescence Microscopy (XFM) to indirectly and directly track the subcellular distribution of NCs. Finally, we demonstrate how the Bionanoprobe, a novel high-resolution XFM apparatus that can scan whole-mounted, frozen-hydrated cells at multiple angles can be used to verify the subcellular distribution of B-loop NCs.

  15. Four Dimensional (4-D BioChemInfoPhysics Models of Cardiac Cellular and Sub-Cellular Vibrations (Oscillations

    Directory of Open Access Journals (Sweden)

    Chang-Hua Zou

    2009-01-01

    Full Text Available Problem statement: Cardiovascular Diseases (CVD continued to be the leading cause of death. Failure or abnormal cardiac cellular or sub-cellular vibrations (oscillations could lead failure or abnormal heart beats that could cause CVD. Understanding the mechanisms of the vibrations (oscillations could help to prevent or to treat the diseases. Scientists have studied the mechanisms for more than 100 years. To our knowledge, the mechanisms are still unclear today. In this investigation, based on published data or results, conservation laws of the momentum as well as the energy, in views of biology, biochemistry, informatics and physics (BioChemInfoPhysics, we proposed our models of cardiac cellular and sub-cellular vibrations (oscillations of biological components, such as free ions in Biological Fluids (BF, Biological Membranes (BM, Ca++H+ (Ca++ and Na+K+ ATPases, Na+Ca++ exchangers (NCX, Ca++ carriers and myosin heads. Approach: Our models were described with 4-D (x, y, z, t or r, ?, z, t momentum transfer equations in mathematical physics. Results: The momentum transfer equations were solved with free and forced, damped, un-damped and over-damped, vibrations (oscillations. The biological components could be modeled as resonators or vibrators (oscillators, such as liquid plasmas, membranes, active springs, passive springs and active swings. Conclusion: We systematically provided new insights of automation (ignition and maintain, transportation, propagation and orientation of the cardiac cellular and sub-cellular vibrations (oscillations and resonances, with our BioChemInfoPhysics models of 4-D momentum transfer equations. Our modeling results implied: Auto-rhythmic cells (Sinoatrial Node Cells (SANC, Atrioventricular Node Cells (AVNC, Purkinje fibers, non-Auto-rhythmic ventricular myocytes and their Sarcoplasmic Reticulums (SR work as Biological Liquid Plasma Resonators (BLPR. The resonators were

  16. Subcellular localizations of Arabidopsis myotubularins MTM1 and MTM2 suggest possible functions in vesicular trafficking between ER and cis-Golgi.

    Science.gov (United States)

    Nagpal, Akanksha; Ndamukong, Ivan; Hassan, Ammar; Avramova, Zoya; Baluška, František

    2016-08-01

    The two Arabidopsis genes AtMTM1 and AtMTM2 encode highly similar phosphoinositide 3-phosphatases from the myotubularin family. Despite the high-level conservation of structure and biochemical activities, their physiological roles have significantly diverged. The nature of a membrane and the concentrations of their membrane-anchored substrates (PtdIns3P or PtdIns3,5P2) and/or products (PtdIns5P and PtdIns) are considered critical for determining the functional specificity of myotubularins. We have performed comprehensive analyses of the subcellular localization of AtMTM1 and AtMTM2 using a variety of specific constructs transiently expressed in Nicotiana benthamiana leaf epidermal cells under the control of 35S promoter. AtMTM1 co-localized preferentially with cis-Golgi membranes, while AtMTM2 associated predominantly with ER membranes. In a stark contrast with animal/human MTMs, neither AtMTM1 nor AtMTM2 co-localizes with early or late endosomes or with TGN/EE compartments, making them unlikely participants in the endosomal trafficking system. Localization of the AtMTM2 is sensitive to cold and osmotic stress challenges. In contrast to animal myotubularins, Arabidopsis myotubularins do not associate with endosomes. Our results suggest that Arabidopsis myotubularins play a role in the vesicular trafficking between ER exit sites and cis-Golgi elements. The significance of these results is discussed also in the context of stress biology and plant autophagy. PMID:27340857

  17. Subcellular location of secretory proteins retained in the liver during the ethanol-induced inhibition of hepatic protein secretion in the rat

    International Nuclear Information System (INIS)

    Ethanol administration inhibits the secretion of proteins by the liver, resulting in their hepatocellular retention. Experiments were designed in this study to determine the subcellular location of the retained secretory proteins. Ethanol was administered acutely to nonfasted rats by gastric intubation, whereas control animals received an isocaloric dose of glucose. Two hours after intubation, when maximum blood ethanol levels (45 mM) were observed, [3H]leucine and [14C]fucose were injected simultaneously into the dorsal vein of the penis. The labelling of secretory proteins was determined in the liver and plasma at various time periods after label injection. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. At the time of maximum inhibition of secretion, leucine labeled secretory proteins located in the Golgi apparatus represented about 50% of the accumulated secretory proteins in the livers of the ethanol-treated rats, whereas the remainder was essentially equally divided among the rough and smooth endoplasmic reticulum and cytosol. Because fucose is incorporated into secretory proteins almost exclusively in the Golgi complex, fucose-labeled proteins accumulated in the livers of the ethanol-treated rats mainly in the Golgi apparatus, with the remainder located in the cytosol. These results show that ethanol administration causes an impaired movement of secretory proteins along the secretory pathway, and that secretory proteins accumulate mainly, but not exclusively, in the Golgi apparatus

  18. Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

    2015-08-21

    GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association with the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.

  19. Understanding subcellular function on the nanometer scale in real time: Single-molecule imaging in living bacteria

    Science.gov (United States)

    Biteen, Julie

    It has long been recognized that microorganisms play a central role in our lives. By beating the diffraction limit that restricts traditional light microscopy, single-molecule fluorescence imaging is a precise, noninvasive way to sensitively probe position and dynamics, even in living cells. We are pioneering this super-resolution imaging method for unraveling important biological processes in live bacteria, and I will discuss how we infer function from subcellular dynamics (Tuson and Biteen, Analytical Chemistry 2015). In particular, we have understood the mechanism of membrane-bound transcription regulation in the pathogenic Vibrio cholerae, revealed an intimate and dynamic coupling between DNA mismatch recognition and DNA replication, and measured starch utilization in an important member of the human gut microbiome.

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

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Krustrup, Peter; Nybo, Lars; Gunnarsson, Thomas P; Madsen, Klavs; Schrøder, Henrik Daa; Bangsbo, Jens; Ortenblad, Niels

    2012-01-01

    biopsy collected immediately after and 24, 48, 72 and 120 h after a competitive soccer match. Transmission electron microscopy was used to estimate the subcellular distribution of glycogen and individual particle size. During the first day of recovery, glycogen content increased by ~60% in all...

  1. DISPOSITION OF GLYCOSIDASE INHIBITORS IN THE ISOLATED-PERFUSED RAT-LIVER - HEPATOBILIARY AND SUBCELLULAR CONCENTRATION GRADIENTS OF 1-DEOXYMANNOJIRIMYCIN AND N-METHYL-1-DEOXYNOJIRIMYCIN

    NARCIS (Netherlands)

    FABER, ED; PROOST, JH; MEIJER, DKF

    1994-01-01

    The hepatic disposition of two glycosidase inhibitors was studied in the isolated perfused rat liver and after subcellular fractionation. The mannosidase inhibitor 1-deoxymannojirimycin (dMM) and the glucosidase inhibitor N-methyl-1-deoxynojirimycin (MedNM) exhibited minimal binding to albumin and r

  2. Subcellular fractionation of a hypercellulolytic mutant, Trichoderma reesei Rut-C30: localization of endoglucanase in microsomal fraction.

    Science.gov (United States)

    Glenn, M; Ghosh, A; Ghosh, B K

    1985-11-01

    The growing mycelia of Trichoderma reesei Rut-C30 are richly endowed with endoplasmic reticula and a variety of pleomorphic subcellular bodies. Mycelia of the culture growing in presence of avicel pH101 was fractionated in sucrose density gradients, and several morphologically and biochemically distinct fractions were isolated. Mycelia were homogenized in a Bead Beater, and the homogenate was freed of nucleus and wall fragments by low-speed centrifugation before fractionation. Organelle-free cytosol, which did not penetrate the gradient, contained (of the total) 72% of the vanadate-sensitive ATPase, 26% of carboxymethyl cellulase (CMCase), 2% of cytochrome c reductase, and 13% of the protein. Significant fractions separated on a gradient were light vesicles containing heavily stained material inside and ribosomes attached to the outside surface, intact vesicles resembling condensing vacuoles, large vesicles derived from the plasma membrane, and heavy vesicles containing crystalline material. The light-vesicle fraction contained a large portion of the cell-bound CMCase activity. The particle-bound ATPase and cytochrome c reductase activities were concentrated in heavy fractions. The fractionation in the presence of MgCl2 improved the preservation of subcellular bodies derived from the endoplasmic reticula. Although the CMCase activity of the light-vesicle fraction was 4 times higher than the activity in the heavy-vesicle fraction, the CMCase antibody-binding capacities of both fractions were about the same. This discrepancy between the catalytic activity and the antibody-binding capacity suggests that the heavy vesicles might have contained considerable amount of inactive CMCase compared with that present in the light vesicles. PMID:4091550

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-07-01

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

  5. Metabolism of benzo(a)pyrene by aortic subcellular fractions in the setting of abdominal aortic aneurysms.

    Science.gov (United States)

    Ramesh, A; Prins, P A; Perati, P R; Rekhadevi, P V; Sampson, U K

    2016-01-01

    As exposure to polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) have been implicated in cardiovascular diseases, the ability of the aortic tissue to process these toxicants is important from the standpoint of abdominal aortic aneurysms and atherosclerosis. Benzo(a)pyrene (B(a)P), a representative PAH compound is released into the environment from automobile exhausts, industrial emissions, and considerable intake of B(a)P is also expected in people who are smokers and barbecued red meat eaters. Therefore, knowledge of B(a)P metabolism in the cardiovascular system will be of importance in the management of vascular disorders. Toward this end, subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were isolated from the aortic tissues of Apo E mice that received a 5 mg/kg/week of B(a)P for 42 days and 0.71 mg/kg/day for 60 days. The fractions were incubated with 1 and 3 μM B(a)P. Post incubation, samples were extracted with ethyl acetate and analyzed by reverse-phase HPLC. Microsomal B(a)P metabolism was greater than the rest of the fractions. The B(a)P metabolite levels generated by all the subcellular fractions showed a B(a)P exposure concentration-dependent increase for both the weekly and daily B(a)P treatment categories. The preponderance of B(a)P metabolites such as 7,8-dihydrodiol, 3,6-, and 6,12-dione metabolites are interesting due to their reported involvement in B(a)P-induced toxicity through oxidative stress. PMID:26530167

  6. Accumulation and sub-cellular partitioning of metals and As in the clam Venerupis corrugata: Different strategies towards different elements.

    Science.gov (United States)

    Velez, Cátia; Figueira, Etelvina; Soares, Amadeu M V M; Freitas, Rosa

    2016-08-01

    The main goal of the present study was to assess accumulation, tolerance and sub-cellular partitioning of As, Hg, Cd and Pb in Venerupis corrugata. Results showed an increase of elements accumulation in V. corrugata with the increase of exposure. However, organisms presented higher capacity to accumulate Hg, Cd and Pb (BCF ≥ 12.8) than As (BCF ≤ 2.1) and higher accumulation rate for Cd and Pb than for Hg and As. With the increase of Hg exposure concentrations clams tended to increase the amount of metal bound to metal-sensitive fractions, which may explain the mortality recorded at the highest exposure concentration. Cd sub-cellular partitioning showed that with the increase of exposure concentrations V. corrugata increased the amount of metal in the cellular debris fraction, probably bound to the cellular membranes which explain the mortality recorded at the highest concentration. Results on As partitioning demonstrated that most of the metalloid was associated with fractions in the biologically detoxified metal compartment (BDM). Since high mortality was observed in clams exposed to As our results may indicate that this strategy was not enough to prevent clams from toxic effects and mortality occurred. When exposed to Pb most of the metal was in the BDM compartment, but in this case the metal was mostly in the metal-rich granules fraction which seemed to be efficient in preventing clams from toxicity, and no mortality was recorded. Our study further revealed that As and Hg were the most available elements to be biomagnified through the food chain. PMID:27174825

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

    International Nuclear Information System (INIS)

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

  8. Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    Directory of Open Access Journals (Sweden)

    Marinelli Raúl A

    2005-08-01

    Full Text Available Abstract Background Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs, a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures. This cell line has been reported to be a good in vitro model for studying hepatocyte polarity. Results Using RT-PCR, immunoblotting and confocal immunofluorescence, we showed that WIF-B cells express the aquaporin water channels that facilitate the osmotically driven water movements in the liver, i.e. AQP8, AQP9, and AQP0; as well as the key solute transporters involved in the generation of canalicular osmotic gradients, i.e., the bile salt export pump Bsep, the organic anion transporter Mrp2 and the chloride bicarbonate exchanger AE2. The subcellular localization of the AQPs and the solute transporters in WIF-B cells was similar to that in freshly isolated rat hepatocytes and in intact liver. Immunofluorescent costaining studies showed intracellular colocalization of AQP8 and AE2, suggesting the possibility that these transporters are expressed in the same population of pericanalicular vesicles. Conclusion The hepatocyte cell line WIF-B retains the expression and subcellular localization of aquaporin water channels as well as key solute transporters for canalicular bile secretion. Thus, these cells can work as a valuable tool for regulatory and mechanistic studies of the biology of bile formation.

  9. Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

    Science.gov (United States)

    Kapp, Nikki; Barnes, William J; Richard, Tom L; Anderson, Charles T

    2015-07-01

    Lignin is a complex polyphenolic heteropolymer that is abundant in the secondary cell walls of plants and functions in growth and defence. It is also a major barrier to the deconstruction of plant biomass for bioenergy production, but the spatiotemporal details of how lignin is deposited in actively lignifying tissues and the precise relationships between wall lignification in different cell types and developmental events, such as flowering, are incompletely understood. Here, the lignin-detecting fluorogenic dye, Basic Fuchsin, was adapted to enable comparative fluorescence-based imaging of lignin in the basal internodes of three Brachypodium distachyon ecotypes that display divergent flowering times. It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls. Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon. Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses. This study provides a new comparative tool for imaging lignin in plants and helps inform our views of how lignification proceeds in grasses. PMID:25922482

  10. Nano-hydroxyapatite and nano-titanium dioxide exhibit different subcellular distribution and apoptotic profile in human oral epithelium.

    Science.gov (United States)

    Tay, Chor Yong; Fang, Wanru; Setyawati, Magdiel Inggrid; Chia, Sing Ling; Tan, Kai Soo; Hong, Catherine Hsu Ling; Leong, David Tai

    2014-05-14

    Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2. PMID:24734929

  11. Subcellular localization and responses of superoxide dismutase isoforms in local wheat varieties subjected to continuous soil drought.

    Science.gov (United States)

    Huseynova, Irada M; Aliyeva, Durna R; Aliyev, Jalal A

    2014-08-01

    Water is a key factor influencing the yield and quality of crops. One of the parameters of plant biological tolerance to constantly changing environmental conditions is the change of activities and numerous molecular forms of antioxidant enzymes. Two durum (Triticum durum Desf.) wheat varieties contrasting for drought tolerance, such as Barakatli-95 (drought tolerant) and Garagylchyg-2 (drought sensitive) were grown over a wide area in the field. Experiments were carried out to study the effect of soil drought on changes in activities and subcellular localization of superoxide dismutase isoforms. The levels of malondialdehyde, glycine betaine and total proteins were also analyzed. The level of the enzyme activity appeared to depend on the wheat varieties, duration of drought and stages of leaf development. Native polyacrylamide gel electrophoresis (PAGE) revealed the presence of 9 isoenzymes of superoxide dismutase in wheat leaves during drought. Mn-SOD was found in the mitochondrial fractions, Fe-SOD in the chloroplast fraction and Cu/Zn-SOD is localized in all subcellular fractions. Wheat leaves contain three different isoforms of SOD (Mn-, Fe-, Cu/Zn-SOD). Three isoforms of Mn-SOD, one isoform of Fe-SOD and five of Cu/Zn-SOD were observed in wheat leaves using 3 mM KCN and 5 mM H2O2 as selective inhibitors. The expression of Mn-SOD was preferentially enhanced by drought stress. It seems that Mn-SOD isoforms more than SOD ones play a major role in the scavenging of superoxide radicals. The observed data showed that status of antioxidant enzymes such as SOD could provide a meaningful tool for depicting drought tolerance of wheat genotype. PMID:24560039

  12. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels; Westerblad, Hakan

    distribution by transmission electron microscopy. At fatigue, tetanic [Ca(2+)]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P < 0.01, n = 9) and LIF (P < 0.01, n = 5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60-75% lower than in rested control...

  13. Uptake,Subcellular Distribution,and Chemical Forms of Cadmium in Wild-Type and Mutant Rice

    Institute of Scientific and Technical Information of China (English)

    HE Jun-Yu; ZHU Cheng; REN Yan-Fang; YAN Yu-Ping; CHENG Chang; JIANG De-An; SUN Zong-Xiu

    2008-01-01

    Wild-type (Zhonghua 11) and mutant rice (Oryza sativa L.) plants were used to investigate the effect of cadmium (Cd) application on biomass production,to characterize the influx of Cd from roots to shoots,and to determine the form,content,and subcellular distribution of Cd in the roots,leaf sheaths,and leaves of the rice plants.Seedlings were cultivated in a nutrient solution and were treated with 0.5 mmol L-1 of Cd2+ for 14 d.The sensitivity of rice plants to Cd toxicity was tested by studying the changes in biomass production and by observing the onset of toxicity symptoms in the plants.Both the wild-type and mutant rice plants developed symptoms of Cd stress.In addition,Cd application significantly (P ≤ 0.01) decreased dry matter production of roots,leaf sheaths,and leaves of both types,especially the mutant.The Cd content in roots of the mutant was significantly (P≤0.05) higher than that of the wild-type rice.However,there was no significant difference in the Cd content of roots,leaf sheaths,and leaves between the wild-type and mutant rice.Most of the Cd was bound to the cell wall of the roots,leaf sheaths,and leaves,and the mutant had greater Cd content in cell organelles than the wild type.The uneven subcellular distribution could be responsible for the Cd sensitivity of the mutant rice.Furthermore,different chemical forms of Cd were found to occur in the roots,leaf sheaths,and leaves of both types of rice plants.Ethanol-,water-,and NaCl-extractable Cd had greater toxicity than the other forms of Cd and induced stunted growth and chlorosls in the plants.The high Cd content of the toxic forms of Cd in the cell organelles could seriously damage the cells and the metabolic processes in mutant rice plants.

  14. In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1

    International Nuclear Information System (INIS)

    In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1, 2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran(12)-1-yl-methoxyl]-1,3 -propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 μg B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 1012 n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when comparing

  15. In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1.

    Science.gov (United States)

    Tilly, N; Olsson, P; Hartman, T; Coderre, J; Makar, M; Malmquist, J; Sjöberg, S; Pettersson, J; Carlsson, J; Glimelius, B

    1996-01-01

    In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when

  16. Characterization, sub-cellular localization and expression profiling of the isoprenylcysteine methylesterase gene family in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Ma Wujun

    2010-09-01

    Full Text Available Abstract Background Isoprenylcysteine methylesterases (ICME demethylate prenylated protein in eukaryotic cell. Until now, knowledge about their molecular information, localization and expression pattern is largely unavailable in plant species. One ICME in Arabidopsis, encoded by At5g15860, has been identified recently. Over-expression of At5g15860 caused an ABA hypersensitive phenotype in transgenic Arabidopsis plants, indicating that it functions as a positive regulator of ABA signaling. Moreover, ABA induced the expression of this gene in Arabidopsis seedlings. The current study extends these findings by examining the sub-cellular localization, expression profiling, and physiological functions of ICME and two other ICME-like proteins, ICME-LIKE1 and ICME-LIKE2, which were encoded by two related genes At1g26120 and At3g02410, respectively. Results Bioinformatics investigations showed that the ICME and other two ICME-like homologs comprise a small subfamily of carboxylesterase (EC 3.1.1.1 in Arabidopsis. Sub-cellular localization of GFP tagged ICME and its homologs showed that the ICME and ICME-like proteins are intramembrane proteins predominantly localizing in the endoplasmic reticulum (ER and Golgi apparatus. Semi-quantitative and real-time quantitative PCR revealed that the ICME and ICME-like genes are expressed in all examined tissues, including roots, rosette leaves, cauline leaves, stems, flowers, and siliques, with differential expression levels. Within the gene family, the base transcript abundance of ICME-LIKE2 gene is very low with higher expression in reproductive organs (flowers and siliques. Time-course analysis uncovered that both ICME and ICME-like genes are up-regulated by mannitol, NaCl and ABA treatment, with ICME showing the highest level of up-regulation by these treatments. Heat stress resulted in up-regulation of the ICME gene significantly but down-regulation of the ICME-LIKE1 and ICME-LIKE2 genes. Cold and dehydration

  17. Quantitative imaging of subcellular calcium stores in mammalian LLC-PK1 epithelial cells undergoing mitosis by SIMS ion microscopy.

    Science.gov (United States)

    Chandra, Subhash

    2005-09-01

    Quantitative 3-D total calcium gradients, representing subcellular stored calcium, were imaged with a CAMECA IMS-3f SIMS ion microscope in cryogenically prepared frozen freeze-dried LLC-PK1 cells captured in interphase and various stages of mitosis. 39K and 23Na concentrations were also measured in the same cells. Correlative optical (or SEM) and SIMS analysis of cells revealed a redistribution of the interphase Golgi calcium store in prophase and prometaphase cells. In metaphase cells, simultaneous SIMS imaging of total calcium in both the spindle and the non-spindle cytoplasm of individual cells revealed a gradual and dynamic alignment of calcium stores in both half-spindles prior to the onset of anaphase. The anaphase cells revealed the highest local total calcium concentrations in the spindle regions behind the daughter chromosomes and the lowest in the central spindle region. The pericentriolar material in telophase cells contained calcium stores. Quantitatively, a typical metaphase cell with well-aligned calcium stores in the spindle region contained 1.1 mM total calcium in each half-spindle, 0.8 mM total calcium in the non-spindle cytoplasm, and 0.5mM total calcium in the chromosomes. At the submicron scale, the distribution of total calcium was heterogeneous in the chromosomes, metaphase spindle, and non-spindle cytoplasm. An increased binding of calcium to chromosomes is not a physiological requirement for chromosomal condensation in mitosis, since interphase nuclei and mitotic chromosomes contained comparable total calcium concentrations measured per unit volume. A significant reduction of total calcium in the non-spindle cytoplasm was observed in the metaphase, anaphase, and telophase cells, which is indicative of the limited storage of the releasable calcium pool in these specific stages of mitosis. Direct total calcium measurements in subcellular regions confirmed that both the spindle and the non-spindle cytoplasm of metaphase cells contained inositol

  18. Absorbed dose at subcellular level by Monte Carlo simulation for a 99mTc-peptide with nuclear internalization

    International Nuclear Information System (INIS)

    The utility of radiolabeled peptides for the early and specific diagnosis of cancer is being investigated around the world. Recent investigations have demonstrated the specificity of 99mTc-bombesin conjugates to target breast and prostate cancer cells. The novel idea of adding the Tat (49-57) peptide to the radiopharmaceutical in order to penetrate the cell nucleus is a new proposal for therapy at cellular level. 99mTc radionuclide produces Auger energy of 0.9 keV/decay and internal conversion electron energy of 15.4 keV/decay, which represent 11.4% of the total 99mTc energy released per decay. It is expected that the dose delivered at specific microscopic levels in cancer cells induce a therapeutic effect. The aim of this research was to assess in vitro internalization kinetics in breast and prostate cancer cells of 99mTc-Tat(49-57)-bombesin and to evaluate the radiation absorbed dose at subcellular level simulating the electron transport. The pen main program from the 2006 version of the Penelope code was used to simulate and calculate the absorbed dose by Auger and internal conversion electron contribution in the membrane, cytoplasm and nucleus of Pc-3 prostate cancer and MCF7 and MDA human breast cancer cell lines. Nuclear data were obtained from the 2002 BNM-LNHB 99mTc decay scheme. The spatial distribution of the absorbed doses to the membrane, cytoplasm and nucleus were calculated using a geometric model built from real images of cancer cells. The elemental cell composition was taken from the literature. The biokinetic data were obtained evaluating total disintegrations in each subcellular compartment by integration of the time-activity curves acquired from experimental data. Results showed that 61, 63 and 46% of total disintegrations per cell-bound 99mTc-Tat-Bn activity unit occurred in the nucleus of Pc-3, MCF7 and MDA-MB231 respectively. 99mTc--Tat-Bn absorbed doses were 1.78, 5.76 and 2.59 Gy/Bq in the nucleus of Pc-3, MCF7 and MDA-MB231 correspondingly

  19. Discrete Element Framework for Modelling Extracellular Matrix, Deformable Cells and Subcellular Components

    OpenAIRE

    Gardiner, Bruce S.; Wong, Kelvin K. L.; Joldes, Grand R.; Rich, Addison J.; Tan, Chin Wee; Burgess, Antony W.; Smith, David W.

    2015-01-01

    This paper presents a framework for modelling biological tissues based on discrete particles. Cell components (e.g. cell membranes, cell cytoskeleton, cell nucleus) and extracellular matrix (e.g. collagen) are represented using collections of particles. Simple particle to particle interaction laws are used to simulate and control complex physical interaction types (e.g. cell-cell adhesion via cadherins, integrin basement membrane attachment, cytoskeletal mechanical properties). Particles may ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Plenin, A.E.

    1973-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Previous studies have shown that skeletal muscle glycogen and mitochondria are distributed in distinct subcellular localizations, but the role and regulation of these subcellular localizations are unclear. In the present study, we used transmission electron microscopy to investigate the effect of...... disuse and aging on human skeletal muscle glycogen and mitochondria content in subsarcolemmal (SS), intermyofibrillar (IMF), and intramyofibrillar (intra) localizations. Five young (∼23 yr) and five old (∼66 yr) recreationally active men had their quadriceps muscle immobilized for 2 wk by whole leg...... casting. Biopsies were obtained from m. vastus lateralis before and after the immobilization period. Immobilization induced a decrement of intra glycogen content by 54% (P <0.001) in both age groups and in two ultrastructurally distinct fiber types, whereas the content of IMF and SS glycogen remained...

  2. The effects of γ-ray irradiation on the cellular and subcellular structures of apical meristem in garlic (Allium sativum) and onion (Allium cepal)

    International Nuclear Information System (INIS)

    Electronic microscopic study revealed that 2 ∼ 30 krads of γ-ray irradiation to garlic and onion could cause various damages to cellular and subcellular structures of the shoot apical meristem. Among the various oganelles, the vacuoles showed the highest radio-sensitivity while mitochondria and nucleus seemed to be most resistant to irradiation. The irradiated cells did not show any visible structural damages until the dormancy ended, suggesting that metabolism played an important role in the structural damages. The study also suggested that even after the irradiation which caused intensive subcellular structural damages, the tissues could survive. However, the potency of mitosis in the apex was lost, resulting in the inhibition of sprouting

  3. Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy

    International Nuclear Information System (INIS)

    The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement

  4. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    International Nuclear Information System (INIS)

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of Mr = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind [3H]flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing [3H]flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated

  5. Exogenous Nitric Oxide Involved in Subcellular Distribution and Chemical Forms of Cu2+Under Copper Stress in Tomato Seedlings

    Institute of Scientific and Technical Information of China (English)

    DONG Yu-xiu; WANG Xiu-feng; CUI Xiu-min

    2013-01-01

    Nitric oxide (NO), a bioactive signaling molecule, serves as an antioxidant and anti-stress agent under abiotic stress. A hydroponics experiment was conducted to investigate the effects of sodium nitroprusside (SNP), a NO donor, on tomato seedlings exposed to 50 µmol L-1 CuCl2. The results show that copper is primarily stored in the soluble cell sap fraction in the roots, especially after treatment with Cu+SNP treatment, which accounted for 66.2%of the total copper content. The copper concentration gradually decreased from the roots to the leaves. In the leaves, exogenous NO induces the storage of excess copper in the cell walls. Copper stress decreases the proportion of copper integrated with pectates and proteins, but exogenous NO remarkably reverses this trend. The alleviating effect of NO is blocked by hemoglobin. Thus, exogenous NO is likely involved in the regulation of the subcellular copper concentrations and its chemical forms under copper stress. Although exogenous NO inhibited the absorption and transport of excess copper to some extent, the copper accumulation in tomato seedlings signiifcantly increased under copper stress. The use of exogenous NO to enhance copper tolerance in some plants is a promising method for copper remediation.

  6. Subcellular distribution and uptake mechanism of di-n-butyl phthalate in roots of pumpkin (Cucurbita moschata) seedlings.

    Science.gov (United States)

    Lin, Qingqi; Yang, Xiuhong; Huang, Xiongfei; Wang, Shizhong; Chao, Yuanqing; Qiu, Rongliang

    2016-01-01

    Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins. PMID:26304812

  7. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    Science.gov (United States)

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  8. Doxorubicin delivered to MCF-7 cancer cells by superparamagnetic iron oxide nanoparticles: effects on subcellular distribution and cytotoxicity

    International Nuclear Information System (INIS)

    The clinical use of the anticancer drug doxorubicin (DOX) is limited by strong side effects and phenomena of cell resistance. Drug targeting by binding DOX to nanoparticles could overcome these limitations. We recently described a method to associate DOX to superparamagnetic iron oxide nanoparticles (SPION) in view of magnetic drug targeting (Munnier et al. in Int J Pharm 363:170–176, 2008). DOX is bound to the nanoparticle surface through a pre-formed DOX–Fe2+ complex. The DOX-loaded SPION present interesting properties in terms of drug loading and biological activity in vitro. The purpose of this study is to explore the possible mechanisms of the in vitro cytotoxicity of DOX-loaded SPION. The uptake of SPION was followed qualitatively by conventional optical microscopy after Prussian blue staining and quantitatively by iron determination by atomic absorption spectroscopy. The subcellular distribution of intrinsically fluorescent DOX was followed by confocal spectral imaging (CSI) and the subsequent cytotoxicity by the MTT method. We reveal modifications of DOX intracellular interactions for SPION-delivered drug and increased cytotoxicity. These results are discussed in terms of internalization route of the drug and of a potential role of iron oxide nanoparticles in the observed cytotoxicity.

  9. The diversification of the LIM superclass at the base of the metazoa increased subcellular complexity and promoted multicellular specialization.

    Directory of Open Access Journals (Sweden)

    Bernard J Koch

    Full Text Available BACKGROUND: Throughout evolution, the LIM domain has been deployed in many different domain configurations, which has led to the formation of a large and distinct group of proteins. LIM proteins are involved in relaying stimuli received at the cell surface to the nucleus in order to regulate cell structure, motility, and division. Despite their fundamental roles in cellular processes and human disease, little is known about the evolution of the LIM superclass. RESULTS: We have identified and characterized all known LIM domain-containing proteins in six metazoans and three non-metazoans. In addition, we performed a phylogenetic analysis on all LIM domains and, in the process, have identified a number of novel non-LIM domains and motifs in each of these proteins. Based on these results, we have formalized a classification system for LIM proteins, provided reasonable timing for class and family origin events; and identified lineage-specific loss events. Our analysis is the first detailed description of the full set of LIM proteins from the non-bilaterian species examined in this study. CONCLUSION: Six of the 14 LIM classes originated in the stem lineage of the Metazoa. The expansion of the LIM superclass at the base of the Metazoa undoubtedly contributed to the increase in subcellular complexity required for the transition from a unicellular to multicellular lifestyle and, as such, was a critically important event in the history of animal multicellularity.

  10. Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation.

    Science.gov (United States)

    Shelby, J Patrick; Edgar, J Scott; Chiu, Daniel T

    2005-01-01

    This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure. PMID:15850426

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-03-01

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

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

    International Nuclear Information System (INIS)

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

  13. Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

    International Nuclear Information System (INIS)

    Cadmium distribution was studied in different subcellular fractions of gill and hepatopancreas tissues of eastern oysters Crassostrea virginica. Oysters were exposed for up to 21 days to low sublethal Cd concentrations (25 μg L-1). Gill and hepatopancreas tissues were sampled and divided into organelle fractions and cytosol by differential centrifugation. Organelle content of different fractions was verified by activities of marker enzymes, citrate synthase and acid phosphatase for mitochondria and lysosomes, respectively. In both tissue types, there was a significant accumulation of cadmium in cytosol reaching 230-350 ng mg-1 protein. Among organelles, mitochondria were the main target for Cd bioaccumulation in gills (250-300 ng mg-1 protein), whereas in hepatopancreas tissues, the highest cadmium accumulation occurred in lysosomes (90-94 ng mg-1 protein). Although 75-83% of total cadmium burden was associated with the cytosol reflecting high volume fraction of this compartment, Cd concentrations in organelle fractions reached levels that could cause dysfunction of mitochondria and lysosomes. Organ- and organelle-specific patterns of cadmium bioaccumulation support our previous in vivo studies, which showed adverse effects of cadmium exposures on mitochondrial oxidation in gills and on the lysosomal system of hepatopancreas. This may have important implications for the development of biomarkers of effect for heavy metals and for understanding the mechanisms of toxic effects of metals

  14. The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

    DEFF Research Database (Denmark)

    Tchórzewski, Marek; Krokowski, Dawid; Rzeski, Wojciech;

    2003-01-01

    The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribos......The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached...... to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have...... used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly...

  15. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii

    Energy Technology Data Exchange (ETDEWEB)

    Qiu Rongliang, E-mail: eesqrl@mail.sysu.edu.cn [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Guangdong Provincial Key Lab of Environmental Pollution Control and Remediation Technology, Guangzhou 510275 (China); Thangavel, Palaniswamy; Hu Pengjie; Senthilkumar, Palaninaicker; Ying Rongrong [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Tang Yetao [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Guangdong Provincial Key Lab of Environmental Pollution Control and Remediation Technology, Guangzhou 510275 (China)

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii.

  16. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii.

    Science.gov (United States)

    Qiu, Rong-Liang; Thangavel, Palaniswamy; Hu, Peng-Jie; Senthilkumar, Palaninaicker; Ying, Rong-Rong; Tang, Ye-Tao

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii. PMID:21211902

  17. Immunocytochemical analysis of the subcellular distribution of ferritin in Imperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant.

    Science.gov (United States)

    de la Fuente, Vicenta; Rodríguez, Nuria; Amils, Ricardo

    2012-05-01

    Ferritin is of interest at the structural and functional level not only as storage for iron, a critical element, but also as a means to prevent cell damage produced by oxidative stress. The main objective of this work was to confirm by immunocytochemistry the presence and the subcellular distribution of the ferritin detected by Mösbauer spectroscopy in Imperata cylindrica, a plant which accumulates large amounts of iron. The localization of ferritin was performed in epidermal, parenchymal and vascular tissues of shoots and leaves of I. cylindrica. The highest density of immunolabeling in shoots appeared in the intracellular space of cell tissues, near the cell walls and in the cytoplasm. In leaves, ferritin was detected in the proximity of the dense network of the middle lamella of cell walls, following a similar path to that observed in shoots. Immunolabeling was also localized in chloroplasts. The abundance of immunogold labelling in mitochondria for I. cylindrica was rather low, probably because the study dealt with tissues from old plants. These results further expand the localization of ferritin in cell components other than chloroplasts and mitochondria in plants. PMID:21764425

  18. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

    Science.gov (United States)

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  19. Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules.

    Directory of Open Access Journals (Sweden)

    Gregory R Johnson

    2015-12-01

    Full Text Available Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins and clinical research (e.g. identification of cancer biomarkers. Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply "vesicular". We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors.

  20. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP with nucleolin.

    Directory of Open Access Journals (Sweden)

    Mohamed S Taha

    Full Text Available Fragile X mental Retardation Protein (FMRP is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa in the cytosol, and a low molecular weight complex (∼200 kDa in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs.

  1. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Czajkowski, C.M.

    1987-01-01

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of M{sub r} = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind ({sup 3}H)flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing ({sup 3}H)flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated.

  2. Activation and subcellular distribution of ERK1/2 following cerebral ischemia/reperfusion in rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    WANG Rui-min; ZHANG Guang-yi; ZHANG Quan-guang; YANG Fang; MA Wen-dong; LI Qi-jia

    2006-01-01

    Objective:To investigate the activation (phosphorylation) and subcellular localization of extracellular signal-regulated kinase(ERK1/2), as well as the possible mechanism, following cerebral ischemia and ischemia/reperfusion in rat hippocampus. Methods: Transient brain ischemia was induced by the four-vessel occlusion method in Sprague-Dawley rats. Western blot analysis. Results: During cerebral ischemia without reperfusion ERK1/2 activation immediately increased with a peak at 5 min and then decreased in the cytosol fraction, which was paralleled by the increase of ERK1/2 activation in the nucleus fraction. During reperfusion, ERK1/2 was activated with peaks occurring at 10 min in the cytosol and at 30 min in the nucleus, respectively. Under those conditions, the protein expressions had no significant change. In order to clarify the possible mechanism of ERK1/2 activation, the rats were intraperitoneally administrated with N-methyl D aspartate (NMDA) receptor antagonist dextromethorphan(DM), L-type voltage-gated Ca2+ channel (L-VGCC) antagonist nifedipine (ND) 20 min before ischemia, finding that DM and ND markedly prevented ERK1/2 activation of nucleus fraction induced by reperfusion, not by ischemia. Conclusion: These results suggested that the nuclear translocation mainly occurred during is chemia, while ischemia-reperfusion induced ERK1/2 activation both in the cytosol and the nucleus. Two type calcium channels contributed, at least partially, to the activation of ERK1/2.

  3. Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages.

    Science.gov (United States)

    Huang, Ming-Der; Huang, Anthony H C

    2016-07-01

    Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species. PMID:27208281

  4. Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages1[OPEN

    Science.gov (United States)

    2016-01-01

    Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species. PMID:27208281

  5. Dietary toxicity of field-contaminated invertebrates to marine fish: effects of metal doses and subcellular metal distribution.

    Science.gov (United States)

    Dang, Fei; Rainbow, Philip S; Wang, Wen-Xiong

    2012-09-15

    There is growing awareness of the toxicological effects of metal-contaminated invertebrate diets on the health of fish populations in metal-contaminated habitats, yet the mechanisms underlying metal bioaccumulation and toxicity are complex. In the present study, marine fish Terapon jurbua terepon were fed a commercial diet supplemented with specimens of the polychaete Nereis diversicolor or the clam Scrobicularia plana, collected from four metal-impacted estuaries (Tavy, Restronguet Creek, West Looe, Gannel) in southwest England, as environmentally realistic metal sources. A comparative toxicological evaluation of both invertebrates showed that fish fed S. plana for 21 d exhibited evident mortality compared to those fed N. diversicolor. Furthermore, a spatial effect on mortality was observed. Differences in metal doses rather than subcellular metal distributions between N. diversicolor and S. plana appeared to be the cause of such different mortalities. Partial least squares regression was used to evaluate the statistical relationship between multiple-metal doses and fish mortality, revealing that Pb, Fe, Cd and Zn in field-collected invertebrates co-varied most strongly with the observed mortality. This study provides a step toward exploring the underlying mechanism of dietary toxicity and identifying the potential causality in complex metal mixture exposures in the field. PMID:22579710

  6. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  7. Subcellular partitioning of cadmium in the freshwater bivalve, Pyganodon grandis, after separate short-term exposures to waterborne or diet-borne metal

    International Nuclear Information System (INIS)

    The dynamics of cadmium uptake and subcellular partitioning were studied in laboratory experiments conducted on Pyganodon grandis, a freshwater unionid bivalve that shows promise as a biomonitor for metal pollution. Bivalves were collected from an uncontaminated lake, allowed to acclimate to laboratory conditions (≥25 days), and then either exposed to a low, environmentally relevant, concentration of dissolved Cd (5 nM; 6, 12 and 24 h), or fed Cd-contaminated algae (∼70 nmol Cd g-1 dry weight; 4 x 4 h). In this latter case, the bivalves were allowed to depurate for up to 8 days after the end of the feeding phase. As anticipated, the gills were the main target organ during the aqueous Cd exposure whereas the intestine was the initial site of Cd accumulation during the dietary exposure; during the subsequent depuration period, the dietary Cd accumulated in both the digestive gland and in the gills. For the gills, the distribution of Cd among the subcellular fractions (i.e., granules > heat-denatured proteins (HDP) ∼ heat-stable proteins (HSP) > mitochondria ∼ lysosomes + microsomes) was insensitive to the exposure route; both waterborne and diet-borne Cd ended up largely bound to the granule fraction. The subcellular distribution of Cd in the digestive gland differed markedly from that in the gills (HDP > HSP ∼ granules ∼ mitochondria > lysosomes + microsomes), but as in the case of the gills, this distribution was relatively insensitive to the exposure route. For both the gills and the digestive gland, the subcellular distributions of Cd differed from those observed in native bivalves that are chronically exposed to Cd in the field - in the short-term experimental exposures of P. grandis, metal detoxification was less effective than in chronically exposed native bivalves.

  8. Checkpoints Studies Using the Budding Yeast Saccharomyces cerevisiae: Analysis of changes in protein level and subcellular localization during cell cycle progression

    OpenAIRE

    Wu, Xiaorong; Liu, Lili; HUANG, Mingxia

    2011-01-01

    Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast S. cerevisiae. Cell synchronization is achieved by an α-factor mediated block-and-release protocol. Cells are collected at different time points for the first two cell cycles upon release. Cellular DNA contents are analyzed by flow cytometry. Trichloroacetic acid protein precipitates are prepared for monitoring levels of cell cycle regulated protei...

  9. Apical Membrane Localization of the Adenomatous Polyposis Coli Tumor Suppressor Protein and Subcellular Distribution of the β-Catenin Destruction Complex in Polarized Epithelial Cells

    OpenAIRE

    Reinacher-Schick, Anke; Gumbiner, Barry M.

    2001-01-01

    The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of β-catenin as part of a high molecular weight complex known as the β-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significant...

  10. Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS.

    Science.gov (United States)

    Penen, Florent; Malherbe, Julien; Isaure, Marie-Pierre; Dobritzsch, Dirk; Bertalan, Ivo; Gontier, Etienne; Le Coustumer, Philippe; Schaumlöffel, Dirk

    2016-09-01

    Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1nm was used for imaging the ultrastructure of algae cells which have diameters of 5-10μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast. PMID:27288221

  11. Experimental study of Americium-241 biokinetics in Homarus Gammarus lobster. Analysis of the accumulation and detoxication mechanisms at the sub-cellular level

    International Nuclear Information System (INIS)

    The Americium 241 radioelement accumulation and elimination rate and mechanisms in the lobster organism have been experimentally studied; incorporation and detoxification capacities of each organ are evaluated. The existence of various biological compartments is shown; the major role of the digestive gland in accumulation of the radioelement, its distribution towards the various organs, and its resorption is comprehensively described, with an analysis at the subcellular and molecular levels. 401 p., 65 fig., 43 tab., 428 ref

  12. Predicting the subcellular localization of mycobacterial proteins by incorporating the optimal tripeptides into the general form of pseudo amino acid composition.

    Science.gov (United States)

    Zhu, Pan-Pan; Li, Wen-Chao; Zhong, Zhe-Jin; Deng, En-Ze; Ding, Hui; Chen, Wei; Lin, Hao

    2015-02-01

    Mycobacterium tuberculosis is a bacterium that causes tuberculosis, one of the most prevalent infectious diseases. Predicting the subcellular localization of mycobacterial proteins in this bacterium may provide vital clues for the prediction of protein function as well as for drug discovery and design. Therefore, a computational method that can predict the subcellular localization of mycobacterial proteins with high precision is highly desirable. We propose a computational method to predict the subcellular localization of mycobacterial proteins. An objective and strict benchmark dataset was constructed after collecting 272 non-redundant proteins from the universal protein resource (the UniProt database). Subsequently, a novel feature selection strategy based on binomial distribution was used to optimize the feature vector. Finally, a subset containing 219 chosen tripeptide features was imported into a support vector machine-based method to estimate the performance of the dataset in accurately and sensitively identifying these proteins. We found that the proposed method gave a maximum overall accuracy of 89.71% with an average accuracy of 81.12% in the jackknife cross-validation. The results indicate that our prediction method gave an efficient and powerful performance when compared with other published methods. We made the proposed method available on a purpose built Web server called MycoSub that is freely accessible at . We anticipate that MycoSub will become a useful tool for studying the functions of mycobacterial proteins and for designing and developing anti-mycobacterium drugs. PMID:25437899

  13. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    International Nuclear Information System (INIS)

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs

  14. SEPPA 2.0—more refined server to predict spatial epitope considering species of immune host and subcellular localization of protein antigen

    Science.gov (United States)

    Qi, Tao; Qiu, Tianyi; Zhang, Qingchen; Tang, Kailin; Fan, Yangyang; Qiu, Jingxuan; Wu, Dingfeng; Zhang, Wei; Chen, Yanan; Gao, Jun; Zhu, Ruixin; Cao, Zhiwei

    2014-01-01

    Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported. PMID:24838566

  15. Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

    Science.gov (United States)

    Ramos, Inés; Cisint, Susana B; Crespo, Claudia A; Medina, Marcela F; Fernández, Silvia N

    2009-08-01

    The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process. PMID:19397840

  16. NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

    Science.gov (United States)

    Zimmermann, Kirstin; Opitz, Nils; Dedio, Jürgen; Renné, Christoph; Müller-Esterl, Werner; Oess, Stefanie

    2002-01-01

    Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS–NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. PMID:12446846

  17. Differential regulation of gene products in newly synthesized Brassica napus allotetraploids is not related to protein function nor subcellular localization

    Directory of Open Access Journals (Sweden)

    Valot Benoît

    2007-02-01

    Full Text Available Abstract Background Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation in synthetic B. napus. Results The complete set of non-additive proteins (335 in the stem and 205 in the root, as well as a subset of additive polypeptides (200 per organ, was identified by mass spectrometry. Several protein isoforms were found, and most of them (~55% displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which ~50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under

  18. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae

    Science.gov (United States)

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

  19. Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

    Institute of Scientific and Technical Information of China (English)

    BAI Xueyuan; CHEN Xiangmei; FEN Zhe; WU Di; HOU Kai; CHENG Genyang; PENG Lixia

    2004-01-01

    Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been integrated into the genome of HKC. Western blot indicates that the fusion proteins have been expressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics approach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for expression and the trans-membrane currents are measured by using two-microelectrode voltage-clamp technique. Na+ inward currents are present on cellular membrane of the injected oocytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lumen membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane localization signal of the SDCT1 protein maybe locate in the C-terminal sequence.

  20. Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

    Science.gov (United States)

    Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

    2016-08-01

    We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. PMID:26708578

  1. Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection.

    Science.gov (United States)

    He, Mingyang; Xu, Yan; Cao, Jiangling; Zhu, Ziguo; Jiao, Yuntong; Wang, Yuejin; Guan, Xin; Yang, Yazhou; Xu, Weirong; Fu, Zhenfang

    2013-02-01

    Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant-pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection. PMID:22327469

  2. The Subcellular Localization and Blue-Light-Induced Movement of Phototropin 1-GFP in Etiolated Seedlings of Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Ying-Lang Wan; William Eisinger; David Ehrhardt; Ulrich Kubitscheck; Frantisek Baluska; Winslow Briggs

    2008-01-01

    Phototropin 1 (phot1) is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOT1::GFP (Sakamoto and Briggs,2002), we investigated the pattern of cellular and subcellular localization of phot1 in 3-4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOT1::GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOT1::GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescence-a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOT1::GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced phot1 relocalization modulates blue-light-activated signal transduction.

  3. Alzheimer's Disease as Subcellular `Cancer' --- The Scale-Invariant Principles Underlying the Mechanisms of Aging ---

    Science.gov (United States)

    Murase, M.

    1996-01-01

    with self-organization, has been thought to underlie `creative' aspects of biological phenomena such as the origin of life, adaptive evolution of viruses, immune recognition and brain function. It therefore must be surprising to find that the same principles will also underlie `non-creative' aspects, for example, the development of cancer and the aging of complex organisms. Although self-organization has extensively been studied in nonliving things such as chemical reactions and laser physics, it is undoubtedly true that the similar sources of the order are available to living things at different levels and scales. Several paradigm shifts are, however, required to realize how the general principles of natural selection can be extensible to non-DNA molecules which do not possess the intrinsic nature of self-reproduction. One of them is, from the traditional, genetic inheritance view that DNA (or RNA) molecules are the ultimate unit of heritable variations and natural selection at any organization level, to the epigenetic (nongenetic) inheritance view that any non-DNA molecule can be the target of heritable variations and molecular selection to accumulate in certain biochemical environment. Because they are all enriched with a β-sheet content, ready to mostly interact with one another, different denatured proteins like β-amyloid, PHF and prions can individually undergo self-templating or self-aggregating processes out of gene control. Other paradigm shifts requisite for a break-through in the etiology of neurodegenerative disorders will be discussed. As it is based on the scale-invariant principles, the present theory also predicts plausible mechanisms underlying quite different classes of disorders such as amyotrophic lateral sclerosis (ALS), atherosclerosis, senile cataract and many other symptoms of aging. The present theory, thus, provides the consistent and comprehensive account to the origin of aging by means of natural selection and self-organization.

  4. Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

    Directory of Open Access Journals (Sweden)

    Ildiko M L Somorjai

    Full Text Available BACKGROUND: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. METHODOLOGY/PRINCIPAL FINDINGS: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10 displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155 caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155 produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10. These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and

  5. Subcellular localisation of Epac

    NARCIS (Netherlands)

    Zhao, Jun

    2006-01-01

    Cyclic adenosine 3', 5'- monophosphate (cAMP) is a second messenger that functions through binding to its downstream targets protein kinase A (PKA), cyclic-regulated ion channels (CNG channels) and Epac1 (exchange protein directly activated by cAMP). Epac1 is guanine nucleotide exchange factor towar

  6. Testing for Subcellular Randomness

    CERN Document Server

    Okunoye, Babatunde O

    2008-01-01

    Statistical tests were conducted on 1,000 numbers generated from the genome of Bacteriophage T4, obtained from GenBank with accession number AF158101.The numbers passed the non-parametric, distribution-free tests.Deoxyribonucleic acid was discovered to be a random number generator, existent in nature.

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

    highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug’s properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization

  10. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

    Directory of Open Access Journals (Sweden)

    Ying-Jung J Lai

    Full Text Available The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB, is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.

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

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    development of subcellularly targeted DDSs that will deliver specific drugs to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs. PMID:26731220

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-12-07

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

  13. Subcellular distribution of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells within subventricular zone of adult rats

    Institute of Scientific and Technical Information of China (English)

    Zhining Li; Wenlong Lü; Hongyan Dong; Hongbin Fan; Ruiguo Dong; Tiejun Xu

    2011-01-01

    The subcellular localization of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells of the subventricular zone of adult rats was detected using electron microscopy, following immunohistochemistry and immunogold-silver double staining. Results confirmed the presence of neural stem cells in the subventricular zone, which is a key neurogenic region in the central nervous system of adult mammals. The expression of N-methyl-D-aspartic acid receptor subunit 1 was higher than that of nestin and mainly distributed in the cell membrane, cytoplasm, rough endoplasmic reticulum and Golgi complex of neural stem cells.

  14. Subcellular localization of the Cherax quadricarinatus photoreceptor Gqα subunit%红螯螯虾感光器Gqα的亚细胞定位

    Institute of Scientific and Technical Information of China (English)

    许燕; 赵云龙; 张慧琦

    2006-01-01

    The subcellular localization of the alpha subunit of the visual Gqα protein of Cherax quadricarinatus photoreceptors was investigated by immunoelectron microscopy. The Gqα was specifically localized in the rhabdom as a membrane - bound form and in the cytoplasm as a soluble form. The localization of Gqα changed under different light conditions. In the dark, more immunogold particles were detected in the rhabdom than in the cytoplasm. The density ratio in the rhabdom vs. the cytoplasm of immunogold particles within a photoreceptor cell was 1.44 ± 0.074. In sunlight, this density ratio decreased to 0.61 ±0.021. The localization of Gqα was found to be different under various light conditions. The density ratio was the highest when exposed to green light ( 1.64 ± 0.046 ); this ratio decreased upon exposure to the following light condition: red light (1.28 ±0.036), blue light (1.16 ±0.111) and yellow light (0.96 ± 0.099). The light - modulated translocation possibly controls the amount of Gqα that can be activated by rhodopsin. The study suggested that different wavelengths of light can influence the ratio of membranebound and soluble forms of Gqα; which could be a result of the amount of photoactivated rhodopsin.%利用免疫细胞化学的方法和电镜技术观察红螯螯虾感光器中Gq蛋白α亚基的亚细胞定位,结果表明:Gqα以膜结合形式定位于感杆束和以可溶性形式定位于细胞质中,Gqα定位的变化依赖于不同的光照条件.在暗适应后,Gqα主要分布于感杆束,细胞质中很少,感杆束与细胞质中胶体金颗粒的密度比为1.44±0.074;在(日)光适应后,密度比减少为0.61±0.021;在不同波长的光照处理后,Gqα定位有差异,感杆束与细胞质中胶体金颗粒的密度比由高至低,依次为:绿光(1.64±0.046)、红光(1.28±0.036)、蓝光(1.16±0.111)和黄光(0.96±0.099).光调节的Gqα转位可能控制了能被视紫红质激活的Gq蛋白的数量.研究表明:光波

  15. Carboxylated multi-walled carbon nanotubes aggravated biochemical and subcellular damages in leaves of broad bean (Vicia faba L.) seedlings under combined stress of lead and cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chengrun, E-mail: chengrunwang@163.com [School of Life Science, Huainan Normal University, Huainan 232001 (China); Liu, Haitao; Chen, Jinyun [School of Life Science, Huainan Normal University, Huainan 232001 (China); Tian, Yuan [Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824 (United States); Shi, Jian; Li, Dongdong; Guo, Chen; Ma, Qingping [School of Life Science, Huainan Normal University, Huainan 232001 (China)

    2014-06-01

    Highlights: • MWCNTs-COOH disturb mineral elements and cause oxidative damages in the leaves. • Cd and Pb combination result in reduction of mineral elements and enrichment of Na, involving in toxicity mechanisms. • MWCNTs-COOH facilitate Cd and Pb uptake, and aggravate biochemical and subcellular damages. - Abstract: Increasing industrialization of multi-walled carbon nanotubes (MWCNTs) would inevitably lead to their release into the environment and combination with heavy metals. However, studies concerning the combined effects of MWCNTs and heavy metals on agricultural crops are limited. Herein, effects and mechanisms of carboxylated MWCNTs (MWCNTs-COOH) (2.5, 5 and 10 mg/L) and their combination with 20 μM Pb and 5 μM Cd (shortened as Pb + Cd) on Vicia faba L. seedlings were investigated. The results showed that the MWCNTs-COOH disturbed the imbalance of nutrient elements, and caused oxidative stress and damages in the leaves. Additionally, the combination of MWCNTs-COOH with Pb + Cd resulted in enrichment of Pb and Cd, and deterioration of oxidative damages compared with the treatments of MWCNTs-COOH or Pb + Cd alone in the leaves. As the results, the concentrations of MWCNTs-COOH not only caused oxidative stress, but also exacerbated the biochemical and subcellular damages due to the treatment of Pb + Cd in the leaves. It also suggests that persistent release of MWCNTs-COOH into the environment may cause phytotoxicity and aggravate ecological risks due to combination of heavy metals.

  16. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Kostsin, Dzmitry G. [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya Str. 27, Minsk 220072 (Belarus); Kashiwayama, Yoshinori [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Takanashi, Kojiro; Yazaki, Kazufumi [Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoko University, Uji, Kyoto 611-0011 (Japan); Imanaka, Tsuneo, E-mail: imanaka@pha.u-toyama.ac.jp [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Morita, Masashi [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2014-10-24

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

  17. An experimental study of americium-241 biokinetics in the Lobster Homarus Gammarus. Analysis of the accumulation/storage and detoxification processes at the subcellular level

    International Nuclear Information System (INIS)

    An experimental study of americium-241 kinetics has been conducted in the lobster Homarus gammmarus. The investigations were conducted at all the levels from the whole body to the subcellular and molecular levels. The animals were contaminated by a single or chronic ingestion of 241 Am labelled mussels. Assessments of accumulation, elimination and distribution of the radionuclide were established on organisms kept in the laboratory; they made it possible to demonstrate the importance of the digestive gland in the radionuclide transfer pathways. The preliminary results led to structural then ultrastructural investigations of the digestive gland in association with radioautographic studies and cellular extractions methods. Four cellular types were demonstrated, only two of them being implied in the radionuclide retention, the former being responsible for americium intake and the latter for its long-term retention. By means of biochemical techniques, subcellular accumulation was studied and the organelles implied in the nuclide retention were specified. Finally, a method of cellular nuclei dissociation was developed; it made it possible to analyse the molecular nature of americium ligands and to demonstrate the function of the protein nuclear matrix in the nuclide retention

  18. Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy.

    Science.gov (United States)

    Thai, Truc Quynh; Nguyen, Huy Bang; Saitoh, Sei; Wu, Bao; Saitoh, Yurika; Shimo, Satoshi; Elewa, Yaser Hosny Ali; Ichii, Osamu; Kon, Yasuhiro; Takaki, Takashi; Joh, Kensuke; Ohno, Nobuhiko

    2016-09-01

    Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine. PMID:26867664

  19. Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle.

    Science.gov (United States)

    Massarweh, Ahmad; Bosco, Michaël; Iatmanen-Harbi, Soria; Tessier, Clarice; Auberger, Nicolas; Busca, Patricia; Chantret, Isabelle; Gravier-Pelletier, Christine; Moore, Stuart E H

    2016-06-01

    Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction. PMID:27037250

  20. Multiple molecular forms of adaptor protein Ruk/CIN85 specifically associate with different subcellular compartments in human breast adenocarcinoma MCF-7 cells.

    Science.gov (United States)

    Vynnytska-Myronovska, B O; Bobak, Ya P; Pasichnyk, G V; Igumentseva, N I; Samoylenko, A A; Drobot, L B

    2014-01-01

    Ruk/CIN85 is a receptor-proximal 'signalling' adaptor that possesses three SH3 domains, Pro- and Ser-rich regions and C-terminal coiled-coil domain. It employs distinct domains and motifs to act as a transducer platform in intracellular signaling. Based on cDNA analysis, various isoforms of Ruk/CIN85 with different combination of protein-protein interaction domains as well as additional Ruk/CIN85 forms that are the products of post-translational modifications have been demonstrated. Nevertheless, there is no precise information regarding both the subcellular distribution and the role of Ruk/CIN85 multiple molecular forms in cellular responses. Using MCF-7 human breast adenocarcinoma cells and cell fractionation technique, specific association of Ruk/CIN85 molecular forms with different subcellular compartments was demonstrated. Induction of apoptosis of MCF-7 cells by doxorubicin treatment or by serum deprivation resulted in the system changes of Ruk/CIN85 molecular forms intracellular localization as well as their ratio. The data obtained provide a new insight into potential physiological significance of Ruk/CIN85 molecular forms in the regulation of various cellular functions. PMID:25816594

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

    International Nuclear Information System (INIS)

    Highlights: • ABCD proteins classifies based on with or without NH2-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH2-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH2-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH2-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH2-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH2-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH2-terminal H0 motif in organelle targeting is widely conserved in living organisms

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

    Directory of Open Access Journals (Sweden)

    Jianjun He

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

  3. Sub-cellular partitioning of essential and non-essential metals in a freshwater mollusc, Pyganodon grandis, collected in the field along a polymetallic environmental gradient

    Science.gov (United States)

    Bonneris, E.; Giguère, A.; Masson, S.; Campbell, P. G. C.

    2003-05-01

    The cellular alterations normally induced by metals at high concentrations can be prevented by detoxification processes [1] such as sequestration into cellular compartments (calcium concretions, lysosomes, etc.) or their binding to specifie cellular ligands like metallothionein [2]. The aim of this project was to study and compare the subcellular partitioning of three metals (Cd, Cu, Zn) in gills of a freshwater mollusc, Pyganodon grandis, collected along a polymetallic environmental gradient (nine lakes in the Rouyn-Noranda area, Abitibi, QC, Canada). Differential centrifugation was used to partition metals among different subcellular fractions. In the gills, along the environmental metal gradient, total tissue metal concentrations were positively correlated with concentrations in the granule fraction; gill tissues contained high amounts of calcium concretions, which acted as preferential sites for sequestration of the three metals. An increase in Cd concentration was observed in the heat stable proteins fraction (including metallothionein), but not in the heat-denatured proteins fraction, suggesting that Cd-induced cell injury could be prevented by the involement of multiple cellular compartments in a protective role.

  4. Dosimetry at the sub-cellular scale of Auger-electron emitter (99m)Tc in a mouse single thyroid follicle.

    Science.gov (United States)

    Taborda, A; Benabdallah, N; Desbrée, A

    2016-02-01

    The Auger-electrons emitted by (99m)Tc have been recently associated with the induction of thyroid stunning in in vivo experiments in mice, making the dosimetry at the sub-cellular level of (99m)Tc a pertinent and pressing subject. The S-values for (99m)Tc were calculated using MCNP6, which was first validated for studies at the sub-cellular scale and for low energies electrons. The calculation was then performed for (99m)Tc within different cellular compartments in a single mouse thyroid follicle model, considering the radiative and non-radiative transitions of the (99m)Tc radiation spectrum. It was shown that the contribution of the (99m)Tc Auger and low energy electrons to the absorbed dose to the follicular cells' nucleus is important, being at least of the same order of magnitude compared to the emitted photons' contribution and cannot be neglected. The results suggest that Auger-electrons emitted by (99m)Tc play a significant role in the occurrence of the thyroid stunning effect in mice. PMID:26704702

  5. Subcellular distribution of cadmium in two aquatic invertebrates: change over time and relationship to Cd assimilation and loss by a predatory insect.

    Science.gov (United States)

    Dubois, Maï'tée; Hare, Landis

    2009-01-15

    We set out to determine if the efficiency of cadmium (Cd) assimilation and loss by a freshwater predator (the alderfly Sialis velata) differs when it is exposed, for various lengths of time, to Cd in either an insect (Chironomus riparius) or a worm (Tubifex tubifex). Prey were exposed to Cd in sediments for up to 28 days and then fractionated to measure Cd distributions in their cells. Cadmium subcellular distributions varied little over time for a given preytype but differed substantially between the two prey species; for example, the cytosol comprised a larger proportion of Cd in the insect (76%) than in the worm (34%). The predator assimilated proportionally more Cd from the insect (72%) than from the worm (46%) and these assimilation efficiencies were similar to the proportion of prey Cd that would theoretically be available to it (cytosolic Cd + organelle Cd). However, measurements of Cd in the predator's feces confirmed that to obtain an exact 1:1 relationship between predator assimilation efficiency and prey subcellular distribution we had to assume that approximately 50% of the Cd associated with the organelle fraction of T. tubifex was unavailable for digestion by the predator. Losses of Cd from the predator also varied depending on the type of prey that were the source of its Cd. PMID:19238964

  6. Toxicity of selenite in the unicellular green alga Chlamydomonas reinhardtii: Comparison between effects at the population and sub-cellular level

    International Nuclear Information System (INIS)

    The toxicity of selenium in aquatic ecosystems is mainly linked to its uptake and biotransformation by micro-organisms, and its subsequent transfer upwards into the food chain. Thus, organisms at low trophic level, such as algae, play a crucial role. The aim of our study was to investigate the biological effects of selenite on Chlamydomonas reinhardtii, both at the sub-cellular level (effect on ultrastructure) and at the population level (effect on growth). The cells were grown under batch culture conditions in well-defined media and exposed to waterborne selenite at concentrations up to 500 μM; i.e. up to lethal conditions. Based on the relationship between Se concentration and cell density achieved after a 96 h exposure period, an EC50 of 80 μM with a 95% confidence interval ranging between 64 and 98 μM was derived. No adaptation mechanisms were observed: the same toxicity was quantified for algae pre-contaminated with Se. The inhibition of growth was linked to impairments observed at the sub-cellular level. The intensity of the ultrastructural damages caused by selenite exposure depended on the level and duration of exposure. Observations by TEM suggested chloroplasts as the first target of selenite cytotoxicity, with effects on the stroma, thylakoids and pyrenoids. At higher concentrations, we could observe an increase in the number and volume of starch grains. For cells collected at 96 h, electron-dense granules were observed. Energy-dispersive X-ray microanalysis revealed that these granules contained selenium and were also rich in calcium and phosphorus. This study confirms that the direct toxicity of selenite on the phytoplankton biomass is not likely to take place at concentrations found in the environment. At higher concentrations, the link between effects at the sub-cellular and population levels, the over-accumulation of starch, and the formation of dense granules containing selenium are reported for the first time in the literature for a

  7. A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Information of subcellular locations of proteins is important for in-depth studies of cell biology. It is very useful for proteomics, system biology and drug development as well. However, most existing methods for predicting protein subcellular location can only cover 5 to 12 location sites. Also, they are limited to deal with single-location proteins and hence failed to work for multiplex proteins, which can simultaneously exist at, or move between, two or more location sites. Actually, multiplex proteins of this kind usually posses some important biological functions worthy of our special notice. A new predictor called "Euk-mPLoc 2.0" is developed by hybridizing the gene ontology information, functional domain information, and sequential evolutionary information through three different modes of pseudo amino acid composition. It can be used to identify eukaryotic proteins among the following 22 locations: (1 acrosome, (2 cell wall, (3 centriole, (4 chloroplast, (5 cyanelle, (6 cytoplasm, (7 cytoskeleton, (8 endoplasmic reticulum, (9 endosome, (10 extracell, (11 Golgi apparatus, (12 hydrogenosome, (13 lysosome, (14 melanosome, (15 microsome (16 mitochondria, (17 nucleus, (18 peroxisome, (19 plasma membrane, (20 plastid, (21 spindle pole body, and (22 vacuole. Compared with the existing methods for predicting eukaryotic protein subcellular localization, the new predictor is much more powerful and flexible, particularly in dealing with proteins with multiple locations and proteins without available accession numbers. For a newly-constructed stringent benchmark dataset which contains both single- and multiple-location proteins and in which none of proteins has pairwise sequence identity to any other in a same location, the overall jackknife success rate achieved by Euk-mPLoc 2.0 is more than 24% higher than those by any of the existing predictors. As a user-friendly web-server, Euk-mPLoc 2.0 is freely accessible at http

  8. Subcellular localization of chlorosome proteins in Chlorobium tepidum and characterization of three new chlorosome proteins: CsmF, CsmH, and CsmX

    DEFF Research Database (Denmark)

    Vassilieva, Elena V; Stirewalt, Veronica L; Jakobs, Christiane U;

    2002-01-01

    water-soluble form after overproduction in E. coli. Interestingly, this protein contains an N-terminal domain that is similar to CsmB/D, while its C-terminal domain is related to CsmC/D. The sequence relationships indicate that, although the protein composition of Chlorobium-type chlorosomes is...... protein in various cellular fractions. Ten chlorosome proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) copurified in a constant proportion together with bacteriochlorophyll c, and none of these 10 proteins was found in substantial amounts in other subcellular fractions. An...

  9. Sub-cellular partitioning of Zn, Cu, Cd and Pb in the digestive gland of native Octopus vulgaris exposed to different metal concentrations (Portugal)

    Energy Technology Data Exchange (ETDEWEB)

    Raimundo, J. [National Institute for Agronomy and Fisheries Research - IPIMAR, Av. Brasilia, 1449-006 Lisbon (Portugal)], E-mail: jraimundo@ipimar.pt; Vale, C. [National Institute for Agronomy and Fisheries Research - IPIMAR, Av. Brasilia, 1449-006 Lisbon (Portugal); Duarte, R.; Moura, I. [REQUIMTE - CQFB, Department of Chemistry, Faculty of Sciences and Technology, New University of Lisbon, Qta Torre, 2829-516 Monte da Caparica (Portugal)

    2008-02-15

    Concentrations of Zn, Cu, Cd and Pb and their sub-cellular distributions were determined in composite samples of digestive glands of the common octopus, Octopus vulgaris caught from two areas of the Portuguese coast characterised by contrasting metal contamination. Minor contents of Zn (1%), Cu (2%), Cd (6%) and Pb (7%) were found in the insoluble fraction, consisting of nuclei, mitochondria, lysosomes and microsome operationally separated from the whole digestive gland through a sequential centrifugation. A tendency for linear relationships between metal concentrations in nuclei, mitochondria, lysosomes and whole digestive gland was observed. These relationships suggest that despite low metal content organelles responded to the increasing accumulated metals, which means that detoxifying mechanism in cytosol was incomplete. Poorer correlations between microsome and whole digestive gland did not point to metal toxicity in the analysed compartments. However, the high accumulated Cd indicated that O. vulgaris is an important vehicle of this element to its predators in the coastal environment.

  10. The relative importance of water and diet for uptake and subcellular distribution of cadmium in the deposit-feeding polychaete, Capitella sp I

    DEFF Research Database (Denmark)

    Selck, Henriette; Forbes, Valery E.

    2004-01-01

    The impact of dietary and water exposure on the accumulation and distribution of cadmium (Cd) in subcellular components of the polychaete Capitella sp. I was investigated. Worms were exposed to either dissolved Cd alone ('Water-Only' treatments; WO) or diet-bound Cd alone ('Algae-bound Only......' treatments; AO). Thus, WO worms were starved and AO worms were fed. Differential centrifugation was used to fractionate worm homogenates into debris- (DE), mitochondrial- (MI), microsomal- (MC) and cytosolic- (CY) fractions, and the concentration of Cd in these fractions was quantified by radiometric......, starvation likewise influenced the distribution of protein between mitochondria and cytosol. Cutaneous uptake and accumulation of Cd from the water was related to surface area while dietary uptake was influenced by the amount of sediment passing through the gut. Irrespective of exposure route, Cd was...

  11. Absolute zinc quantification at the sub-cellular level by combined use of hard X-ray fluorescence and phase contrast imaging techniques

    International Nuclear Information System (INIS)

    Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected zinc mass fraction in whole cell of PC12 cell lines. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements while quantitative phase contrast imaging provides maps of the projected mass. The combined method was validated on calibration samples by comparison with other alternative techniques such as Atomic Force Microscopy (AFM) and Scanning Transmission Ion Microscopy (STIM). Up to now, absolute quantification at the sub-cellular level was impossible using X-ray fluorescence microscopy but can be reached for the first time with the use of the proposed approach

  12. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    Science.gov (United States)

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN. PMID:27059238

  13. Quantification of cellular volume and sub-cellular density fluctuations: comparison of normal peripheral blood cells and circulating tumor cells identified in a breast cancer patient

    Directory of Open Access Journals (Sweden)

    KevinGregoryPhillips

    2012-08-01

    Full Text Available Cancer metastasis, the leading cause of cancer-related deaths, is facilitated in part by the hematogenous transport of circulating tumor cells (CTCs through the vasculature. Clinical studies have demonstrated that CTCs circulate in the blood of patients with metastatic disease across the major types of carcinomas, and that the number of CTCs in peripheral blood is correlated with overall survival in metastatic breast, colorectal, and prostate cancer. While the potential to monitor metastasis through CTC enumeration exists, the basic physical features of CTCs remain ill defined and moreover, the corresponding clinical utility of these physical parameters is unknown. To elucidate the basic physical features of CTCs we present a label-free imaging technique utilizing differential interference contrast (DIC microscopy to measure cell volume and to quantify sub-cellular mass-density variations as well as the size of subcellular constituents from mass-density spatial correlations. DIC measurements were carried out on CTCs identified in a breast cancer patient using the high-definition (HD CTC detection assay. We compared the biophysical features of HD-CTC to normal blood cell subpopulations including leukocytes, platelets, and red blood cells. HD-CTCs were found to possess larger volumes, decreased mass-density fluctuations, and shorter-range spatial density correlations in comparison to leukocytes. Our results suggest that HD-CTCs exhibit biophysical signatures that might be used to potentially aid in their detection and to monitor responses to treatment in a label-free fashion. The biophysical parameters reported here can be incorporated into computational models of CTC-vascular interactions and in vitro flow models to better understand metastasis.

  14. Study of chromium-containing proteins in subcellular fractions of rat liver by enriched stable isotopic tracer technique and gel filtration chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Weiyue; Li, Bai; Liu, Jing; Chai, Zhifang; Zhang, Peiqun; Gao, Yuxi; Zhao, Jiujiang [Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics, Chinese Academy of Sciences, P.O. Box 918, 100039, Beijing (China)

    2003-02-01

    Ten male Wistar rats were intravenously injected with a single approximately physiological dose of enriched stable isotopic Cr-50 tracer solution (200 ng {sup 50}Cr{sup 3+}/100 g body wt). The fundamental distribution patterns of the chromium-containing proteins in the nucleic, mitochondrial, lysosomal, microsomal, and cytosolic subcellular fractions of the rat liver were investigated by means of Sephadex G-100 gel chromatography combined with neutron activation analysis via {sup 50}Cr (n, {gamma}) {sup 51}Cr reaction. In total, nine kinds of Cr-containing proteins were found in the five subcellular fractions, whose relative molecular masses were 96.6{+-}6.2, 68.2{+-}1.4, 57.9{+-}4.7, 36.6{+-}1.2, 24.2{+-}1.8, 14.0{+-}1.5, 8.8{+-}0.6, 6.9{+-}0.4, and 4.2{+-}0.4 kDa. Approximately 64.5% of Cr proteins accumulated in the cytosolic fraction. The second enriched part was the nucleic fraction; about 12.2% Cr proteins were stored in this section. The 4.2-kDa molecular mass might contain the so-called low molecular weight chromium-containing substance; however, in this research, it was only observed in the mitochondria, lysosome, and microsome. In the mitochondrial fraction, most of the Cr proteins were present as relatively low molecular weight substances: about 56% of chromium-containing proteins had molecular masses {<=}6.9 kDa. Nevertheless, more than 69% of Cr-containing proteins were observed with molecular masses {>=}57.9 kDa in the liver cytosolic fraction. (orig.)

  15. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

    Science.gov (United States)

    Koukourakis, Michael I.; Kalamida, Dimitra; Giatromanolaki, Alexandra; Zois, Christos E.; Sivridis, Efthimios; Pouliliou, Stamatia; Mitrakas, Achilleas; Gatter, Kevin C.; Harris, Adrian L.

    2015-01-01

    LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies. PMID:26378792

  16. Characteristics of the effect of insecticide karate on the metabolism of the nucleic acids and proteins in the subcellular fractions of the liver and intestine

    Directory of Open Access Journals (Sweden)

    Mukaddas Khamrakulova

    2012-12-01

    Full Text Available Karate is contact insecticide from the group of pyrethroids, its chemical formulae C23H19ClF3NO3, molecular mass 449.9. (1-RS-cis-2.2-dimethyl-3-(3.3.3-triphtoro-2chloropropanyl-cyclopropan of the carbonic acid-1 (RS-α-cyano-3-phenoxybenzolic ether. Purpose of this work was to study effect of insecticide Karate on the contents of DNA and RNA and total protein in the subcellular elements of the liver and intestinal mucosa in white rats.Experiments were performed on the outbred white rats of mass 120-250 g in 2 stages. Analysis of the results obtained showed that in acute poisoning with pesticide Karate the acute decrease in RNA and DNA occurred in the subcellular elements of liver and intestine mucosa during the first 7 days of experiment with subsequent normalization of the levels during the following 7 and 15 days. In chronic poisoning the number of RNA in the studied hepatic biomedium decreased in the homogenate to 66.47 and 55%, in the nucleic fractions to 64-62.5%, and in the supernatant fluid - to 52.6-48%; and in the small intestine - from 55.6% to 64.6%, respectively, according to the time of experiment. The DNA level reduced from 20% to 55%. In acute and chronic poisoning with pesticides of group pyrethroids, concentration of nucleic acids sharply reduced in the elements of liver and intestinal mucosa, that indicated about possibility of the inhibition of the DNA and RNA synthesis, which effect on the synthesis of protein in the cells.

  17. Robustness and adaptation reveal plausible cell cycle controlling subnetwork in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Jiun-Yan; Huang, Chi-Wei; Kao, Kuo-Ching; Lai, Pik-Yin

    2013-04-10

    Biological systems are often organized spatially and temporally by multi-scale functional subsystems (modules). A specific subcellular process often corresponds to a subsystem composed of some of these interconnected modules. Accurate identification of system-level modularity organization from the large scale networks can provide valuable information on subsystem models of subcellular processes or physiological phenomena. Computational identification of functional modules from the large scale network is the key approach to solve the complexity of modularity in the past decade, but the overlapping and multi-scale nature of modules often renders unsatisfactory results in these methods. Most current methods for modularity detection are optimization-based and suffered from the drawback of size resolution limit. It is difficult to trace the origin of the unsatisfactory results, which may be due to poor data, inappropriate objective function selection or simply resulted from natural evolution, and hence no system-level accurate modular models for subcellular processes can be offered. Motivated by the idea of evolution with robustness and adaption as guiding principles, we propose a novel approach that can identify significant multi-scale overlapping modules that are sufficiently accurate at the system and subsystem levels, giving biological insights for subcellular processes. The success of our evolution strategy method is demonstrated by applying to the yeast protein-protein interaction network. Functional subsystems of important physiological phenomena can be revealed. In particular, the cell cycle controlling network is selected for detailed discussion. The cell cycle subcellular processes in yeast can be successfully dissected into functional modules of cell cycle control, cell size check point, spindle assembly checkpoint, and DNA damage check point in G2/M and S phases. The interconnections between check points and cell cycle control modules provide clues on the

  18. Chronic nickel bioaccumulation and sub-cellular fractionation in two freshwater teleosts, the round goby and the rainbow trout, exposed simultaneously to waterborne and dietborne nickel.

    Science.gov (United States)

    Leonard, Erin M; Banerjee, Upasana; D'Silva, Joshua J; Wood, Chris M

    2014-09-01

    Rainbow trout and round goby were exposed for 30 days to waterborne and dietary Ni in combination at two waterborne concentration ranges (6.2-12 μmol/L, 68-86 μmol/L), the lower of which is typical of contaminated environments. The prey (black worms; Lumbriculus variegatus) were exposed for 48 h in the effluent of the fish exposure tanks before being fed to the fish (ration=2% body weight/day). Ni in gills, gut, and prey was fractionated into biologically inactive metal [BIM=metal-rich granules (MRG) and metallothionein-like proteins (MT)] and biologically active metal [BAM=organelles (ORG) and heat-denaturable proteins (HDP)]. Gobies were more sensitive than trout to chronic Ni exposure. Possibly, this greater sensitivity may have been due to the goby's pre-exposure to pollutants at their collection site, as evidenced by ∼2-fold greater initial Ni concentrations in both gills and gut relative to trout. However, this was followed by ∼2-16× larger bioaccumulation in both the gills and the gut during the experimental exposure. On a subcellular level, ∼3-40× more Ni was associated with the BAM fraction of goby in comparison to trout. Comparison of the fractional distribution of Ni in the prey versus the gut tissue of the predators suggested that round goby were more efficient than rainbow trout in detoxifying Ni taken up from the diet. Assessing sub-cellular distribution of Ni in the gills and gut of two fish of different habitat and lifestyles revealed two different strategies of Ni bioaccumulation and sub-cellular distribution. On the one hand, trout exhibited an ability to regulate gill Ni bioaccumulation and maintain the majority of the Ni in the MT fraction of the BIM. In contrast goby exhibited large Ni spillovers to both the HDP and ORG fractions of the BAM in the gill. However, the same trend was not observed in the gut, where the potential acclimation of goby to pollutants from their collection site may have aided their ability to regulate Ni

  19. Distinct subcellular localization of alternative splicing variants of EFA6D, a guanine nucleotide exchange factor for Arf6, in the mouse brain.

    Science.gov (United States)

    Fukaya, Masahiro; Ohta, Shingo; Hara, Yoshinobu; Tamaki, Hideaki; Sakagami, Hiroyuki

    2016-09-01

    EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc. PMID:27241101

  20. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses

    International Nuclear Information System (INIS)

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L∞) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently

  1. Subcellular Distribution of Cadmium in Mining Ecotype Sedum alfredii%镉在东南景天中的亚细胞分配

    Institute of Scientific and Technical Information of China (English)

    倪天华; 魏幼璋

    2003-01-01

    The mining ecotype Sedum alfredii Hance could tolerate and grow normally in a nutritivesolution containing cadmium (Cd) as high as 400 μmol/L. Under such a high Cd concentration, the subcel-lular accumulation of Cd in root, stem and leaf of this plant was found to be the highest in the cell wall, lessin the soluble fraction and lowest in the cell organs. The mode of subcellular distribution of Cd in themining ecotype S. alfredii was similar to other hyper accumulators of heavy metals, in which Cd wasdistributed more in the aerial part of plant. The results suggest that the mining ecotype S. alfredii is a newspecies of Cd hyperaccumulator.%采用差速离心技术,比较研究了Cd在矿山生态型东南景天(Sedum alfredii Hance)根、茎、叶中的亚细胞分布.结果表明:矿山生态型东南景天对Cd具有很好的忍受耐和累积能力,非矿山生态型东南景天则不具有这种能力.Cd在矿山生态型东南景天根、茎、叶各部分的亚细胞分配满足F1(细胞壁部分)>F3(可溶部分)>F2(细胞器及膜)规律,且Cd在细胞壁部分的分配占绝对优势;同时矿山生态型东南景天地上部有很好的Cd累积能力.与众多超累积植物相似的Cd亚细胞分配规律和地上部的良好的Cd累积能力.对此植物的Cd亚细胞分布完全不同于非矿山生态型东南景天的情况作了比较分析,结果显示矿山生态型东南景天很可能是一种新的镉超累积种质资源.

  2. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris

    Energy Technology Data Exchange (ETDEWEB)

    Qian Haifeng; Chen Wei; Sheng, G. Daniel; Xu Xiaoyan; Liu Weiping [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Fu Zhengwei [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China)], E-mail: azwfu2003@yahoo.com.cn

    2008-07-30

    Greater exposure to herbicide increases the likelihood of harmful effects in humans and the environment. Glufosinate, a non-selective herbicide, inhibits glutamine synthetase (GS) and thus blocks ammonium assimilation in plants. In the present study, the aquatic unicellular alga Chlorella vulgaris was chosen to assess the effects of acute glufosinate toxicity. We observed physiological changes during 12-96 h of exposure, and gene transcription during 6-48 h of exposure. Exposure to glufosinate increased malondialdehyde content by up to 2.73 times compared with the control, suggesting that there was some oxidative damage. Electron microscopy also showed that there were some chloroplast abnormalities in response to glufosinate. The activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) also increased markedly in the presence of glufosinate. Maximum activities of SOD, POD, and CAT were 2.90, 2.91, and 2.48 times that of the control, respectively. These elevated activities may help alleviate oxidative damage. A real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL. The results showed that glufosinate reduced the transcript abundances of the three genes after 12 h exposure. The lowest abundances of psaB, psbC and rbcL transcripts in response to glufosinate exposure were 38%, 16% and 43% of those of the control, respectively. Our results demonstrate that glufosinate affects the activities of antioxidant enzymes, disrupts chloroplast ultrastructure, and reduces transcription of photosynthesis-related genes in C. vulgaris.

  3. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris

    International Nuclear Information System (INIS)

    Greater exposure to herbicide increases the likelihood of harmful effects in humans and the environment. Glufosinate, a non-selective herbicide, inhibits glutamine synthetase (GS) and thus blocks ammonium assimilation in plants. In the present study, the aquatic unicellular alga Chlorella vulgaris was chosen to assess the effects of acute glufosinate toxicity. We observed physiological changes during 12-96 h of exposure, and gene transcription during 6-48 h of exposure. Exposure to glufosinate increased malondialdehyde content by up to 2.73 times compared with the control, suggesting that there was some oxidative damage. Electron microscopy also showed that there were some chloroplast abnormalities in response to glufosinate. The activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) also increased markedly in the presence of glufosinate. Maximum activities of SOD, POD, and CAT were 2.90, 2.91, and 2.48 times that of the control, respectively. These elevated activities may help alleviate oxidative damage. A real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL. The results showed that glufosinate reduced the transcript abundances of the three genes after 12 h exposure. The lowest abundances of psaB, psbC and rbcL transcripts in response to glufosinate exposure were 38%, 16% and 43% of those of the control, respectively. Our results demonstrate that glufosinate affects the activities of antioxidant enzymes, disrupts chloroplast ultrastructure, and reduces transcription of photosynthesis-related genes in C. vulgaris

  4. Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Larissa Reifur

    2012-09-01

    Full Text Available Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

  5. Multiple heat priming enhances thermo-tolerance to a later high temperature stress via improving subcellular antioxidant activities in wheat seedlings

    DEFF Research Database (Denmark)

    Wang, Xiao; Cai, Jian; Liu, Fulai;

    2014-01-01

    Seedlings of winter wheat (Triticum aestivum L.) were firstly twice heat-primed at 32/24 °C, and subsequently subjected to a more severe high temperature stress at 35/27 °C. The later high temperature stress significantly decreased plant biomass and leaf total soluble sugars concentration. Howeve....... Collectively, heat priming effectively improved thermo-tolerance of wheat seedlings subjected to a later high temperature stress, which could be largely ascribed to the enhanced anti-oxidation at the subcellular level.......Seedlings of winter wheat (Triticum aestivum L.) were firstly twice heat-primed at 32/24 °C, and subsequently subjected to a more severe high temperature stress at 35/27 °C. The later high temperature stress significantly decreased plant biomass and leaf total soluble sugars concentration. However......, plants experienced priming (PH) up-regulated the Rubisco activase B encoding gene RcaB, which was in accordance with the higher photosynthesis rate in relation to the non-primed plants (NH) under the later high temperature stress. In relation to NH, the major chlorophyll a/b-binding protein gene Cab was...

  6. Engineering metal-binding sites of bacterial CusF to enhance Zn/Cd accumulation and resistance by subcellular targeting.

    Science.gov (United States)

    Yu, Pengli; Yuan, Jinhong; Zhang, Hui; Deng, Xin; Ma, Mi; Zhang, Haiyan

    2016-01-25

    The periplasmic protein CusF acts as a metallochaperone to mediate Cu resistance in Escherichia coli. CusF does not contain cysteine residues and barely binds to divalent cations. Here, we addressed effects of cysteine-substitution mutant (named as mCusF) of CusF on zinc/cadmium (Zn/Cd) accumulation and resistance. We targeted mCusF to different subcellular compartments in Arabidopsis. We found that plants expressing vacuole-targeted mCusF were more resistant to excess Zn than WT and plants with cell wall-targeted or cytoplasmic mCusF. Under long-term exposure to excess Zn, all transgenic lines accumulated more Zn (up to 2.3-fold) in shoots than the untransformed plants. Importantly, plants with cytoplasmic mCusF showed higher efficiency of Zn translocation from root to shoot than plants with secretory pathway-targeted-mCusF. Furthermore, the transgenic lines exhibited enhanced resistance to Cd and significant increase in root-to-shoot Cd translocation. We also found all transgenic plants greatly improved manganese (Mn) and iron (Fe) homeostasis under Cd exposure. Our results demonstrate heterologous expression of mCusF could be used to engineer a new phytoremediation strategy for Zn/Cd and our finding also deepen our insights into mechanistic basis for relieving Cd toxicity in plants through proper root/shoot partitioning mechanism and homeostatic accumulation of Mn and Fe. PMID:26476315

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

    Science.gov (United States)

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

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence. PMID:13679851

  8. GOASVM: a subcellular location predictor by incorporating term-frequency gene ontology into the general form of Chou's pseudo-amino acid composition.

    Science.gov (United States)

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

    2013-04-21

    Prediction of protein subcellular localization is an important yet challenging problem. Recently, several computational methods based on Gene Ontology (GO) have been proposed to tackle this problem and have demonstrated superiority over methods based on other features. Existing GO-based methods, however, do not fully use the GO information. This paper proposes an efficient GO method called GOASVM that exploits the information from the GO term frequencies and distant homologs to represent a protein in the general form of Chou's pseudo-amino acid composition. The method first selects a subset of relevant GO terms to form a GO vector space. Then for each protein, the method uses the accession number (AC) of the protein or the ACs of its homologs to find the number of occurrences of the selected GO terms in the Gene Ontology annotation (GOA) database as a means to construct GO vectors for support vector machines (SVMs) classification. With the advantages of GO term frequencies and a new strategy to incorporate useful homologous information, GOASVM can achieve a prediction accuracy of 72.2% on a new independent test set comprising novel proteins that were added to Swiss-Prot six years later than the creation date of the training set. GOASVM and Supplementary materials are available online at http://bioinfo.eie.polyu.edu.hk/mGoaSvmServer/GOASVM.html. PMID:23376577

  9. Itm2a expression in the developing mouse first lower molar, and the subcellular localization of Itm2a in mouse dental epithelial cells.

    Directory of Open Access Journals (Sweden)

    Makiko Kihara

    Full Text Available Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6 cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.

  10. Subcellular distribution of heavy metals in liver and kidney of a narwhal whale (Monodon monoceros): an evaluation for the presence of metallothionein.

    Science.gov (United States)

    Wagemann, R; Hunt, R; Klaverkamp, J F

    1984-01-01

    The subcellular distribution of Zn, Cd, Cu and Hg in liver and kidney from a narwhal was determined by ultracentrifugation and gel filtration. Most of the total mercury in the liver and kidney was bound by the cellular pellet (88 and 73%, respectively). Of the total mercury, 7 and 11% was in the form of methylmercury in the liver and kidney, respectively. More than half (74%) of the total Zn and Cu in the kidney was in the cytosol and somewhat less than this was in the cytosol of the liver. Almost all of the cadmium in liver and kidney (88 and 92%, respectively) was in cytosol. Cytosolic fractions from liver and kidney were evaluated for the presence of metallothionein by analysing for Zn, Cd, Hg, Cu, Fe and--SH groups, by molecular weight estimation and by u.v. absorption spectra. Metallothionein was found in these organs in estimated concentrations similar to those present in terrestrial and other marine mammals. PMID:6149069

  11. Direct mapping of 19F in 19FDG-6P in brain tissue at subcellular resolution using soft X-ray fluorescence

    International Nuclear Information System (INIS)

    Low energy x-ray fluorescence (LEXRF) detection was optimized for imaging cerebral glucose metabolism by mapping the fluorine LEXRF signal of 19F in 19FDG, trapped as intracellular 19F-deoxyglucose-6-phosphate (19FDG-6P) at 1μm spatial resolution from 3μm thick brain slices. 19FDG metabolism was evaluated in brain structures closely resembling the general cerebral cytoarchitecture following formalin fixation of brain slices and their inclusion in an epon matrix. 2-dimensional distribution maps of 19FDG-6P were placed in a cytoarchitectural and morphological context by simultaneous LEXRF mapping of N and O, and scanning transmission x-ray (STXM) imaging. A disproportionately high uptake and metabolism of glucose was found in neuropil relative to intracellular domains of the cell body of hypothalamic neurons, showing directly that neurons, like glial cells, also metabolize glucose. As 19F-deoxyglucose-6P is structurally identical to 18F-deoxyglucose-6P, LEXRF of subcellular 19F provides a link to in vivo 18FDG PET, forming a novel basis for understanding the physiological mechanisms underlying the 18FDG PET image, and the contribution of neurons and glia to the PET signal

  12. Mutation of light-dependent phosphorylation sites of the Drosophila transient receptor potential-like (TRPL) ion channel affects its subcellular localization and stability.

    Science.gov (United States)

    Cerny, Alexander C; Oberacker, Tina; Pfannstiel, Jens; Weigold, Sebastian; Will, Carina; Huber, Armin

    2013-05-31

    The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation. PMID:23592784

  13. Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability*

    Science.gov (United States)

    Cerny, Alexander C.; Oberacker, Tina; Pfannstiel, Jens; Weigold, Sebastian; Will, Carina; Huber, Armin

    2013-01-01

    The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation. PMID:23592784

  14. A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS

    Directory of Open Access Journals (Sweden)

    Guo Anping

    2010-06-01

    Full Text Available Abstract Background Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS. Results Based on the reported Borax/PVPP/Phenol (BPP protocol, we developed an efficient method for protein preparation from different latex subcellular fractions and constructed high-resolution reference 2-DE maps. The obtained proteins from both total latex and C-serum fraction with this protocol generate more than one thousand protein spots and several hundreds of protein spots from rubber particles as well as lutoid fraction and its membranes on the CBB stained 2-DE gels. The identification of 13 representative proteins on 2-DE gels by MALDI TOF/TOF MS/MS suggested that this method is compatible with MS. Conclusion The proteins extracted by this method are compatible with 2-DE and MS. This protein preparation protocol is expected to be used in future comparative proteomic analysis for natural rubber latex.

  15. Transformation of Arabidopsis by Rice OsWRKY78:: GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

    Institute of Scientific and Technical Information of China (English)

    Shunzhi LIU; Mei ZHANG; Xin TANG; Xiaolan WANG

    2012-01-01

    [Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid ex- pression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The re- combinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tume- faciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related sig- nal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

  16. Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

    Science.gov (United States)

    Van Malderen, Stijn J M; Vergucht, Eva; De Rijcke, Maarten; Janssen, Colin; Vincze, Laszlo; Vanhaecke, Frank

    2016-06-01

    This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 μg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions. PMID:27149342

  17. Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations

    Directory of Open Access Journals (Sweden)

    Shaoliang Chen

    2014-04-01

    Full Text Available Energy-dispersive X-ray microanalysis (EDX is a technique for determining the distribution of elements in various materials. Here, we report a protocol for high-spatial-resolution X-ray elemental imaging and quantification in plant tissues at subcellular levels with a scanning transmission electron microscope (STEM. Calibration standards were established by producing agar blocks loaded with increasing KCl or NaCl concentrations. TEM-EDX images showed that the salts were evenly distributed in the agar matrix, but tended to aggregate at high concentrations. The mean intensities of K+, Cl−, and Na+ derived from elemental images were linearly correlated to the concentrations of these elements in the agar, over the entire concentration range tested (R > 0.916. We applied this method to plant root tissues. X-ray images were acquired at an actual resolution of 50 nm ´ 50 nm to 100 nm ´ 100 nm. We found that cell walls exhibited higher elemental concentrations than vacuoles. Plants exposed to salt stress showed dramatic accumulation of Na+ and Cl− in the transport tissues, and reached levels similar to those applied in the external solution (300 mM. The advantage of TEM-EDX mapping was the high-spatial-resolution achieved for imaging elemental distributions in a particular area with simultaneous quantitative analyses of multiple target elements.

  18. Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase Cα activation and subcellular translocation of Smac in human small cell lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    Desheng Xiao; Kuansong Wang; Jianhua Zhou; Huiqiu Cao; Zhenghao Deng; Yongbin Hu; Xiahui Qu; Jifang Wen

    2008-01-01

    To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation,apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation.Survivin expression induced by FGF-2 and protein kinase Cα (PKCα) translocation was detected by subcellular fractionation and Western blot analysis. In addition, FGF-2-induced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence.FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dosedependent and time-dependent manner, and inhibited caspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKCα translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKCα regulated FGF-2-induced survivin expression. Thus, survivin, Smac,and PKCα might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.

  19. Seasonal and size-related variation of subcellular biomarkers in quagga mussels (Dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants.

    Science.gov (United States)

    Ács, A; Vehovszky, Á; Győri, J; Farkas, A

    2016-07-01

    The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals. PMID:27329477

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-01

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

  1. Changes in the Subcellular Distribution of NADPH Oxidase Subunit p47phox in Dendrites of Rat Dorsomedial Nucleus Tractus Solitarius Neurons in Response to Chronic Administration of Hypertensive Agents

    OpenAIRE

    Glass, Michael J.; Chan, June; Frys, Kelly A.; Oselkin, Martin; Tarsitano, M. Jacqueline; Iadecola, Costantino; Pickel, Virginia M.

    2007-01-01

    NADPH oxidase-generated superoxide can modulate crucial intracellular signaling cascades in neurons of the nucleus tractus solitarius (NTS), a brain region that plays an important role in cardiovascular processes. Modulation of NTS signaling by superoxide may be linked to the subcellular location of the mobile NADPH oxidase p47phox subunit, which is known to be present in dendrites of NTS neurons. It is not known, however, if hypertension can produce changes in the trafficking of p47phox in d...

  2. Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chunying; Zhao, Jiujiang; Zhang, Peiqun; Chai, Zhifang [Institute of High Energy Physics and Laboratory of Nuclear Analytical Techniques, Chinese Academy of Sciences, Beijing (China)

    2002-02-01

    Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle. (orig.)

  3. The PanK2 Genes of Mouse and Human Specify Proteins with DistinctSubcellular Locations

    Energy Technology Data Exchange (ETDEWEB)

    Leonardi, Roberta; Zhang, Yong-Mei; Lydikis, Athanasios; Stevens,Robert D.; Ilkayeva, Olga R.; Wenner, Brett R.; Bain, James R.; Newgard,Christopher B.; Rock, Charles O.; Jackowski, Suzanne

    2007-05-01

    Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency. (c) 2007 Federation of European Biochemical Societies.Published by Elsevier B.V.

  4. Changes in subcellular distribution of n-octanoyl or n-decanoyl ghrelin in ghrelin-producing cells

    Directory of Open Access Journals (Sweden)

    YoshihiroNishi

    2013-07-01

    Results: Immunoelectron microscopic analysis revealed the co-existence of C8- and C10-ghrelin within the same secretory granules (mixed-type in ghrelin-producing cells. Compared to control mice fed standard chow, the ratio of C10-type secretory granules increased significantly after ingestion of C10-MCT, whereas that of C8-type granules declined significantly under the same treatment. After ingestion of C8-MCT, the proportion of C8-type secretory granules increased significantly. Within the mixed-type granules the ratio of ir-C10-ghrelin increased significantly and that of ir-C8-ghrelin decreased significantly upon fasting. Conclusions: These findings confirmed that C10-ghrelin, another acyl-form of active ghrelin, is stored within the same secretory granules as C8-ghrelin, and suggested that the types of medium-chain acyl-molecules surrounding and available to the ghrelin-GOAT system may affect the physiological processes of ghrelin acylation.

  5. Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

    Science.gov (United States)

    Sarria, Ignacio; Orlandi, Cesare; McCall, Maureen A; Gregg, Ronald G; Martemyanov, Kirill A

    2016-03-01

    In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling. PMID:26961947

  6. Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Zhongli; Chattopadhyay, Niladri; Kwon, Yongkyu Luke [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Pignol, Jean-Philippe [Department of Radiation Oncology, University of Toronto, Toronto, Ontario M4N 3M5, Canada and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Lechtman, Eli [Department of Medical Biophysics, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Reilly, Raymond M. [Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, Ontario M5S 3E2 (Canada); Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4 (Canada)

    2013-11-15

    Purpose: The authors’ aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF).Methods: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10–100 keV), an x-ray beam (100 kVp), and {sup 125}I and {sup 103}Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared.Results: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2

  7. Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation

    International Nuclear Information System (INIS)

    Purpose: The authors’ aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF).Methods: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10–100 keV), an x-ray beam (100 kVp), and 125I and 103Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared.Results: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2) using 30 nm

  8. Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7

    Science.gov (United States)

    Chen, Hao; Babino, Darwin; Schoenbichler, Stefan A.; Arkhipova, Valeryia; Töchterle, Sonja; Martin, Fabian; Huck, Christian W.; von Lintig, Johannes; Meyer, Dirk

    2015-01-01

    Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation. PMID:26618989

  9. Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

    Directory of Open Access Journals (Sweden)

    Hao Chen

    Full Text Available Retinol binding proteins (Rbps are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

  10. Subcellular partitioning of non-essential trace metals (Ag, As, Cd, Ni, Pb, and Tl) in livers of American (Anguilla rostrata) and European (Anguilla anguilla) yellow eels

    Energy Technology Data Exchange (ETDEWEB)

    Rosabal, Maikel [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Pierron, Fabien [Université de Bordeaux, UMR EPOC CNRS 5805, F-33400 Talence (France); CNRS, EPOC, UMR 5805, F-33400 Talence (France); Couture, Patrice [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Baudrimont, Magalie [Université de Bordeaux, UMR EPOC CNRS 5805, F-33400 Talence (France); CNRS, EPOC, UMR 5805, F-33400 Talence (France); Hare, Landis [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada)

    2015-03-15

    Highlights: • Handling of hepatic metals consistently involved cytosolic, thermostable ligands. • Granule-like fractions are also involved in the detoxification of Ni, Pb, and Tl. • Despite these sequestration mechanisms, metal detoxification is incomplete. • Along the metal gradient, concentrations increase in metal-sensitive fractions. • This increase could represent a toxicological risk for the yellow eels. - Abstract: We determined the intracellular compartmentalization of the trace metals Ag, As, Cd, Ni, Pb, and Tl in the livers of yellow eels collected from the Saint Lawrence River system in Canada (Anguilla rostrata) and in the area of the Gironde estuary in France (Anguilla anguilla). Differential centrifugation, NaOH digestion and thermal shock were used to separate eel livers into putative “sensitive” fractions (heat-denatured proteins, mitochondria and microsomes + lysosomes) and detoxified metal fractions (heat-stable peptides/proteins and granules). The cytosolic heat-stable fraction (HSP) was consistently involved in the detoxification of all trace metals. In addition, granule-like structures played a complementary role in the detoxification of Ni, Pb, and Tl in both eel species. However, these detoxification mechanisms were not completely effective because increasing trace metal concentrations in whole livers were accompanied by significant increases in the concentrations of most trace metals in “sensitive” subcellular fractions, that is, mitochondria, heat-denatured cytosolic proteins and microsomes + lysosomes. Among these “sensitive” fractions, mitochondria were the major binding sites for As, Cd, Pb, and Tl. This accumulation of non-essential metals in “sensitive” fractions likely represents a health risk for eels inhabiting the Saint Lawrence and Gironde environments.

  11. Developmental alterations in expression and subcellular localization of antizyme and antizyme inhibitor and their functional importance in the murine mammary gland.

    Science.gov (United States)

    Murakami, Y; Suzuki, J; Samejima, K; Oka, T

    2010-02-01

    Ornithine decarboxylase (ODC), antizyme (AZ), and antizyme inhibitor (AIn) play a key role in regulation of intracellular polyamine levels by forming a regulatory circuit through their interactions. To gain insight into their functional importance in cell growth and differentiation, we systematically examined the changes of their expression, cellular polyamine contents, expression of genes related to polyamine metabolism, and beta-casein gene expression during murine mammary gland development. The activity of ODC and AZ1 as well as putrescine level were low in the virgin and involuting stages, but they increased markedly during late pregnancy and early lactation when mammary cells proliferate extensively and begin to augment their differentiated function. The level of spermidine and expression of genes encoding spermidine synthase and AIn increased in a closely parallel manner with that of casein gene expression during pregnancy and lactation. On the other hand, the level of spermidine/spermine N(1)-acetyltransferase (SSAT) mRNA and AZ2 mRNA decreased during those periods. Immunohistochemical analysis showed the translocation of ODC and AIn between the nucleus and cytoplasm and the continuous presence of AZ in the nucleus during gland development. Reduction of AIn by RNA interference inhibited expression of beta-casein gene stimulated by lactogenic hormones in HC11 cells. In contrast, reduction of AZ by AZsiRNA resulted in the small increase of beta-casein gene expression. These results suggested that AIn plays an important role in the mammary gland development by changing its expression, subcellular localization, and functional interplay with AZ. PMID:19997757

  12. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

    International Nuclear Information System (INIS)

    We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates

  13. The high-mobility group box 1 cytokine induces transporter-mediated release of glutamate from glial subcellular particles (gliosomes) prepared from in situ-matured astrocytes.

    Science.gov (United States)

    Bonanno, Giambattista; Raiteri, Luca; Milanese, Marco; Zappettini, Simona; Melloni, Edon; Pedrazzi, Marco; Passalacqua, Mario; Tacchetti, Carlo; Usai, Cesare; Sparatore, Bianca

    2007-01-01

    The multifunctional protein high-mobility group box 1 (HMGB1) is expressed in restricted areas of adult brain where it can act as a proinflammatory cytokine. We report here that HMGB1 affects CNS transmission by inducing glutamatergic release from glial (gliosomes) but not neuronal (synaptosomes) resealed subcellular particles isolated from mouse cerebellum and hippocampus. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins such as GFAP and S-100, but not with neuronal proteins such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several approximately 30-nm nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin or ATP, by mechanisms involving extracellular Ca(2+) and Ca(2+) release from intracellular stores. HMGB1-induced release of the stable glutamate analogue [(3)H]d-aspartate and endogenous glutamate form gliosomes, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of glutamate was independent on modifications of cytosolic Ca(2+) concentration, but it was blocked by dl-threo-beta-benzyloxyaspartate, suggesting the involvement of transporter-mediated release mechanisms. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 does not block the HMGB1 effect, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. HMGB1 bind to gliosomes but not to synaptosomes and can physically interact with GLAST and receptor for advanced glycation end products (RAGE). Taken together, these results suggest that the HMGB1 cytokine

  14. In-situ second harmonic generation by cancer cell targeting ZnO nanocrystals to effect photodynamic action in subcellular space.

    Science.gov (United States)

    Gu, Bobo; Pliss, Artem; Kuzmin, Andrey N; Baev, Alexander; Ohulchanskyy, Tymish Y; Damasco, Jossana A; Yong, Ken-Tye; Wen, Shuangchun; Prasad, Paras N

    2016-10-01

    This paper introduces the concept of in-situ upconversion of deep penetrating near infrared light via second harmonic generation from ZnO nanocrystals delivered into cells to effect photo activated therapies, such as photodynamic therapy, which usually require activation by visible light with limited penetration through biological tissues. We demonstrated this concept by subcellular activation of a photodynamic therapy drug, Chlorin e6, excited within its strong absorption Soret band by the second harmonic (SH) light, generated at 409 nm by ZnO nanocrystals, which were targeted to cancer cells and internalized through the folate-receptor mediated endocytosis. By a combination of theoretical modeling and experimental measurements, we show that SH light, generated in-situ by ZnO nanocrystals significantly contributes to activation of photosensitizer, leading to cell death through both apoptotic and necrotic pathways initiated in the cytoplasm. This targeted photodynamic action was studied using label-free Coherent Anti-Stokes Raman Scattering imaging of the treated cells to monitor changes in the distribution of native cellular proteins and lipids. We found that initiation of photodynamic therapy with upconverted light led to global reduction in the intracellular concentration of macromolecules, likely due to suppression of proteins and lipids synthesis, which could be considered as a real-time indicator of cellular damage from photodynamic treatment. In prospective applications this in-situ photon upconversion could be further extended using ZnO nanocrystals surface functionalized with a specific organelle targeting group, provided a powerful approach to identify and consequently maximize a cellular response to phototherapy, selectively initiated in a specific cellular organelle. PMID:27442221

  15. Subcellular partitioning of non-essential trace metals (Ag, As, Cd, Ni, Pb, and Tl) in livers of American (Anguilla rostrata) and European (Anguilla anguilla) yellow eels

    International Nuclear Information System (INIS)

    Highlights: • Handling of hepatic metals consistently involved cytosolic, thermostable ligands. • Granule-like fractions are also involved in the detoxification of Ni, Pb, and Tl. • Despite these sequestration mechanisms, metal detoxification is incomplete. • Along the metal gradient, concentrations increase in metal-sensitive fractions. • This increase could represent a toxicological risk for the yellow eels. - Abstract: We determined the intracellular compartmentalization of the trace metals Ag, As, Cd, Ni, Pb, and Tl in the livers of yellow eels collected from the Saint Lawrence River system in Canada (Anguilla rostrata) and in the area of the Gironde estuary in France (Anguilla anguilla). Differential centrifugation, NaOH digestion and thermal shock were used to separate eel livers into putative “sensitive” fractions (heat-denatured proteins, mitochondria and microsomes + lysosomes) and detoxified metal fractions (heat-stable peptides/proteins and granules). The cytosolic heat-stable fraction (HSP) was consistently involved in the detoxification of all trace metals. In addition, granule-like structures played a complementary role in the detoxification of Ni, Pb, and Tl in both eel species. However, these detoxification mechanisms were not completely effective because increasing trace metal concentrations in whole livers were accompanied by significant increases in the concentrations of most trace metals in “sensitive” subcellular fractions, that is, mitochondria, heat-denatured cytosolic proteins and microsomes + lysosomes. Among these “sensitive” fractions, mitochondria were the major binding sites for As, Cd, Pb, and Tl. This accumulation of non-essential metals in “sensitive” fractions likely represents a health risk for eels inhabiting the Saint Lawrence and Gironde environments

  16. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    Directory of Open Access Journals (Sweden)

    Amirali eSattarzadeh

    2013-10-01

    Full Text Available Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the PAL sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-06-01

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

  19. Subcellular Fractionation of Human Neutrophils and Analysis of Subcellular Markers

    DEFF Research Database (Denmark)

    Clemmensen, Stine Novrup; Udby, Lene; Borregaard, Niels

    2014-01-01

    The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its pass...

  20. Toponome imaging system: in situ protein network mapping in normal and cancerous colon from the same patient reveals more than five-thousand cancer specific protein clusters and their subcellular annotation by using a three symbol code.

    Science.gov (United States)

    Bhattacharya, Sayantan; Mathew, George; Ruban, Ernie; Epstein, David B A; Krusche, Andreas; Hillert, Reyk; Schubert, Walter; Khan, Michael

    2010-12-01

    In a proof of principle study, we have applied an automated fluorescence toponome imaging system (TIS) to examine whether TIS can find protein network structures, distinguishing cancerous from normal colon tissue present in a surgical sample from the same patient. By using a three symbol code and a power of combinatorial molecular discrimination (PCMD) of 2(21) per subcellular data point in one single tissue section, we demonstrate an in situ protein network structure, visualized as a mosaic of 6813 protein clusters (combinatorial molecular phenotype or CMPs), in the cancerous part of the colon. By contrast, in the histologically normal colon, TIS identifies nearly 5 times the number of protein clusters as compared to the cancerous part (32 009). By subcellular visualization procedures, we found that many cell surface membrane molecules were closely associated with the cell cytoskeleton as unique CMPs in the normal part of the colon, while the same molecules were disassembled in the cancerous part, suggesting the presence of dysfunctional cytoskeleton-membrane complexes. As expected, glandular and stromal cell signatures were found, but interestingly also found were potentially TIS signatures identifying a very restricted subset of cells expressing several putative stem cell markers, all restricted to the cancerous tissue. The detection of these signatures is based on the extreme searching depth, high degree of dimensionality, and subcellular resolution capacity of TIS. These findings provide the technological rationale for the feasibility of a complete colon cancer toponome to be established by massive parallel high throughput/high content TIS mapping. PMID:20822185

  1. Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short- or long-term dietary exposure

    Directory of Open Access Journals (Sweden)

    Rousseau-Ralliard Delphine

    2009-03-01

    Full Text Available Abstract Background Previous work showed that the functional cardiac effect of dietary alpha-linolenic acid (ALA in rats requires a long feeding period (6 months, although a docosahexaenoic (DHA acid-supply affects cardiac adrenergic response after 2 months. However, the total cardiac membrane n-3 polyunsaturated fatty acid (PUFA composition remained unchanged after 2 months. This delay could be due to a specific reorganization of the different subcellular membrane PUFA profiles. This study was designed to investigate the evolution between 2 and 6 months of diet duration of the fatty acid profile in sarcolemmal (SL, mitochondrial (MI, nuclear (NU and sarcoplasmic reticulum (SR membrane fractions. Methods Male Wistar rats were randomly assigned to 3 dietary groups (n = 10/diet/period, either n-3 PUFA-free diet (CTL, or ALA or DHA-rich diets. After 2 or 6 months, the subcellular cardiac membrane fractions were separated by differential centrifugations and sucrose gradients. Each membrane profile was analysed by gas chromatography (GC after lipid extraction. Results As expected the n-3 PUFA-rich diets incorporated n-3 PUFA instead of n-6 PUFA in all the subcellular fractions, which also exhibited individual specificities. The diet duration increased SFA and decreased PUFA in SL, whereas NU remained constant. The SR and MI enriched in n-3 PUFA exhibited a decreased DHA level with ageing in the DHA and CTL groups. Conversely, the n-3 PUFA level remained unchanged in the ALA group, due to a significant increase in docosapentaenoic acid (DPA. N-3 PUFA rich diets lead to a better PUFA profile in all the fractions and significantly prevent the profile modifications induced by ageing. Conclusion With the ALA diet the n-3 PUFA content, particularly in SR and SL kept increasing between 2 and 6 months, which may partly account for the delay to achieve the modification of adrenergic response.

  2. Two AAA family peroxins, PpPex1p and PpPex6p, interact with each other in an ATP-dependent manner and are associated with different subcellular membranous structures distinct from peroxisomes.

    Science.gov (United States)

    Faber, K N; Heyman, J A; Subramani, S

    1998-02-01

    Two peroxins of the AAA family, PpPex1p and PpPex6p, are required for peroxisome biogenesis in the yeast Pichia pastoris. Cells from the corresponding deletion strains (Pp delta pex1 and Pp delta pex6) contain only small vesicular remnants of peroxisomes, the bulk of peroxisomal matrix proteins is mislocalized to the cytosol, and these cells cannot grow in peroxisome-requiring media (J. A. Heyman, E. Monosov, and S. Subramani, J. Cell Biol. 127:1259-1273, 1994; A. P. Spong and S. Subramani, J. Cell Biol. 123:535-548, 1993). We demonstrate that PpPex1p and PpPex6p interact in an ATP-dependent manner. Genetically, the interaction was observed in a suppressor screen with a strain harboring a temperature-sensitive allele of PpPEX1 and in the yeast two-hybrid system. Biochemially, these proteins were coimmunoprecipitated with antibodies raised against either of the proteins, but only in the presence of ATP. The protein complex formed under these conditions was 320 to 400 kDa in size, consistent with the formation of a heterodimeric PpPex1p-PpPex6p complex. Subcellular fractionation revealed PpPex1p and PpPex6p to be predominantly associated with membranous subcellular structures distinct from peroxisomes. Based on their behavior in subcellular fractionation experiments including flotation gradients and on the fact that these structures are also present in a Pp delta pex3 strain in which no morphologically detectable peroxisomal remnants have been observed, we propose that these structures are small vesicles. The identification of vesicle-associated peroxins is novel and implies a role for these vesicles in peroxisome biogenesis. We discuss the possible role of the ATP-dependent interaction between PpPex1p and PpPex6p in regulating peroxisome biogenesis events. PMID:9447990

  3. The Vacuolar ATPase from Entamoeba histolytica: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

    Directory of Open Access Journals (Sweden)

    Luna-Arias Juan

    2008-12-01

    Full Text Available Abstract Background Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. Results We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. Conclusion We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits

  4. Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

    Directory of Open Access Journals (Sweden)

    Hughes Richard K

    2007-11-01

    Full Text Available Abstract Background Hydroperoxide lyase (HPL is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively. Results To study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF between cytosol and lipid droplets (LD whereas, as expected, M.truncatula 13-HPL (HPLE was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF. Conclusion We provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme

  5. Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

    Directory of Open Access Journals (Sweden)

    Kluge Christoph

    2004-08-01

    Full Text Available Abstract Background Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET. Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i a less conserved cytoplasmically orientated N-terminus and (ii a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP in transfected cells. Conclusions All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA

  6. Variation in cadmium accumulation among 30 cultivars and cadmium subcellular distribution in 2 selected cultivars of water spinach (Ipomoea aquatica Forsk.).

    Science.gov (United States)

    Wang, Junli; Yuan, Jiangang; Yang, Zhongyi; Huang, Baifei; Zhou, Yihui; Xin, Junliang; Gong, Yulian; Yu, Hui

    2009-10-14

    To reduce the influx of cadmium (Cd), a toxic heavy metal, into the human food chain through vegetable intake, a pot experiment for the selection of a pollution-safe cultivar (PSC) of water spinach (Ipomoea aquatica Forsk.) was carried out. The experiment with 30 tested cultivars revealed that the maximum differences in Cd concentration between the cultivars containing the highest and the lowest Cd were 3.0-3.9-fold under low-Cd treatment (soil Cd = 0.593 mg kg(-1)), 2.7-3.5-fold under middle-Cd treatment (soil Cd = 1.091 mg kg(-1)), and 2.6-2.7-fold under high-Cd treatment (soil Cd = 1.824 mg kg(-1)), large enough to define the Cd-PSCs. Concentrations of Cd in edible parts of six cultivars, cv. Daxingbaigu, Huifengqing, Qiangkunbaigu, Qiangkunqinggu, Shenniuliuye, and Xingtianqinggu, were lower than 0.2 mg kg(-1), the maximum level (ML) of Cd allowed by the Codex Alimentarius Commission (CAC) standard, even under middle-Cd treatment. Accordingly, these cultivars were treated as typical Cd-PSCs. Four cultivars, cv. Jieyangbaigeng, Xianggangdaye, Sannongbaigeng, and Taiwan 308, contained Cd in edible parts exceeding the ML even under low-Cd treatment, and they were defined as typical non-Cd-PSCs. The correlations of the Cd concentrations among the tested cultivars between the three treatments were significant at the p < 0.05 level. A conspicuous difference in Cd subcellular distribution in hydroponic plant tissues between cv. Qiangkunqinggu (a typical Cd-PSC) and cv. Taiwan 308 (a typical non-Cd-PSC) were observed. Cd absorbed by cv. Qiangkunqinggu seemed to be well-compartmentalized in root and in cell wall fragment, which may be one of the mechanisms leading to its low Cd accumulating property. The results indicated that water spinach, a leafy vegetable, could be easily polluted by soils contaminated with Cd, as 80% of the tested cultivars had exceeded the ML of Cd according to the CAC standard even under the middle-Cd treatment. Much of the evidence obtained from

  7. Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth.

    Science.gov (United States)

    Balla, Jozef; Kalousek, Petr; Reinöhl, Vilém; Friml, Jiří; Procházka, Stanislav

    2011-02-01

    Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds. PMID:21219506

  8. Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

    Science.gov (United States)

    Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

    1994-08-01

    As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear

  9. Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled.positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells

    DEFF Research Database (Denmark)

    Flebus, Luca; Lombart, Francois; Sevrin, Chantal;

    2015-01-01

    ) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein.The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and h...... a minor cytotoxicity compared to the higher ones.As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes....

  10. Subcellular metal partitioning in larvae of the insect Chaoborus collected along an environmental metal exposure gradient (Cd, Cu, Ni and Zn)

    Energy Technology Data Exchange (ETDEWEB)

    Rosabal, Maikel; Hare, Landis [Institut national de la Recherche scientifique, Centre Eau Terre Environnement (INRS-ETE), 490 de la Couronne, Quebec, Quebec, G1K 9A9 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [Institut national de la Recherche scientifique, Centre Eau Terre Environnement (INRS-ETE), 490 de la Couronne, Quebec, Quebec, G1K 9A9 (Canada)

    2012-09-15

    Highlights: Black-Right-Pointing-Pointer Midge larvae were collected from 12 lakes representing Cd, Cu, Ni and Zn gradients. Black-Right-Pointing-Pointer Along the gradients, the heat-stable protein fractions increased for Cd, Ni and Cu. Black-Right-Pointing-Pointer However, this metal detoxification response was incomplete for Cd and Ni. Black-Right-Pointing-Pointer Concentrations of these two metals increased in putative metal-sensitive fractions. Black-Right-Pointing-Pointer Metal detoxification is Chaoborus is compared to that in other freshwater animals. - Abstract: Larvae of the phantom midge Chaoborus are common and widespread in lakes contaminated by metals derived from mining and smelting activities. To explore how this insect is able to cope with potentially toxic metals, we determined total metal concentrations and subcellular metal partitioning in final-instar Chaoborus punctipennis larvae collected from 12 lakes situated along gradients in aqueous Cd, Cu, Ni and Zn concentrations. Concentrations of the non-essential metals Cd and Ni were more responsive to aqueous metal gradients than were larval concentrations of the essential metals Cu and Zn; these latter metals were better regulated and exhibited only 2-3-fold increases between the least and the most contaminated lakes. Metal partitioning was determined by homogenization of larvae followed by differential centrifugation, NaOH digestion and heat denaturation steps so as to separate the metals into operationally defined metal-sensitive fractions (heat-denaturable proteins (HDP), mitochondria, and lysosomes/microsomes) and metal-detoxified fractions (heat stable proteins (HSP) and NaOH-resistant or granule-like fractions). Of these five fractions, the HSP fraction was the dominant metal-binding compartment for Cd, Ni and Cu. The proportions and concentrations of these three metals in this fraction increased along the metal bioaccumulation gradient, which suggests that metallothionein-like proteins

  11. Influence of a step-change in metal exposure (Cd, Cu, Zn) on metal accumulation and subcellular partitioning in a freshwater bivalve, Pyganodon grandis: A long-term transplantation experiment between lakes with contrasting ambient metal levels

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Sophie [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada); Bonneris, Emmanuelle [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada) and Bayer S.A.S., Bayer CropScience, 16 Rue Jean-Marie Leclair, CP 90106, F 69266 Lyon Cedex 09 (France); Michaud, Annick [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada) and Direction des Évaluations environnementales, Ministère du Développement durable, de l’Environnement et des Parcs, 675, boul. René-Lévesque Est, 6e étage, Québec, QC G1R 5V7 (Canada); Pinel-Alloul, Bernadette [Groupe de Recherche Interuniversitaire en Limnologie et Environnement Aquatique (GRIL), Département de Sciences Biologiques, Université de Montréal, C.P. 6128, Montréal, QC H3C 3J7 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada)

    2013-05-15

    Highlights: ? We transferred freshwater bivalves from a reference lake to a Cd and Zn contaminated lake. ? Changes in metal accumulation and subcellular partitioning were followed over time (up to 860 d). ? Metal detoxification strategies differed between target organs (gills vs. digestive gland). ? The ability to handle Cd is inherent in P. grandis, not a trait acquired after long-term adaptation. -- Abstract: The objective of the present field experiment was to identify detoxification responses in the gills and digestive gland of a freshwater unionid bivalve, Pyganodon grandis, subjected to a step-change in metal exposure. Adult bivalves were transferred from a reference site (Lake Opasatica) and a metal-contaminated lake (Lake Héva) to a second contaminated lake (Lake Vaudray) in northwestern Quebec, Canada. Changes in organ metal concentrations, in the subcellular distribution of metals and in metallothionein concentrations were followed over time (t = 0, 132, (400) and 860 days). At each collection time and for each bivalve, the gills and digestive gland were excised and gently homogenized; six sub-cellular fractions were separated by differential centrifugation and analyzed for their Cd, Cu and Zn content, and metallothionein was quantified independently. Metal detoxification strategies were shown to differ between target organs: in the gills, incoming metals were sequestered largely in the granules, whereas in the digestive gland the same metals primarily accumulated in the cytosol, in the metallothionein-like protein fraction. These metal-handling strategies, as employed by the metal-naïve bivalves originating in the reference lake, closely resemble those identified in free-living P. grandis chronically exposed in the metal-contaminated lake, suggesting that the ability to handle incoming metals (Cd in particular) is inherent in P. grandis and is not a trait acquired after long-term adaptation of the bivalve to metal-contaminated environments. The

  12. Influence of a step-change in metal exposure (Cd, Cu, Zn) on metal accumulation and subcellular partitioning in a freshwater bivalve, Pyganodon grandis: A long-term transplantation experiment between lakes with contrasting ambient metal levels

    International Nuclear Information System (INIS)

    Highlights: ► We transferred freshwater bivalves from a reference lake to a Cd and Zn contaminated lake. ► Changes in metal accumulation and subcellular partitioning were followed over time (up to 860 d). ► Metal detoxification strategies differed between target organs (gills vs. digestive gland). ► The ability to handle Cd is inherent in P. grandis, not a trait acquired after long-term adaptation. -- Abstract: The objective of the present field experiment was to identify detoxification responses in the gills and digestive gland of a freshwater unionid bivalve, Pyganodon grandis, subjected to a step-change in metal exposure. Adult bivalves were transferred from a reference site (Lake Opasatica) and a metal-contaminated lake (Lake Héva) to a second contaminated lake (Lake Vaudray) in northwestern Quebec, Canada. Changes in organ metal concentrations, in the subcellular distribution of metals and in metallothionein concentrations were followed over time (t = 0, 132, (400) and 860 days). At each collection time and for each bivalve, the gills and digestive gland were excised and gently homogenized; six sub-cellular fractions were separated by differential centrifugation and analyzed for their Cd, Cu and Zn content, and metallothionein was quantified independently. Metal detoxification strategies were shown to differ between target organs: in the gills, incoming metals were sequestered largely in the granules, whereas in the digestive gland the same metals primarily accumulated in the cytosol, in the metallothionein-like protein fraction. These metal-handling strategies, as employed by the metal-naïve bivalves originating in the reference lake, closely resemble those identified in free-living P. grandis chronically exposed in the metal-contaminated lake, suggesting that the ability to handle incoming metals (Cd in particular) is inherent in P. grandis and is not a trait acquired after long-term adaptation of the bivalve to metal-contaminated environments. The

  13. Types of ectomycorrhiza of mature beech and spruce at ozone-fumigated and control forest plots

    OpenAIRE

    Grebenc, Tine; Kraigher, Hojka

    2015-01-01

    In the Kranzberg forest near Freising (Germany) a novel “Free-Air Canopy O3 Exposure” system has been employed for analysing O3-induced responses from sub-cellular to ecosystem levels that are relevant for carbon balance and CO2 demand of 60-year-old beech trees. The below-ground ectomycorrhizal community was studied in two-fold ambient O3 concentrations (five cores per sampling) and in a control plot with an ambient O3 concentration (four cores per sampling). Five samplings were taken throug...

  14. Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alpha- synuclein

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Xiaoping Liao; Guoqiang Wen; Yidong Deng; Min Guo; Zhigang Long; Feng Ouyang

    2012-01-01

    The specific and effective α-synuclein RNA interference (RNAi) plasmids, and the α-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 (HEK293) cells using the lipofectamine method. Using an inverted fluorescence microscope, α-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type α-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies (Lewy body-like inclusions) in the cytoplasm of some cells transfected with α-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type α-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type α-synuclein in mitochondria.

  15. Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast Comportamento agregatório das plaquetas incubadas com frações subcelulares de sinciciotrofoblasto humano normal e chagásico

    Directory of Open Access Journals (Sweden)

    A.R. Eynard

    1993-06-01

    Full Text Available The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an e