WorldWideScience

Sample records for situ subcellular identification

  1. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

    Science.gov (United States)

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

  2. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Science.gov (United States)

    Lake, Michael P; Bouchard, Louis-S

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  3. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Michael P Lake

    Full Text Available Transmission electron microscopy (TEM can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  4. In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

    2010-01-01

    A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

  5. In situ spatiotemporal mapping of flow fields around seeded stem cells at the subcellular length scale.

    Directory of Open Access Journals (Sweden)

    Min Jae Song

    2010-09-01

    Full Text Available A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  7. Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells

    OpenAIRE

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conj...

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

    Science.gov (United States)

    Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2016-10-01

    Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform

  10. Noninvasive identification of subcellular organization and nuclear morphology features associated with leukemic cells using light-scattering spectroscopy

    Science.gov (United States)

    Hsiao, Austin; Hunter, Martin; Greiner, Cherry; Gupta, Sharad; Georgakoudi, Irene

    2011-03-01

    Leukemia is the most common and deadly cancer among children and one of the most prevalent cancers among adults. Improvements in its diagnosis and monitoring of leukemic patients could have a significant impact in their long-term treatment. We demonstrate that light-scattering spectroscopy (LSS)-based approaches could serve as a tool to achieve this goal. Specifically, we characterize the light scattering properties of leukemic (NALM-6) cells and compare them to those of normal lymphocytes and granulocytes in the 440-710 nm range, over +/-4 deg about the exact backscattering direction. We find that the LSS spectra are well described by an inverse power-law wavelength dependence, with a power exponent insensitive to the scattering angle but significantly higher for leukemic cells than for normal leukocytes. This is consistent with differences in the subcellular morphology of these cells, detected in differential interference contrast images. Furthermore, the residual light-scattering signal, extracted after subtracting the inverse power-law fit from the data, can be analyzed assuming a Gaussian distribution of spherical scatterers using Mie theory. This analysis yields scatterer sizes that are consistent with the diameters of cell nuclei and allows the detection of the larger nuclei of NALM-6 cells compared to those of lymphocytes and granulocytes.

  11. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  12. Identification of molecular pathways facilitating glioma cell invasion in situ.

    Directory of Open Access Journals (Sweden)

    Ido Nevo

    Full Text Available Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

  13. In-situ identification of marine organisms using high frequency, wideband ultrasound

    DEFF Research Database (Denmark)

    Pham, An Hoai

    methods. Conventional acoustical methods use frequencies in the range of 10 to 500 kHz and give reasonable estimations of size distribution, if the species is known, but can only significantly support the determination of the actual species, if there are only a few known species available. It is expected...... that higher frequencies and broader bandwidths than used until now will give more information useful for fish species identification. The objective of this Ph.D. study has been to develop a method to investigate the possibility of in-situ identification of fish with high-frequency, wideband ultrasound...... and the fish bodies. The frequencies are 2, 3.5, and 6 MHz. The angles are -30°, -15°, 0°, 15°, and 30°. The results show that even though there are variations, a scan of the ultrasound backscatter along a fish of a specific species contains patterns that are characteristic for that species. This is true...

  14. Dual-Remote Raman Technology for In-Situ Identification of Tank Waste - 13549

    International Nuclear Information System (INIS)

    Bryan, Sam; Levitskaia, Tatiana; Lines, Amanda; Smith, Frannie; Josephson, Gary; Bello, Job

    2013-01-01

    A new Raman spectroscopic system for in-situ identification of the composition of solid nuclear tank waste is being developed by collaborative effort between Pacific Northwest National Laboratory (PNNL) and EIC Laboratories, Inc. The recent advancements in Raman technology allow probing the chemical composition of the tank waste without sample collection. In the newly tested configuration, the Raman probe is installed on the top of the tank riser and sends the incident laser beam to the bottom of the tank, 10 - 70 feet away. The returning light containing chemical information is collected by the Raman probe and is transmitted via fiber optic cable to the spectrometer located outside the tank farm area. This dual remote technology significantly expands currently limited options for the safe rapid in-situ identification of the solid tank waste needed for the retrieval decisions. The developed Raman system was extensively tested for acceptability prior to tank farm deployment. This testing included calibration of the system with respect of the distance between the Raman probe and the sample, incident laser beam angle, and presence of the optical interferences. The Raman system was successfully deployed on Tank C-111 at the US DOE Hanford site. As the result of this deployment, the composition of the hardpan at the bottom of C-111 tank was identified. Further development of the dual-remote Raman technology will provide a significant safety enhancement eliminating the potential of personnel radiation exposure associated with the grab sample collection and expands options of the rapid and cost-effective in-situ chemical analysis of the tank waste. (authors)

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

    Science.gov (United States)

    Xu, Zong-Chang; Kong, Yingzhen

    2017-06-20

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

  16. Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants.

    Science.gov (United States)

    Yang, Yanru; Zarda, Annatina; Zeyer, Josef

    2003-12-01

    One of the central topics in environmental bioremediation research is to identify microorganisms that are capable of degrading the contaminants of interest. Here we report application of combined microautoradiography (MAR) and fluorescence in situ hybridization (FISH). The method has previously been used in a number of systems; however, here we demonstrate its feasibility in studying the degradation of xenobiotic compounds. With a model system (coculture of Pseudomonas putida B2 and Sphingomonas stygia incubated with [14C] o-nitrophenol), combination of MAR and FISH was shown to be able to successfully identify the microorganisms degrading o-nitrophenol. Compared with the conventional techniques, MAR-FISH allows fast and accurate identification of the microorganisms involved in environmental contaminant degradation.

  17. Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

    DEFF Research Database (Denmark)

    Pinto, Rita; Carvalho, Ana S; Conze, Tim

    2012-01-01

    Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these b......Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification...... of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA......) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a) ) and Sialyl-Le(x) (SLe(x) ) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla...

  18. A drilling tool design and in situ identification of planetary regolith mechanical parameters

    Science.gov (United States)

    Zhang, Weiwei; Jiang, Shengyuan; Ji, Jie; Tang, Dewei

    2018-05-01

    The physical and mechanical properties as well as the heat flux of regolith are critical evidence in the study of planetary origin and evolution. Moreover, the mechanical properties of planetary regolith have great value for guiding future human planetary activities. For planetary subsurface exploration, an inchworm boring robot (IBR) has been proposed to penetrate the regolith, and the mechanical properties of the regolith are expected to be simultaneously investigated during the penetration process using the drilling tool on the IBR. This paper provides a preliminary study of an in situ method for measuring planetary regolith mechanical parameters using a drilling tool on a test bed. A conical-screw drilling tool was designed, and its drilling load characteristics were experimentally analyzed. Based on the drilling tool-regolith interaction model, two identification methods for determining the planetary regolith bearing and shearing parameters are proposed. The bearing and shearing parameters of lunar regolith simulant were successfully determined according to the pressure-sinkage tests and shear tests conducted on the test bed. The effects of the operating parameters on the identification results were also analyzed. The results indicate a feasible scheme for future planetary subsurface exploration.

  19. Identification of biomarkers in ductal carcinoma in situ of the breast with microinvasion

    International Nuclear Information System (INIS)

    Okumura, Yasuhiro; Iwase, Hirotaka; Yamamoto, Yutaka; Zhang, Zhenhuan; Toyama, Tatsuya; Kawasoe, Teru; Ibusuki, Mutsuko; Honda, Yumi; Iyama, Ken-ichi; Yamashita, Hiroko

    2008-01-01

    Widespread use of mammography in breast cancer screening has led to the identification of increasing numbers of patients with ductal carcinoma in situ (DCIS). DCIS of the breast with an area of focal invasion 1 mm or less in diameter is defined as DCIS with microinvasion, DCIS-Mi. Identification of biological differences between DCIS and DCIS-Mi may aid in understanding of the nature and causes of the progression of DCIS to invasiveness. In this study, using resected breast cancer tissues, we compared pure DCIS (52 cases) and DCIS-Mi (28 cases) with regard to pathological findings of intraductal lesions, biological factors, apoptosis-related protein expression, and proliferative capacity through the use of immunohistochemistry and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. There were no differences in biological factors between DCIS and DCIS-Mi, with respect to levels of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2. The frequency of necrosis and positive expression ratio of survivin and Bax were significantly higher in DCIS-Mi than in DCIS. In addition, apoptotic index, Ki-67 index, and positive Bcl-2 immunolabeling tended to be higher in DCIS-Mi than in DCIS. Multivariate analysis revealed that the presence of necrosis and positive survivin expression were independent factors associated with invasion. Compared with DCIS, DCIS-Mi is characterized by a slightly elevated cell proliferation capacity and enhanced apoptosis within the intraductal lesion, both of which are thought to promote the formation of cell necrotic foci. Furthermore, the differential expression of survivin may serve in deciding the response to therapy and may have some prognostic significance

  20. Phytoplankton Group Identification Using Simulated and In situ Hyperspectral Remote Sensing Reflectance

    Directory of Open Access Journals (Sweden)

    Hongyan Xi

    2017-08-01

    Full Text Available In the present study we investigate the bio-geo-optical boundaries for the possibility to identify dominant phytoplankton groups from hyperspectral ocean color data. A large dataset of simulated remote sensing reflectance spectra, Rrs(λ, was used. The simulation was based on measured inherent optical properties of natural water and measurements of five phytoplankton light absorption spectra representing five major phytoplankton spectral groups. These simulated data, named as C2X data, contain more than 105 different water cases, including cases typical for clearest natural waters as well as for extreme absorbing and extreme scattering waters. For the simulation the used concentrations of chlorophyll a (representing phytoplankton abundance, Chl, are ranging from 0 to 200 mg m−3, concentrations of non-algal particles, NAP, from 0 to 1,500 g m−3, and absorption coefficients of chromophoric dissolved organic matter (CDOM at 440 nm from 0 to 20 m−1. A second, independent, smaller dataset of simulated Rrs(λ used light absorption spectra of 128 cultures from six phytoplankton taxonomic groups to represent natural variability. Spectra of this test dataset are compared with spectra from the C2X data in order to evaluate to which extent the five spectral groups can be correctly identified as dominant under different optical conditions. The results showed that the identification accuracy is highly subject to the water optical conditions, i.e., contribution of and covariance in Chl, NAP, and CDOM. The identification in the simulated data is generally effective, except for waters with very low contribution by phytoplankton and for waters dominated by NAP, whereas contribution by CDOM plays only a minor role. To verify the applicability of the presented approach for natural waters, a test using in situ Rrs(λ dataset collected during a cyanobacterial bloom in Lake Taihu (China is carried out and the approach predicts blue cyanobacteria to be dominant

  1. Plasma effects on subcellular structures

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  2. A new wireless detection device for the in-situ identification of Salmonella Typhimurium

    Science.gov (United States)

    Chai, Yating; Wikle, Howard C.; Park, Mi-kyung; Horikawa, Shin; Hong, Xie; Chin, Bryan A.

    2013-05-01

    This paper presents a new device and method for the in-situ detection of Salmonella Typhimurium on tomato surfaces. This real-time in-situ detection was accomplished with phage-based magnetoelastic (ME) biosensors on fresh food surfaces. The E2 phage from a landscape phage library serves as the bio-recognition element that has the capability of binding specifically with S. Typhimurium. This mass-sensitive ME biosensor is wirelessly actuated into mechanical resonance by an externally applied time-varying magnetic field. When the biosensor binds with S. Typhimurium, the mass of the sensor increases, resulting in a decrease in the sensor's resonant frequency. Until now, ME sensors had to be collected from the tomato surface where they are exposed to S. Typhimurium and inserted into a measurement coil for the detection of the bacterium. In contrast, the newly designed test device allows the whole detection process to take place directly on the tomato. Changes in resonant frequency over time due to the accumulation of S. Typhimurium on the sensor were measured and are presented. Real-time in-situ detection of 20 minutes was achieved. In addition, this new methodology effectively decreases the measurement error and enables the simultaneous detection of multiple pathogens.

  3. In situ identification of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process.

    Science.gov (United States)

    Gagliano, Maria Cristina; Braguglia, Camilla Maria; Rossetti, Simona

    2014-09-01

    Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of thermophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  4. Chromosome identification by new molecular markers and genomic in situ hybridization in the Triticum-Secale-Thinopyrum trigeneric hybrids.

    Science.gov (United States)

    Dai, Yi; Duan, Yamei; Chi, Dawn; Liu, Huiping; Huang, Shuai; Cao, Wenguang; Gao, Yong; Fedak, George; Chen, Jianmin

    2017-08-01

    It is very important to use chromosome-specific markers for identifying alien chromosomes in advanced generations of distant hybridization. The chromosome-specific markers of rye and Thinopyrum elongatum, as well as genomic in situ hybridization, were used to identify the alien chromosomes in eight lines that were derived from the crossing between Triticum trititrigia (AABBEE) and triticale (AABBRR). The results showed that four lines contained all rye chromosomes but no Th. elongatum chromosomes. The line RE36-1 contained all of the rye chromosomes except for chromosome 2R. The lines RE33-2 and RE62-1 contained all rye chromosomes and 1E and 5E translocated chromosome, respectively. The line RE24-4 contained 12 rye chromosomes plus a 7E chromosome or 12 rye chromosomes plus one R-E translocated chromosome. Chromosome identification in the above lines was consistent using chromosome-specific markers and genomic in situ hybridization. These chromosome-specific markers provide useful tools for detecting alien chromosomes in trigeneric hybrids, and these lines could be utilized as valuable germplasm in wheat improvement.

  5. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    Directory of Open Access Journals (Sweden)

    Christy Catherine

    Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

  6. Identification of chemical processes influencing constituent mobility during in-situ uranium leaching

    International Nuclear Information System (INIS)

    Sherwood, D.R.; Hostetler, C.J.; Deutsch, W.J.

    1984-07-01

    In-situ leaching of uranium has become a widely accepted method for production of uranium concentrate from ore zones that are too small, too deep, and/or too low in grade to be mined by conventional techniques. One major environmental concern that exists with in-situ leaching of uranium is the possible adverse effects mining might have on regional ground water quality. The leaching solution (lixiviant), which extracts uranium from the ore zone, might also mobilize other potential contaminants (As, Se, Mo, and SO 4 ) associated with uranium ore. Column experiments were performed to investigate the geochemical interactions between a lixiviant and a uranium ore during in-situ leaching and to identify chemical processes that might influence contaminant mobility. The analytical composition data for selected column effluents were used with the MINTEQ code to develop a computerized geochemical model of the system. MINTEQ was used to calculate saturation indices for solid phases based on the composition of the solution. A potential constraint on uranium leaching efficiency appears to be the solubility control of schoepite. Gypsum and powellite solubilities may limit the mobilities of sulfate and molybdenum, respectively. In contrast, the mobilities of arsenic and selenium were not limited by solubility constraints, but were influenced by other chemical interaction between the solution and sediment, perhaps adsorption. Bulk chemical and mineralogical analyses were performed on both the original and leached ores. Using these analyses together with the column effluent data, mass balance calculations were performed on five constituents based on solution chemical analysis and bulk chemical and γ-spectroscopy analysis for the sediment. 6 references, 10 figures, 10 tables

  7. Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

    Science.gov (United States)

    Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

    2017-03-01

    This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

  8. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    Science.gov (United States)

    Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

  9. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    Science.gov (United States)

    Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

  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. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization.

    Science.gov (United States)

    Chen, H; Tuck-Muller, C M; Batista, D A; Wertelecki, W

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype.

  12. In situ Raman identification of corrosion products on galvanized steel sheets

    International Nuclear Information System (INIS)

    Bernard, M.C.; Hugot le Goff, A.; Massinon, D.; Phillips, N.; Thierry, D.

    1992-01-01

    In situ Raman spectroscopy was used to identify corrosion products on zinc immersed in chloride solutions. In aerated 0,03 M NaCl solution, zinc carbonate was identified as the main corrosion product. Even with higher chloride concentrations, for which zinc hydroxychloride was also detected, the carbon dioxide concentration is likely to be the rate controlling factor of the corrosion process. In a confinement experiment, Raman analysis revealed that the upper face of the sample was covered with zinc carbonate, whereas hydroxychlorides were identified on the confined face. This result confirmed the hypothesis of a differential aeration mechanism responsible for the formation of zinc hydroxychloride. This is in good agreement with Raman spectroscopy results obtained in the case of painted galvanized steel

  13. Identification of genes differentially expressed in testes containing carcinoma in situ

    DEFF Research Database (Denmark)

    Hoei-Hansen, C E; Nielsen, J E; Almstrup, K

    2004-01-01

    Virtually all testicular germ cell tumours originate from a common precursor, the carcinoma in situ (CIS) cell. The precise nature of the molecular mechanisms leading to CIS remains largely unknown. We performed the first systematic analysis of gene expression in testis with CIS compared to normal...... the novel expressed sequence tag (EST) OIC1 (Overexpressed In CIS). The genes could be grouped functionally into genes involved in cell growth, proliferation, differentiation, immunological response, and genes with unknown biological function. Examples of overexpressed genes are SFRP1 that is involved...... to testicular development (e.g. DCN, IGFBP6, SFRP1, SALL1), supporting our hypothesis that the origin of CIS is probably associated with disturbances of the fetal development of the testis....

  14. Identification of natural fractures and in situ stress at Rantau Dedap geothermal field

    Science.gov (United States)

    Artyanto, Andika; Sapiie, Benyamin; Idham Abdullah, Chalid; Permana Sidik, Ridwan

    2017-12-01

    Rantau Dedap Area is a geothermal field which is located in Great Sumatra Fault (GSF). The fault and fracture are main factor in the permeability of the geothermal system. However, not all faults and fractures have capability of to flow the fluids. Borehole image log is depiction of the borehole conditions, it is used to identify the natural fractures and drilling induced fracture. Both of them are used to identify the direction of the fracture, direction of maximum horizontal stress (SHmax), and geomechanics parameters. The natural fractures are the results of responses to stress on a rock and permeability which controlling factor in research area. Breakouts is found in this field as a trace of drilling induced fracture due to in situ stress work. Natural fractures are strongly clustered with true strike trending which first, second, and third major direction are N170°E - N180°E (N-S), N60°E - N70°E (NE-SW), and N310°E - N320°E (NW-SE), while the dominant dip is 80° -90°. Based on borehole breakout analysis, maximum horizontal stress orientation is identified in N162°E - N204°E (N-S) and N242°E (NE-SW) direction. It’s constantly similar with regional stress which is affected by GSF. Several parameters have been identified and analyzed are SHmax, SHmin, and Sy. It can be concluded that Rantau Dedap Geothermal Field is affected by strike-slip regime. The determination of in situ stress and natural fractures are important to study the pattern of permeability which is related to the fault in reservoir of this field.

  15. The Bone Black Pigment Identification by Noninvasive, In Situ Infrared Reflection Spectroscopy

    Directory of Open Access Journals (Sweden)

    Alessia Daveri

    2018-01-01

    Full Text Available Two real case studies, an oil painting on woven paper and a cycle of mural paintings, have been presented to validate the use of infrared reflection spectroscopy as suitable technique for the identification of bone black pigment. By the use of the sharp weak band at 2013 cm−1, it has been possible to distinguish animal carbon-based blacks by a noninvasive method. Finally, an attempt for an eventual assignment for the widely used sharp band at 2013 cm−1 is discussed.

  16. Identification of bacteria used for microbial enhanced oil recovery process by fluorescence in situ hybridization technique

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, K.; Tanaka, S.; Otsuka, M. [Kansai Research Institute, Kyoto (Japan). Lifescience Lab.; Yonebayashi, H. [Japan National Oil Corp., Chiba (Japan). Tech. Research Center; Enomoto, H. [Tohoku University, Sendai (Japan). Dept. of Geoscience and Tech.

    2000-01-01

    A fluorescence in situ hybridization (FISH) technique using 16S rRNA-targeted oligonucleotide probes was developed for rapid detection of microorganisms for use in the microbial enhancement of oil recovery (MEOR) process. Two microorganisms, Enterobacter cloacae TRC-322 and Bacillus licheniformis TRC-18-2-a, were selected from a collection of Enterobacter sp. and Bacillus sp. which were screened in previous studies as candidate microorganisms for injection, and were used for this experiment. Oligonucleotide probes, design based on specific sequences in the 16S rRNA gene were labeled with either fluorescein isothiocyanate (FITC), or 6-car-boxy-X-rhodamine (ROX), and were allowed to hybridize with fixed cells of the two microorganisms noted above. The fluorescence signal emitted from each microorganism cells could clearly be detected by an epifluorescence microscope. Moreover, E. cloacae TRC-322 and B, licheniformis TRC-18-2-a, suspended in actual reservoir brine, including inorganic salts, oil and aboriginal cells of the reservoir brine, could be detected directly by this hybridization method, without the need for cultivation and isolation. (author)

  17. The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification.

    Science.gov (United States)

    Swyka, Robert A; Berkowitz, David B

    2017-12-14

    The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The in situ enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol interfaces an organic reaction layer with an adjacent aqueous layer containing reporting enzymes that act upon the organic reaction product, giving rise to a spectroscopic signal. ISES allows the experimentalist to rapidly glean information on the relative rates of a set of parallel organic/organometallic reactions under investigation, without the need to quench the reactions or draw aliquots. In certain cases, the real-time enzymatic readout also provides information on sense and magnitude of enantioselectivity and substrate specificity. This article contains protocols for single-well (relative rate) and double-well (relative rate/enantiomeric excess) ISES, in addition to a colorimetric ISES protocol and a miniaturized double-well procedure. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  18. Combined fluorescent-chromogenic in situ hybridization for identification and laser microdissection of interphase chromosomes.

    Directory of Open Access Journals (Sweden)

    Nerea Paz

    Full Text Available Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.

  19. In situ identification of paper chromatogram spots by surface enhanced Raman scattering

    Energy Technology Data Exchange (ETDEWEB)

    Tran, C D

    1984-01-01

    The use of silver hydrosols to enhance the Raman scattering of paper chromatogram spots has been used successfully. This enhancement technique, which is dependent on the interaction between the substrate, silver particles, and paper fibers, has been applied to detection and identification of ng amounts of crystal violet, malachite green, and basic fuchsin with an argon laser of only 4 mW. This technique enhances the resonance of the Raman scattering so that the Raman cross sections of the spots are approximately 9 to 10 orders of magnitude higher than those observed for non-enhanced systems. The limit of detection of the techniques is defined as the amount of dye spot that yields a signal to noise ratio of 2 when excited with the 4MeV.

  20. In-situ identification of iron electrocoagulation speciation and application for natural organic matter (NOM) removal.

    Science.gov (United States)

    Dubrawski, Kristian L; Mohseni, Madjid

    2013-09-15

    In this work, iron speciation in electrocoagulation (EC) was studied to determine the impact of operating parameters on natural organic matter (NOM) removal from natural water. Two electrochemical EC parameters, current density (i) and charge loading rate (CLR), were investigated. Variation of these parameters led to a near unity current efficiency (φ = 0.957 ± 0.03), at any combination of i in a range of 1-25 mA/cm(2) and CLR in a range of 12-300 C/L/min. Higher i and CLR led to a higher bulk pH and limited the amount of dissolved oxygen (DO) reduced at the cathode surface due to mass transfer limitations. A low i (1 mA/cm(2)) and intermediate CLR (60 C/L/min) resulted in low bulk DO (<2.5 mg/L), where green rust (GR) was identified by in-situ Raman spectroscopy as the primary crystalline electrochemical product. Longer electrolysis times at higher i led to magnetite (Fe3O4) formation. Both higher (300 C/L/min) and lower (12 C/L/min) CLR values led to increased DO and/or increased pH, with lepidocrocite (γ-FeOOH) as the only crystalline species observed. The NOM removal of the three identified species was compared, with conditions leading to GR formation showing the greatest dissolved organic carbon removal, and highest removal of the low apparent molecular weight (<550 Da) chromophoric NOM fraction, determined by high performance size exclusion chromatography. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Identification of groundwater microorganisms capable of assimilating RDX-derived nitrogen during in-situ bioremediation

    International Nuclear Information System (INIS)

    Cho, Kun-Ching; Fuller, Mark E.; Hatzinger, Paul B.; Chu, Kung-Hui

    2016-01-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a nitroamine explosive, is commonly detected in groundwater at military testing and training sites. The objective of this study was to characterize the microbial community capable of using nitrogen derived from the RDX or RDX intermediates during in situ bioremediation. Active groundwater microorganisms capable of utilizing nitro-, ring- or fully-labeled "1"5N-RDX as a nitrogen source were identified using stable isotope probing (SIP) in groundwater microcosms prepared from two wells in an aquifer previously amended with cheese whey to promote RDX biodegradation. A total of fifteen 16S rRNA gene sequences, clustered in Clostridia, β-Proteobacteria, and Spirochaetes, were derived from the "1"5N-labeled DNA fractions, suggesting the presence of metabolically active bacteria capable of using RDX and/or RDX intermediates as a nitrogen source. None of the derived sequences matched RDX-degrading cultures commonly studied in the laboratory, but some of these genera have previously been linked to RDX degradation in site groundwater via "1"3C-SIP. When additional cheese whey was added to the groundwater samples, 28 sequences grouped into Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria were identified. The data suggest that numerous bacteria are capable of incorporating N from ring- and nitro-groups in RDX during anaerobic bioremediation, and that some genera may be involved in both C and N incorporation from RDX. - Highlights: • Cheese whey addition resulted in 28 different clones associated with RDX degradation. • The 28 clones belong to Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria. • SIP identified 15 clones using RDX and/or its metabolites as a nitrogen source. • The clones clustered in Clostridia, β-Proteobacteria, and Spirochaetes

  2. Identification of groundwater microorganisms capable of assimilating RDX-derived nitrogen during in-situ bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Kun-Ching [Zachry Department of Civil Engineering, Texas A& M University, College Station, TX 77843-3136 (United States); Fuller, Mark E.; Hatzinger, Paul B. [CB& I Federal Services, Lawrenceville, NJ 08648 (United States); Chu, Kung-Hui, E-mail: kchu@civil.tamu.edu [Zachry Department of Civil Engineering, Texas A& M University, College Station, TX 77843-3136 (United States)

    2016-11-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a nitroamine explosive, is commonly detected in groundwater at military testing and training sites. The objective of this study was to characterize the microbial community capable of using nitrogen derived from the RDX or RDX intermediates during in situ bioremediation. Active groundwater microorganisms capable of utilizing nitro-, ring- or fully-labeled {sup 15}N-RDX as a nitrogen source were identified using stable isotope probing (SIP) in groundwater microcosms prepared from two wells in an aquifer previously amended with cheese whey to promote RDX biodegradation. A total of fifteen 16S rRNA gene sequences, clustered in Clostridia, β-Proteobacteria, and Spirochaetes, were derived from the {sup 15}N-labeled DNA fractions, suggesting the presence of metabolically active bacteria capable of using RDX and/or RDX intermediates as a nitrogen source. None of the derived sequences matched RDX-degrading cultures commonly studied in the laboratory, but some of these genera have previously been linked to RDX degradation in site groundwater via {sup 13}C-SIP. When additional cheese whey was added to the groundwater samples, 28 sequences grouped into Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria were identified. The data suggest that numerous bacteria are capable of incorporating N from ring- and nitro-groups in RDX during anaerobic bioremediation, and that some genera may be involved in both C and N incorporation from RDX. - Highlights: • Cheese whey addition resulted in 28 different clones associated with RDX degradation. • The 28 clones belong to Bacteroidia, Bacilli, and α-, β-, and γ-Proteobacteria. • SIP identified 15 clones using RDX and/or its metabolites as a nitrogen source. • The clones clustered in Clostridia, β-Proteobacteria, and Spirochaetes.

  3. Image processing for identification and quantification of filamentous bacteria in in situ acquired images.

    Science.gov (United States)

    Dias, Philipe A; Dunkel, Thiemo; Fajado, Diego A S; Gallegos, Erika de León; Denecke, Martin; Wiedemann, Philipp; Schneider, Fabio K; Suhr, Hajo

    2016-06-11

    In the activated sludge process, problems of filamentous bulking and foaming can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy, commonly combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The in situ microscope appears as a suitable tool for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and eliminating sampling, preparation and transport of samples. In this context, a proper image processing algorithm is proposed for automated recognition and measurement of filamentous objects. This work introduces a method for real-time evaluation of images without any staining, phase-contrast or dilution techniques, differently from studies present in the literature. Moreover, we introduce an algorithm which estimates the total extended filament length based on geodesic distance calculation. For a period of twelve months, samples from an industrial activated sludge plant were weekly collected and imaged without any prior conditioning, replicating real environment conditions. Trends of filament growth rate-the most important parameter for decision making-are correctly identified. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72 % of the filaments pixels, with a false positive rate of at most 14 %. An average execution time of 0.7 s per image was achieved. Experiments have shown that the designed algorithm provided a suitable quantification of filaments when compared with human perception and standard methods. The algorithm's average execution time proved its suitability for being optimally mapped into a computational architecture to provide real-time monitoring.

  4. 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

  5. Optogenetic Tools for Subcellular Applications in Neuroscience.

    Science.gov (United States)

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

    2017-11-01

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

  6. In situ identification of CD44+/CD24- cancer cells in primary human breast carcinomas.

    Directory of Open Access Journals (Sweden)

    Giuseppe Perrone

    Full Text Available Breast cancer cells with the CD44+/CD24- phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24- cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24- population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24- cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%-21.23% of the tumour. A strong correlation was found between the percentage of CD44+/CD24- cells in primary tumours and distant metastasis development (p = 0.0001; in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively. No relationship was evident with tumour size (T and regional lymph node (N status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24- cancer cells was an independent factor related to metastasis development (p = 0.004. Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24- tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24- cell percentage.

  7. In Situ Identification of Mineral Resources with an X-Ray-Optical "Hands-Lens" Instrument

    Science.gov (United States)

    Marshall, J.; Koppel, L.; Bratton, C.; Metzger, E.; Hecht, M.

    1999-09-01

    The recognition of material resources on a planetary surface requires exploration strategies not dissimilar to those employed by early field geologists who searched for ore deposits primarily from surface clues. In order to determine the location of mineral ores or other materials, it will be necessary to characterize host terranes at regional or subregional scales. This requires geographically broad surveys in which statistically significant numbers of samples are rapidly scanned from a roving platform. To enable broad-scale, yet power-conservative planetary-surface exploration, we are developing an instrument that combines x-ray diffractometry (XRD), x-ray fluorescence spectrometry (XRF), and optical capabilities; the instrument can be deployed at the end of a rover's robotic arm, without the need for sample capture or preparation. The instrument provides XRD data for identification of mineral species and lithological types; diffractometry of minerals is conducted by ascertaining the characteristic lattice parameters or "d-spacings" of mineral compounds. D-spacings of 1.4 to 25 angstroms can be determined to include the large molecular structures of hydrated minerals such as clays. The XRF data will identify elements ranging from carbon (Atomic Number = 6) to elements as heavy as barium (Atomic Number = 56). While a sample is being x-rayed, the instrument simultaneously acquires an optical image of the sample surface at magnifications from lx to at least 50x (200x being feasible, depending on the sample surface). We believe that imaging the sample is extremely important as corroborative sample-identification data (the need for this capability having been illustrated by the experience of the Pathfinder rover). Very few geologists would rely on instrument data for sample identification without having seen the sample. Visual inspection provides critical recognition data such as texture, crystallinity, granularity, porosity, vesicularity, color, lustre, opacity, and

  8. The computerized semi-quantitative comprehensive identification-evaluation model for the large-sized in-situ leachable sandstone type uranium deposits in Northern Xinjiang, China

    Energy Technology Data Exchange (ETDEWEB)

    Zhengbang, Wang; Mingkuan, Qin; Ruiquan, Zhao; Shenghuang, Tang [Beijing Research Inst. of Uranium Geology, CNNC (China); Baoqun, Wang; Shuangxing, Lin [Geo-prospecting Team No. 216, CNNC (China)

    2001-08-01

    The process of establishment of the model includes following steps: (1) Systematically studying a known typical in-situ leachable sandstone type uranium deposit--Deposit No. 512 in Yili basin, analyzing its controlling factors and establishing its metallogenetic model; (2) Establishing the metallogenetic models of this type of uranium deposit and uranium-bearing area on the basis of comparison study on the deposit No. 512 with the same type uranium deposits in the world; (3) Creating the computerized semi-quantitative comprehensive identification-evaluation model for the large-sized in-situ leachable sandstone type uranium deposits in northern Xinjiang; (4) Determining the standards of giving a evaluation-mark for each controlling factor of in-situ leachable sandstone type uranium deposit and uranium-bearing area; (5) Evaluating uranium potential and prospect of the unknown objective target.

  9. The computerized semi-quantitative comprehensive identification-evaluation model for the large-sized in-situ leachable sandstone type uranium deposits in Northern Xinjiang, China

    International Nuclear Information System (INIS)

    Wang Zhengbang; Qin Mingkuan; Zhao Ruiquan; Tang Shenghuang; Wang Baoqun; Lin Shuangxing

    2001-01-01

    The process of establishment of the model includes following steps: (1) Systematically studying a known typical in-situ leachable sandstone type uranium deposit--Deposit No. 512 in Yili basin, analyzing its controlling factors and establishing its metallogenetic model; (2) Establishing the metallogenetic models of this type of uranium deposit and uranium-bearing area on the basis of comparison study on the deposit No. 512 with the same type uranium deposits in the world; (3) Creating the computerized semi-quantitative comprehensive identification-evaluation model for the large-sized in-situ leachable sandstone type uranium deposits in northern Xinjiang; (4) Determining the standards of giving a evaluation-mark for each controlling factor of in-situ leachable sandstone type uranium deposit and uranium-bearing area; (5) Evaluating uranium potential and prospect of the unknown objective target

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

    Science.gov (United States)

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

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

  11.   In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Dige, Irene; Kilian, Mogens; Nilsson, Holger

    2007-01-01

    Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim...

  12. Bioinformatic identification of FGF, p38-MAPK, and calcium signalling pathways associated with carcinoma in situ in the urinary bladder

    International Nuclear Information System (INIS)

    Herbsleb, Malene; Christensen, Ole F; Thykjaer, Thomas; Wiuf, Carsten; Borre, Michael; Ørntoft, Torben F; Dyrskjøt, Lars

    2008-01-01

    Carcinoma in situ (CIS) is believed to be a precursor of invasive bladder cancer. Identification of CIS is a valuable prognostic factor since radical treatment strategies can be offered these patients before the disease becomes invasive. We developed a pathway based classifier approach to predict presence or absence of CIS in patients suffering from non muscle invasive bladder cancer. From Ingenuity Pathway Analysis we considered four canonical signalling pathways (p38 MAPK, FGF, Calcium, and cAMP pathways) with most coherent expression of transcription factors (TFs) across samples in a set of twenty-eight non muscle invasive bladder carcinomas. These pathways contained twelve TFs in total. We used the expression of the TFs to predict presence or absence of CIS in a Leave-One-Out Cross Validation classification. We showed that TF expression levels in three pathways (FGF, p38 MAPK, and calcium signalling) or the expression of the twelve TFs together could be used to predict presence or absence of concomitant CIS. A cluster analysis based on expression of the twelve TFs separated the samples in two main clusters: one branch contained 11 of the 15 patients without concomitant CIS and with the majority of the genes being down regulated; the other branch contained 10 of 13 patients with concomitant CIS, and here genes were mostly up regulated. The expression in the CIS group was comparable to the expression of twenty-three patients suffering from muscle-invasive bladder carcinoma. Finally, we validated our results in an independent test set and found that prediction of CIS status was possible using TF expression of the p38 MAPK pathway. We conclude that it is possible to use pathway analysis for molecular classification of bladder tumors

  13. Identification of the Infection Source of an Outbreak of Mycobacterium Chelonae Keratitis After Laser in Situ Keratomileusis.

    Science.gov (United States)

    Nascimento, Heloisa; Viana-Niero, Cristina; Nogueira, Christiane Lourenço; Martins Bispo, Paulo José; Pinto, Fernando; de Paula Pereira Uzam, Camila; Matsumoto, Cristianne Kayoko; Oliveira Machado, Antônia Maria; Leão, Sylvia Cardoso; Höfling-Lima, Ana Luisa; de Freitas, Denise

    2018-01-01

    Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

  14. New analytical method for fast nuclide identification in mobile in-situ gamma spectrometers; Neue analytische Methode zur schnellen Nuklididentifikation in mobilen in-situ Gammaspektrometern

    Energy Technology Data Exchange (ETDEWEB)

    Streil, T.; Oeser, V.; Wagner, W. [SARAD GmbH, Dresden (Germany); Doerfel, H.R. [IDEA System GmbH, Karlsruhe (Germany)

    2016-07-01

    Demolition and accidents of nuclear reactors or terroristic attacks may lead to large-area contamination with radionuclides. A suitable mobile measurement equipment should allow a quick overview about the extent of contamination. Recent methods apply for nuclide identification either time-consuming peak-fitting methods inclusive background correction or the so-called trapezoid method determining so-called regions of interest (ROI). Since the nuclide vector is often known, this information can be used as a starting point for the nuclide identification. The presented method uses dynamic smoothing of the registered energy spectrum in accordance with the detector resolution. In this way, noise is effectively suppressed without substantial degradation of the detector resolution. The statistically prepared spectrum is then two-fold differentiated. This provides the peak positions and the two turning points of the found peaks. Nuclide identification is possible using the peak positions, and with the peak and turning point positions and the corresponding values of the spectrum, on may calculate the area of an assumed Gausz distribution without considering the in any case present continuous background. With a 2 x 2'' NaI-detector, as being used in the NucScout device, one can identify a {sup 137}Cs-activity of 200 Bq/kg at distance of 1 m in 10 s. Combined with adapted calibration methods, the algorithm for nuclide identification implemented in the NucScout is also applicable for other geometries, e. g., using a Marinelli cup in the LabScout device.

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

    African Journals Online (AJOL)

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

  16. Subcellular sites for bacterial protein export

    NARCIS (Netherlands)

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

    2004-01-01

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

  17. Subcellular sites for bacterial protein export.

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    African Journals Online (AJOL)

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

  19. In situ hybridisation for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and Mycoplasma hyorhinis in formalin-fixed porcine tissue sections

    DEFF Research Database (Denmark)

    Boye, Mette; Jensen, Tim Kåre; Ahrens, Peter

    2001-01-01

    Oligonucleotide probes targeting 16S ribosomal RNA were designed for species-specific identification of the porcine mycoplasmas Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma hyosynoviae using a fluorescent in situ hybridisation assay. The specificity of the probes was evaluated...... using pure cultures as well as porcine tissue sections with artificial presence of mycoplasma, and the probes were found specific for the target organisms. The assay was applied on sections of 28 tissue samples from pigs infected with one or more of the three Mycoplasma species as determined...

  20. Non-invasive in situ identification and band assignments of diazepam, flunitrazepam and methadone hydrochloride with FT-near-infrared spectroscopy.

    Science.gov (United States)

    Ali, Hassan Refat H

    2011-03-20

    Near-infrared spectroscopy (NIR) has evolved into an important rapid, direct and non-invasive technique in drugs analysis. In this study, the suitability of NIR spectroscopy to identify two benzodiazepine derivatives, diazepam and flunitrazepam, and a synthetic opiate, methadone hydrochloride, inside USP vials and probe the solid-state form of diazepam presents in tablets has been explored. The results show the potential of NIR spectroscopy for rapid, in situ and non-destructive identification of drugs. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Isonychia spp. and macroinvertebrate community responses to stressors in streams utilizing the benthic in situ toxicity identification evaluation (BiTIE) method

    Energy Technology Data Exchange (ETDEWEB)

    Custer, Kevin W. [Department of Earth and Environmental Sciences, Wright State University, 127 Allyn Hall, 3640 Colonel Glenn Highway, Dayton, OH 45435 (United States)], E-mail: custer.4@wright.edu; Burton, G. Allen [Department of Earth and Environmental Sciences, Wright State University, 127 Allyn Hall, 3640 Colonel Glenn Highway, Dayton, OH 45435 (United States)

    2008-01-15

    Exposures of caged organisms in situ have proven to be a useful way to improve exposure realism and link to stressor effects in aquatic assessments of hazard or risk. A novel cage system, the benthic in situ toxicity identification evaluation (BiTIE), was developed for benthic macroinvertebrates (surrogate species, resident populations and communities) to separate low and high flow effects, and major chemical classes of stressors in streams. Three resin types were used to separate the chemical stressors in the streams Honey Creek and Little Beavercreek, Ohio, USA: Dowex{sup TM} Optipore{sup TM} (non-polar organics), zeolite (ammonia), and polywool (control). Isonychia spp. sensitivity was compared to Chironomus tentans, and no significant differences were found (p > 0.05). Isonychia spp. growth (length) showed a stressor response in the zeolite treatments, and community testing revealed improved metric responses in the Dowex{sup TM} treatments. The BiTIE chamber system demonstrated stressor-response relationships using sublethal and multimetric endpoints. - Use of an indigenous aquatic insect and benthic macroinvertebrate communities allows for discerning the stressors in low and high flows with an in situ TIE approach.

  2. Isonychia spp. and macroinvertebrate community responses to stressors in streams utilizing the benthic in situ toxicity identification evaluation (BiTIE) method

    International Nuclear Information System (INIS)

    Custer, Kevin W.; Burton, G. Allen

    2008-01-01

    Exposures of caged organisms in situ have proven to be a useful way to improve exposure realism and link to stressor effects in aquatic assessments of hazard or risk. A novel cage system, the benthic in situ toxicity identification evaluation (BiTIE), was developed for benthic macroinvertebrates (surrogate species, resident populations and communities) to separate low and high flow effects, and major chemical classes of stressors in streams. Three resin types were used to separate the chemical stressors in the streams Honey Creek and Little Beavercreek, Ohio, USA: Dowex TM Optipore TM (non-polar organics), zeolite (ammonia), and polywool (control). Isonychia spp. sensitivity was compared to Chironomus tentans, and no significant differences were found (p > 0.05). Isonychia spp. growth (length) showed a stressor response in the zeolite treatments, and community testing revealed improved metric responses in the Dowex TM treatments. The BiTIE chamber system demonstrated stressor-response relationships using sublethal and multimetric endpoints. - Use of an indigenous aquatic insect and benthic macroinvertebrate communities allows for discerning the stressors in low and high flows with an in situ TIE approach

  3. Subcellular Nanoparticle Distribution from Light Transmission Spectroscopy

    Science.gov (United States)

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

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

  4. Subcellular Iron Localization Mechanisms in Plants

    Directory of Open Access Journals (Sweden)

    Emre Aksoy

    2017-12-01

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

  5. Identification of MYCN gene amplification in neuroblastoma using chromogenic in situ hybridization (CISH): an alternative and practical method.

    Science.gov (United States)

    Bhargava, Rohit; Oppenheimer, Orit; Gerald, William; Jhanwar, Suresh C; Chen, Beiyun

    2005-06-01

    Chromogenic in situ hybridization (CISH) is a recently developed technique, which utilizes the general principles of in situ hybridization and a detection system similar to immunohistochemistry. To assess the utility of CISH for analysis of MYCN gene amplification, we compared this assay with established diagnostic assays such as Southern blot analysis (SB) and fluorescent in situ hybridization (FISH). CISH was performed on 67 cases of neuroblastoma using tissue microarray (65 cases) and whole tissue sections (2 cases). Unequivocal, high-level amplification (> or =10 gene copies per tumor nucleus) was identified in 19 of 67 (28.4%) tumors. Two (3%) tumors showed low-level amplification (6-9 gene copies per tumor nucleus). No amplification was seen in 46 of 67 (68.6%) tumors. SB data were available in 44 tumors. Forty-one of the 44 tumors (93%) showed concordant results between CISH and SB. Three tumors showed MYCN amplification by CISH but no amplification by SB, most likely due to dilution effect of nonneoplastic tissue in the test samples. Two of these three tumors also showed MYCN amplification by FISH, and the third tumor was not analyzed by FISH. FISH data were available in total of 30 tumors. All 30 tumors showed concordant results between CISH and FISH for classifying a tumor as MYCN amplified or not amplified. We conclude that CISH is an accurate method for determining MYCN gene amplification, with added advantages that make it a more practically useful method.

  6. Diversity and subcellular distribution of archaeal secreted proteins.

    Science.gov (United States)

    Szabo, Zalan; Pohlschroder, Mechthild

    2012-01-01

    Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis, and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat) pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as phyla-specific pathways among the archaea. A more comprehensive understanding of the transport pathways used and post-translational modifications of secreted archaeal proteins will also facilitate the identification and heterologous expression of commercially valuable archaeal enzymes.

  7. Seismic performance evaluation of an historical concrete deck arch bridge using survey and drawing of the damages, in situ tests, dynamic identification and pushover analysis

    Science.gov (United States)

    Bergamo, Otello; Russo, Eleonora; Lodolo, Fabio

    2017-07-01

    The paper describes the performance evaluation of a retrofit historical multi-span (RC) deck arch bridge analyzed with in situ tests, dynamic identification and FEM analysis. The peculiarity of this case study lies in the structural typology of "San Felice" bridge, an historical concrete arch bridge built in the early 20th century, a quite uncommon feature in Italy. The preservation and retrofit of historic cultural heritage and infrastructures has been carefully analyzed in the international codes governing seismic response. A complete survey of the bridge was carried out prior to sketching a drawing of the existing bridge. Subsequently, the study consists in four steps: material investigation and dynamic vibration tests, FEM analysis and calibration, retrofit assessment, pushover analysis. The aim is to define an innovative approach to calibrate the FEM analysis through modern experimental investigations capable of taking structural deterioration into account, and to offer an appropriate and cost-effective retrofitting strategy.

  8. Subcellular distribution of curium in beagle liver

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  9. Small supernumerary marker chromosome derived from proximal p-arm of chromosome 2: identification by fluorescent in situ hybridization.

    Science.gov (United States)

    Lasan Trcić, Ruzica; Hitrec, Vlasta; Letica, Ljiljana; Cuk, Mario; Begović, Davor

    2003-08-01

    Conventional cytogenetics detected an interstitial deletion of proximal region of p-arm of chromosome 2 in a 6-month-old boy with a phenotype slightly resembling Down's syndrome. The deletion was inherited from the father, whose karyotype revealed a small ring-shaped marker chromosome, in addition to interstitial deletion. Fluorescence in situ hybridization identified the marker, which consisted of the proximal region of the p-arm of chromosome 2, including a part of its centromere. This case shows that molecular cytogenetic analysis can reveal the mechanism of the formation of the marker chromosome.

  10. Multi-sensor system for in situ shape monitoring and damage identification of high-speed composite rotors

    Science.gov (United States)

    Philipp, K.; Filippatos, A.; Kuschmierz, R.; Langkamp, A.; Gude, M.; Fischer, A.; Czarske, J.

    2016-08-01

    Glass fibre-reinforced polymer (GFRP) composites offer a higher stiffness-to-weight ratio than conventional rotor materials used in turbomachinery. However, the material behaviour of GFRP high-speed rotors is difficult to predict due to the complexity of the composite material and the dynamic loading conditions. Consequently dynamic expansion measurements of GRFP rotors are required in situ and with micron precision. However, the whirling motion amplitude is about two orders of magnitude higher than the desired precision. To overcome this problem, a multi-sensor system capable of separating rotor expansion and whirling motion is proposed. High measurement rates well above the rotational frequency and micron uncertainty are achieved at whirling amplitudes up to 120μm and surface velocities up to 300 m/s. The dynamic elliptical expansion of a GFRP rotor is investigated in a rotor loading test rig under vacuum conditions. In situ measurements identified not only the introduced damage but also damage initiation and propagation.

  11. Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

    Science.gov (United States)

    Syutsubo, K; Kishira, H; Harayama, S

    2001-06-01

    The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.

  12. Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

    Science.gov (United States)

    Yamane, Asaka; Fukui, Mina; Sugimura, Yoshiaki; Itoh, Miho; Alea, Mileidys Perez; Thomas, Vincent; El Alaoui, Said; Akiyama, Masashi; Hitomi, Kiyotaka

    2010-09-01

    Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

  13. In situ spectroscopic identification of neptunium(V) inner-sphere complexes on the hematite-water interface.

    Science.gov (United States)

    Müller, Katharina; Gröschel, Annett; Rossberg, André; Bok, Frank; Franzen, Carola; Brendler, Vinzenz; Foerstendorf, Harald

    2015-02-17

    Hematite plays a decisive role in regulating the mobility of contaminants in rocks and soils. The Np(V) reactions at the hematite-water interface were comprehensively investigated by a combined approach of in situ vibrational spectroscopy, X-ray absorption spectroscopy and surface complexation modeling. A variety of sorption parameters such as Np(V) concentration, pH, ionic strength, and the presence of bicarbonate was considered. Time-resolved IR spectroscopic sorption experiments at the iron oxide-water interface evidenced the formation of a single monomer Np(V) inner-sphere sorption complex. EXAFS provided complementary information on bidentate edge-sharing coordination. In the presence of atmospherically derived bicarbonate the formation of the bis-carbonato inner-sphere complex was confirmed supporting previous EXAFS findings.1 The obtained molecular structure allows more reliable surface complexation modeling of recent and future macroscopic data. Such confident modeling is mandatory for evaluating water contamination and for predicting the fate and migration of radioactive contaminants in the subsurface environment as it might occur in the vicinity of a radioactive waste repository or a reprocessing plant.

  14. Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.

    Science.gov (United States)

    Fazi, Stefano; Amalfitano, Stefano; Pizzetti, Ilaria; Pernthaler, Jakob

    2007-09-01

    We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.

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

    Science.gov (United States)

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

    2018-06-14

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

  16. Diversity and subcellular distribution of archaeal secreted proteins

    Directory of Open Access Journals (Sweden)

    Mechthild ePohlschroder

    2012-07-01

    Full Text Available Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell-wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as archaeal kingdom-specific pathways. A more comprehensive understanding of the transport pathways and post-translational modifications of secreted archaeal proteins will also generate invaluable insights that will facilitate the identification of commercially valuable archaeal enzymes and the development of heterologous systems in which to efficiently express them.

  17. Characterization of hydrometeors in Sahelian convective systems with an X-band radar and comparison with in situ measurements. Part I : Sensitivity of polarimetric radar particle identification retrieval and case study evaluation

    OpenAIRE

    Cazenave, Frédéric; Gosset, Marielle; Kacou, M.; Alcoba, M.; Fontaine, E.; Duroure, C.; Dolan, B.

    2016-01-01

    The particle identification scheme developed by Dolan and Rutledge for X-band polarimetric radar is tested for the first time in Africa and compared with in situ measurements. The data were acquired during the Megha-Tropiques mission algorithm-validation campaign that occurred in Niger in 2010. The radar classification is compared with the in situ observations gathered by an instrumented aircraft for the 13 August 2010 squall-line case. An original approach has been developed for the radar-in...

  18. Identification of Fetal Inflammatory Cells in Eosinophilic/T-cell Chorionic Vasculitis Using Fluorescent In Situ Hybridization.

    Science.gov (United States)

    Katzman, Philip J; Li, LiQiong; Wang, Nancy

    2015-01-01

    Eosinophilic/T-cell chorionic vasculitis (ETCV) is an inflammatory lesion of placental fetal vessels. In contrast to acute chorionic vasculitis, inflammation in ETCV is seen in chorionic vessel walls opposite the amnionic surface. It is not known whether inflammation in ETCV consists of maternal cells from the intervillous space or fetal cells migrating from the vessel. We used fluorescent in situ hybridization (FISH) to differentiate fetal versus maternal cells in ETCV. Placentas with ETCV, previously identified for a published study, were used. Infant sex in each case was identified using the electronic medical record. For male infants, 3-μm sections were cut from archived tissue blocks from placentas involving ETCV and stained with fluorescent X- and Y-chromosome centromeric probes. A consecutive hematoxylin/eosin-stained section was used for correlation. FISH analysis was performed on 400 interphase nuclei at the site of ETCV to determine the proportion of XX, XY, X, and Y cells. Of 31 ETCV cases, 20 were female and 10 were male (1 sex not recorded). Six of 10 cases with male infants had recuts with visible ETCV. In these 6 cases the average percentages (ranges) of XY cells, X-only cells, and Y-only cells in the region of inflammation were 81 (70-90), 11 (6-17), and 8 (2-14), respectively. There was a 2:1 female:male infant ratio in ETCV. Similar to acute chorionic vasculitis, the inflammation in ETCV is of fetal origin. It is still unknown, however, whether the stimulus for ETCV is of fetal or maternal origin.

  19. 671-nm microsystem diode laser based on portable Raman sensor device for in-situ identification of meat spoilage

    Science.gov (United States)

    Sowoidnich, Kay; Schmidt, Heinar; Schwägele, Fredi; Kronfeldt, Heinz-Detlef

    2011-05-01

    Based on a miniaturized optical bench with attached 671 nm microsystem diode laser we present a portable Raman system for the rapid in-situ characterization of meat spoilage. It consists of a handheld sensor head (dimensions: 210 x 240 x 60 mm3) for Raman signal excitation and collection including the Raman optical bench, a laser driver, and a battery pack. The backscattered Raman radiation from the sample is analyzed by means of a custom-designed miniature spectrometer (dimensions: 200 x 190 x 70 mm3) with a resolution of 8 cm-1 which is fiber-optically coupled to the sensor head. A netbook is used to control the detector and for data recording. Selected cuts from pork (musculus longissimus dorsi and ham) stored refrigerated at 5 °C were investigated in timedependent measurement series up to three weeks to assess the suitability of the system for the rapid detection of meat spoilage. Using a laser power of 100 mW at the sample meat spectra can be obtained with typical integration times of 5 - 10 seconds. The complex spectra were analyzed by the multivariate statistical tool PCA (principal components analysis) to determine the spectral changes occurring during the storage period. Additionally, the Raman data were correlated with reference analyses performed in parallel. In that way, a distinction between fresh and spoiled meat can be found in the time slot of 7 - 8 days after slaughter. The applicability of the system for the rapid spoilage detection of meat and other food products will be discussed.

  20. Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

    Science.gov (United States)

    Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

    2007-01-01

    In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

  1. Identification of Streptococcus agalactiae by fluorescent in situ hybridization compared to culturing and the determination of prevalence of Streptococcus agalactiae colonization among pregnant women in Bushehr, Iran.

    Science.gov (United States)

    Tajbakhsh, Saeed; Norouzi Esfahani, Marjan; Emaneini, Mohammad; Motamed, Niloofar; Rahmani, Elham; Gharibi, Somayyeh

    2013-09-08

    Pregnant women colonized by Streptococcus agalactiae (group B streptococci [GBS]) may transfer this microorganism to their newborns. S. agalactiae is an important cause of pneumonia, sepsis, and meningitis in newborns. Fluorescent in situ hybridization (FISH) is considered as a method of identification in the field of diagnostic microbiology. In this paper, we have designed a study to compare the DNA FISH after 7 h Lim broth enrichment and culturing for the identification of S. agalactiae and to determine the prevalence of vaginal colonization by S. agalactiae among pregnant women in Bushehr, Iran. Vaginal swab specimens were obtained from 285 pregnant women at 35 weeks or more than 35 weeks of gestation. The specimens were inoculated into Lim broth. In order to evaluate the sensitivity and specificity of GBS DNA FISH after 7 h Lim broth enrichment, the specimens were tested using both FISH and conventional culture methods. In addition, the prevalence of GBS colonization was determined. Based on the results of this study, both the sensitivity and specificity of FISH were 100%. S. agalactiae was detected by both culture and FISH in 27 of the 285 pregnant women. Thus, the prevalence of GBS colonization was 9.5%. Since short-term (7 h) Lim broth enrichment followed by FISH using oligonucleotide probes showed a high sensitivity and specificity, this protocol is therefore a highly accurate and relatively rapid method for the detection of S. agalactiae. Our analysis suggests that the use of DNA FISH to screen for S. agalactiae colonization in pregnant women may be considered in the absence of GBS culture availability.

  2. Metabolism of polybrominated diphenyl ethers and tetrabromobisphenol A by fish liver subcellular fractions in vitro.

    Science.gov (United States)

    Shen, Mengnan; Cheng, Jie; Wu, Ruohan; Zhang, Shenghu; Mao, Liang; Gao, Shixiang

    2012-06-15

    Polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA) are two major flame retardants that accumulate in fish tissues and are potentially toxic. Their debrominated and oxidated derivatives were also reported in fish tissues although the sources of theses derivatives were unidentified. Our study was to determine whether PBDEs and TBBPA could be metabolized by fish liver subcellular fractions in vitro and to identify what types of metabolites were formed. Liver microsomes and S9 fractions of crucian carp (Carassius auratus) were exposed to 4,4'-dibromodiphenyl ether (BDE 15), 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) or TBBPA solutions for 4h. Exposure of liver subcellular fractions to BDE 15 resulted in the formation of bromophenol and two monohydroxylated dibromodiphenyl ether metabolites. Neither in microsomes nor in S9 studies has revealed the presence of hydroxylated metabolites with BDE 47 exposure which indicated that the oxidation reactions in vitro were hindered by the increased number of bromine substituents on the PBDEs. TBBPA underwent an oxidative cleavage near the central carbon of the molecule, which led to the production of 2,6-dibromo-4-isopropyl-phenol and three unidentified metabolites. Another metabolite of TBBPA characterized as a hexa-brominated compound with three aromatic rings was also found in the liver subcellular fractions. These results suggest that the biotransformation of BDE 15 and TBBPA in fish liver is mediated by cytochrome P450 (CYP450) enzymes, as revealed by the formation of hydroxylated metabolites and oxidative bond cleavage products. Moreover, further studies on the identification of specific CYP450 isozymes involved in the biotransformation revealed that CYP1A was the major enzyme responsible for the biotransformation of BDE 15 and TBBPA in fish liver subcellular fractions and CYP3A4 also played a major role in metabolism of TBBPA. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Microstructure formation and in situ phase identification from undercooled Co-61.8 at.% Si melts solidified on an electromagnetic levitator and an electrostatic levitator

    Energy Technology Data Exchange (ETDEWEB)

    Li Mingjun [National Institute of Advanced Industrial Science and Technology (AIST), Materials Research Institute for Sustainable Development, 2266-98 Shimo-Shidami, Moriyama, Nagoya, Aichi 463-8560 (Japan); Japan Aerospace Exploration Agency (JAXA), Institute of Space and Astronautical Science (ISAS), Tsukuba Space Centre, ISS Science Project Office, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505 (Japan)], E-mail: li.mingjun@aist.go.jp; Nagashio, Kosuke [Japan Aerospace Exploration Agency (JAXA), Institute of Space and Astronautical Science (ISAS), Sagamihara Campus, 3-1-1 Yoshinodai, Sagamihara, Kanagawa 229-8510 (Japan); Ishikawa, Takehiko [Japan Aerospace Exploration Agency (JAXA), Institute of Space and Astronautical Science (ISAS), Tsukuba Space Centre, ISS Science Project Office, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505 (Japan); Mizuno, Akitoshi; Adachi, Masayoshi; Watanabe, Masahito [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima, Tokyo 171-8588 (Japan); Yoda, Shinichi [Japan Aerospace Exploration Agency (JAXA), Institute of Space and Astronautical Science (ISAS), Tsukuba Space Centre, ISS Science Project Office, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505 (Japan); Kuribayashi, Kazuhiko [Japan Aerospace Exploration Agency (JAXA), Institute of Space and Astronautical Science (ISAS), Sagamihara Campus, 3-1-1 Yoshinodai, Sagamihara, Kanagawa 229-8510 (Japan); Katayama, Yoshinori [Japan Atomic Energy Agency (JAEA), 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148 (Japan)

    2008-06-15

    Co-61.8 at.% Si (CoSi-CoSi{sub 2}) eutectic alloys were solidified on an electromagnetic levitator (EML) and an electrostatic levitator (ESL) at different undercooling levels. The results indicated that there is only a single recalescence event at low undercooling with the CoSi intermetallic compound as primary phase, which is independent of processing facilities, on either an EML or an ESL. The microstructure, however, is strongly dependent on the processing facility. The interior melt flow behavior in the sphere solidified at the EML differs substantially from that at the ESL, thus yielding different microstructures. On high undercooling, double recalescence takes place regardless of levitation condition. In situ X-ray diffraction of alloys solidified on the EML demonstrates that the CoSi{sub 2} compound becomes the primary phase upon the first recalescence, and the CoSi intermetallic phase crystallizes during the second recalescence. In addition to phase identification, real-time diffraction patterns can also provide additional evidence of the fragmentation of the primary phase and the ripening feature in the subsequent cooling process in the semisolid state. The phase competition between the CoSi and CoSi{sub 2} compounds is discussed when considering the nucleation barrier. The low interfacial energy of the CoSi{sub 2} phase favors a preferential nucleation event over the CoSi phase, which also plays a critical role in non-reciprocity nucleation and thus yields a double recalescence profile at high undercooling.

  4. Assessment of bioavailability of pesticides in soils and identification of pesticide degradation drivers using the in-situ Mass Distribution Quotient (iMDQ)

    Science.gov (United States)

    Folberth, Christian

    2010-05-01

    The in-situ Mass Distribution Quotient (iMDQ) has recently been shown to reliably describe the bioavailability and mineralization of the widely applied pesticide isoproturon in agricultural soils. It is determined by pore water extraction from previously incubated soil samples and subsequent assessment of the mass distribution between solid and liquid phase. The method was verified by comparing the bioavailability with co-metabolic mineralization in soils under optimum microbial soil conditions (water tension -15 kPa and bulk density 1.3 g cm-3). A comparison of the results with the chemical partitioning assessed by the Kd method has shown a higher accuracy of the new method. By combining the iMDQ/pore water extraction method with mineralization of the pesticide under optimum microbial conditions in the soils, further information about mineralization and degradation processes could be obtained or confirmed: a) Metabolically outstanding soils could be identified due to inconsistency between bioavailability and mineralization when compared to the co-metabolic soils. In a metabolically hampered soil, the mineralization was very low compared to the bioavailability and in a soil with metabolically IPU degrading microorganisms the mineralization was extremely high despite low bioavailability. b) Analysis of metabolite patterns in soil water fractions of a degradation experiment allowed for an additional identification of the metabolic status of the soil. In co-metabolic soils, the diversity of metabolites increased proportionally with the degree of mineralization of the parent compound, whereas in a metabolically hampered soil the metabolite pattern was very diverse despite low mineralization. c) A quite stable fractioning between total mineralization of the parent compound to CO2 and build-up of non-extractable bound residues was found. This is a hint that also during co-metabolic degradation that can up to now not be attributed to a certain group of microorganisms

  5. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity, and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent, subcellular site and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied 109 CdCl 2 or 112 CdSO 4 accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissues followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. No evidence was obtained for the production of volatile Cd complexes in tobacco

  6. Comparison of two threshold detection criteria methodologies for determination of probe positivity for intraoperative in situ identification of presumed abnormal 18F-FDG-avid tissue sites during radioguided oncologic surgery.

    Science.gov (United States)

    Chapman, Gregg J; Povoski, Stephen P; Hall, Nathan C; Murrey, Douglas A; Lee, Robert; Martin, Edward W

    2014-09-13

    Intraoperative in situ identification of (18)F-FDG-avid tissue sites during radioguided oncologic surgery remains a significant challenge for surgeons. The purpose of our study was to evaluate the 1.5-to-1 ratiometric threshold criteria method versus the three-sigma statistical threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ identification of presumed abnormal (18)F-FDG-avid tissue sites in a manner that was independent of the specific type of gamma detection probe used. From among 52 patients undergoing appropriate in situ evaluation of presumed abnormal (18)F-FDG-avid tissue sites during (18)F-FDG-directed surgery using 6 available gamma detection probe systems, a total of 401 intraoperative gamma detection probe measurement sets of in situ counts per second measurements were cumulatively taken. For the 401 intraoperative gamma detection probe measurement sets, probe positivity was successfully met by the 1.5-to-1 ratiometric threshold criteria method in 150/401 instances (37.4%) and by the three-sigma statistical threshold criteria method in 259/401 instances (64.6%) (P < 0.001). Likewise, the three-sigma statistical threshold criteria method detected true positive results at target-to-background ratios much lower than the 1.5-to-1 target-to-background ratio of the 1.5-to-1 ratiometric threshold criteria method. The three-sigma statistical threshold criteria method was significantly better than the 1.5-to-1 ratiometric threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ detection of presumed abnormal (18)F-FDG-avid tissue sites during radioguided oncologic surgery. This finding may be extremely important for reshaping the ongoing and future research and development of gamma detection probe systems that are necessary for optimizing the in situ detection of radioisotopes of higher-energy gamma photon emissions used during radioguided oncologic surgery.

  7. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

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

    2016-01-01

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

  8. 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.

  9. Tau regulates the subcellular localization of calmodulin

    International Nuclear Information System (INIS)

    Barreda, Elena Gomez de; Avila, Jesus

    2011-01-01

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

  10. 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.

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

    International Nuclear Information System (INIS)

    Dang Fei; Wang Wenxiong

    2010-01-01

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

  12. 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.

  13. Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging.

    Science.gov (United States)

    Comolli, L R; Kundmann, M; Downing, K H

    2006-07-01

    We illustrate the combined use of cryo-electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal-to-noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus-rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus-rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo-electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.

  14. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

    Science.gov (United States)

    Csukai, M; Mochly-Rosen, D

    1999-04-01

    Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems. Copyright 1999 The Italian Pharmacological Society.

  17. Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

    Science.gov (United States)

    Vardi, Assaf; Haramaty, Liti; Van Mooy, Benjamin A. S.; Fredricks, Helen F.; Kimmance, Susan A.; Larsen, Aud; Bidle, Kay D.

    2012-01-01

    Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host–virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the “arms race” between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi–EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host–virus interactions in natural systems. PMID:23134731

  18. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

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

  19. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

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

    2009-09-08

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

  20. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    Science.gov (United States)

    Antonacci, Giuseppe; Braakman, Sietse

    2016-11-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p biomechanics and its role in pathophysiology.

  1. Neptunium 237 behaviour in subcellular fractions of rat kidneys

    International Nuclear Information System (INIS)

    Kreslov, V.V.; Maksutova, A.Ya.; Mushkacheva, G.S.

    1978-01-01

    Subcellular distribution of intravenously injected (1 and 0.5 μCi/rat) neptunium nitrate (5- and 6-valent) in kidneys of rat males and females has been investigated. It has been shown that the radionuclide was unevenly distributed within the cell. As early as 24 hours after administration, about 50 per cent of neptunium were concentrated in the mitochondrial fraction. The data are presented on variations in neptunium behaviour within subcellular fractions of rat kidneys depending on the sex of animals, valency and dose of the isotope

  2. 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.

  3. Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

    Science.gov (United States)

    Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

    2015-11-06

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  5. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2015-01-01

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

  6. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    Science.gov (United States)

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

    2016-04-02

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

  7. Tip chip : Subcellular sampling from single cancer cells

    NARCIS (Netherlands)

    Quist, Jos; Sarajlic, Edin; Lai, Stanley C.S.; Lemay, Serge G.

    2016-01-01

    To analyze the molecular content of single cells, cell lysis is typically required, yielding a snapshot of cell behavior only. To follow complex molecular profiles over time, subcellular sampling methods potentially can be used, but to date these methods involve laborious offline analysis. Here we

  8. Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy

    International Nuclear Information System (INIS)

    Bacquart, Thomas; Deves, Guillaume; Ortega, Richard

    2010-01-01

    Identification of arsenic chemical species at a sub-cellular level is a key to understanding the mechanisms involved in arsenic toxicology and antitumor pharmacology. When performed with a microbeam, X-ray absorption near-edge structure (μ-XANES) enables the direct speciation analysis of arsenic in sub-cellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. This methodology couples tracking of cellular organelles in a single cell by confocal or epifluorescence microscopy with local analysis of chemical species by μ-XANES. Here we report the results obtained with a μ-XANES experimental setup based on Kirkpatrick-Baez X-ray focusing optics that maintains high flux of incoming radiation (>10 11 ph/s) at micrometric spatial resolution (1.5x4.0 μm 2 ). This original experimental setup enabled the direct speciation analysis of arsenic in sub-cellular organelles with a 10 -15 g detection limit. μ-XANES shows that inorganic arsenite, As(OH) 3 , is the main form of arsenic in the cytosol, nucleus, and mitochondrial network of cultured cancer cells exposed to As 2 O 3 . On the other hand, a predominance of As(III) species is observed in HepG2 cells exposed to As(OH) 3 with, in some cases, oxidation to a pentavalent form in nuclear structures of HepG2 cells. The observation of intra-nuclear mixed redox states suggests an inter-individual variability in a cell population that can only be evidenced with direct sub-cellular speciation analysis.

  9. Identification of Intermediates in Zeolite-Catalyzed Reactions Using In-situ UV/Vis Micro- Spectroscopy and a Complementary Set of Molecular Simulations

    NARCIS (Netherlands)

    Hemelsoet, K.L.J.; Qian, Q.|info:eu-repo/dai/nl/34138609X; De Meyer, T.; De Wispelaere, K.; De Sterck, B.; Weckhuysen, B.M.|info:eu-repo/dai/nl/285484397; Waroquier, M.; Van Speybroeck, V.

    2013-01-01

    The optical absorption properties of (poly)aromatic hydrocarbons occluded in a nanoporous environment were investigated by theoretical and experimental methods. The carbonaceous species are an essential part of a working catalyst for the methanol-toolefins (MTO) process. In situ UV/Vis microscopy

  10. Protein subcellular localization prediction using artificial intelligence technology.

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2008-01-01

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

  11. Subcellular distribution of styrene oxide in rat liver

    International Nuclear Information System (INIS)

    Pacifici, G.M.; Cuoci, L.; Rane, A.

    1984-01-01

    The subcellular distribution of ( 3 H)-styrene-7,8-oxide was studied in the rat liver. The compound was added to liver homogenate to give a final concentration of 2 X 10(-5); 2 X 10(-4) and 2 X 10(-3) M. Subcellular fractions were obtained by differential centrifugation. Most of styrene oxide (59-88%) was associated with the cytosolic fraction. Less than 15 percent of the compound was retrieved in each of the nuclear, mitochondrial and microsomal fractions. A considerable percentage of radioactivity was found unextractable with the organic solvents, suggesting that styrene oxide reacted with the endogenous compounds. The intracellular distribution of this epoxide was also studied in the perfused rat liver. Comparable results with those previously described were obtained. The binding of styrene oxide to the cytosolic protein was investigated by equilibrium dialysis and ultrafiltration. Only a small percentage of the compound was bound to protein

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

    Science.gov (United States)

    Wang, Xin; Komatsu, Setsuko

    2016-06-30

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

  13. 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

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

    Science.gov (United States)

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

    2011-01-01

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

  15. In situ molecular identification of the Influenza A (H1N1 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City

    Directory of Open Access Journals (Sweden)

    Ocadiz-Delgado Rodolfo

    2013-01-01

    Full Text Available Abstract Background In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City’s hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1 triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1 2009 strains is possible in archival material such as paraffin-embedded lung samples. Methods In order to detect A (H1N1 virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1 Neuraminidase (NA RNA using in situ RT-PCR. Results We detected NA transcripts in 100% of the previously diagnosed A (H1N1-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. Conclusions We have successfully amplified specific influenza A (H1N1 NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1 outbreak in Mexico City.

  16. Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures▿

    Science.gov (United States)

    Morgan, Margie; Marlowe, Elizabeth; Della-Latta, Phyllis; Salimnia, Hossein; Novak-Weekley, Susan; Wu, Fann; Crystal, Benjamin S.

    2010-01-01

    A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques. PMID:20357212

  17. Identification of lymphogranuloma venereum-associated Chlamydia trachomatis serovars by fluorescence in situ hybridisation--a proof-of-principle analysis.

    Science.gov (United States)

    Frickmann, Hagen; Essig, Andreas; Poppert, Sven

    2014-04-01

    We describe a proof-of-principle evaluation of a fluorescence in situ hybridisation (FISH) procedure to identify Chlamydia trachomatis serovars L1-L3, the causative agents of lymphogranuloma venereum, in cell cultures based on newly designed DNA probes. Rapid and easy-to-perform FISH could facilitate the diagnosis of lymphogranuloma venereum without nucleic acid amplification or serotyping, but requires broader evaluation studies, for example, in tropical high-endemicity regions. © 2014 John Wiley & Sons Ltd.

  18. In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS Genes

    Directory of Open Access Journals (Sweden)

    Csengele E. Barta

    2017-10-01

    Full Text Available Mature oak (Quercus spp. leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS from pin oak (Quercus palustris, whose full genome has not yet been sequenced.

  19. In-situ fluorimetry: A powerful non-invasive diagnostic technique for natural dyes used in artefacts. Part II. Identification of orcein and indigo in Renaissance tapestries

    Science.gov (United States)

    Clementi, C.; Miliani, C.; Romani, A.; Santamaria, U.; Morresi, F.; Mlynarska, K.; Favaro, G.

    2009-01-01

    In this paper, three Renaissance tapestries depicting scenes painted by Raffaello Sanzio, conserved at the Vatican Museum, were investigated using in-situ UV-Visible fluorimetric measurements. The results show that this technique is suitable for the detection of natural organic colorants used for dyeing the threads woven in these tapestries. The emission signals detected on red-purple colours were assigned to the colorant orcein and those on different nuances of blue and green colours to indigo by comparison with data from reference laboratory samples. The assignments were supported by chromatographic experiments carried out on threads taken from the back side of the tapestry in the same points analysed by spectrofluorimentry.

  20. Spectroscopic identification of the active site for CO oxidation on Rh/Al{sub 2}O{sub 3} by concentration modulation in situ DRIFTS

    Energy Technology Data Exchange (ETDEWEB)

    Cavers, M.; Davidson, J.M.; Harkness, I.R.; Rees, L.V.C.; McDougall, G.S.

    1999-12-10

    Diffuse reflectance infrared spectroscopy experiments are described in which the concentrations of the reactant gases passing over a solid catalyst are modulated. This simple modification to the normal in situ experiment enables direct correlation of specific surface species with the production of gaseous products. Spectra of solely the active surface intermediates with no contribution from so called spectator species are generated. For CO oxidation over a Rh/alumina catalyst at 576 K, the active CO species is identified as linearly adsorbed CO on oxidized Rh sites ({nu}{sub CO} = 2,100 cm{sup {minus}1}) on a catalyst surface apparently otherwise dominated by CO adsorbed as unreactive geminal dicarbonyl.

  1. Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers

    Directory of Open Access Journals (Sweden)

    Lawrence W. Zettler

    2012-02-01

    Full Text Available Epiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs. We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet – harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill. on a moss substrate – harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.

  2. Specific identification of human papillomavirus type in cervical smears and paraffin sections by in situ hybridization with radioactive probes: a preliminary communication

    International Nuclear Information System (INIS)

    Gupta, J.; Gendelman, H.E.; Naghashfar, Z.; Gupta, P.; Rosenshein, N.; Sawada, E.; Woodruff, J.D.; Shah, K.

    1985-01-01

    Cervical Papanicolaou smears and paraffin sections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using 35 S-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two 35 S-labeled nucleotides. Paraffin sections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material

  3. Interphase fluorescence in situ hybridization in multiple myeloma and monoclonal gammopathy of undetermined significance without and with positive plasma cell identification

    DEFF Research Database (Denmark)

    Christensen, Jacob H; Abildgaard, Niels; Plesner, Torben

    2007-01-01

    identification (PC-ID+); 134 were investigated at diagnosis, 32 at time of progression, 7 at time of relapse, and 9 were investigated with partial remission or no response. The FISH analysis detected 11q23 (n = 61), 13q13 approximately q14 (n = 181), 14q32 (n = 121), 17p13.1 (n = 181), t(4;14) (n = 76), and t(11...

  4. Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.

    Science.gov (United States)

    Lemos, Vanessa; Graczyk, Thaddeus K; Alves, Margarida; Lobo, Maria Luísa; Sousa, Maria C; Antunes, Francisco; Matos, Olga

    2005-12-01

    In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

  5. Subcellular localization of ammonium transporters in Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Davis Carter T

    2008-12-01

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

  6. Dynamic neuroanatomy at subcellular resolution in the zebrafish.

    Science.gov (United States)

    Faucherre, Adèle; López-Schier, Hernán

    2014-01-01

    Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

  7. 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.

  8. Identification of intermediates in zeolite-catalyzed reactions by in situ UV/Vis microspectroscopy and a complementary set of molecular simulations.

    Science.gov (United States)

    Hemelsoet, Karen; Qian, Qingyun; De Meyer, Thierry; De Wispelaere, Kristof; De Sterck, Bart; Weckhuysen, Bert M; Waroquier, Michel; Van Speybroeck, Veronique

    2013-12-02

    The optical absorption properties of (poly)aromatic hydrocarbons occluded in a nanoporous environment were investigated by theoretical and experimental methods. The carbonaceous species are an essential part of a working catalyst for the methanol-to-olefins (MTO) process. In situ UV/Vis microscopy measurements on methanol conversion over the acidic solid catalysts H-SAPO-34 and H-SSZ-13 revealed the growth of various broad absorption bands around 400, 480, and 580 nm. The cationic nature of the involved species was determined by interaction of ammonia with the methanol-treated samples. To determine which organic species contribute to the various bands, a systematic series of aromatics was analyzed by means of time-dependent density functional theory (TDDFT) calculations. Static gas-phase simulations revealed the influence of structurally different hydrocarbons on the absorption spectra, whereas the influence of the zeolitic framework was examined by using supramolecular models within a quantum mechanics/molecular mechanics framework. To fully understand the origin of the main absorption peaks, a molecular dynamics (MD) study on the organic species trapped in the inorganic host was essential. During such simulation the flexibility is fully taken into account and the effect on the UV/Vis spectra is determined by performing TDDFT calculations on various snapshots of the MD run. This procedure allows an energy absorption scale to be provided and the various absorption bands determined from in situ UV/Vis spectra to be assigned to structurally different species. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues (Conference Presentation)

    Science.gov (United States)

    Parker, Lindsay M.; Staikopoulos, Vicky; Cordina, Nicole M.; Sayyadi, Nima; Hutchinson, Mark R.; Packer, Nicolle H.

    2016-03-01

    Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.

  10. Y-chromosome status identification suggests a recipient origin of posttransplant non-small cell lung carcinomas: chromogenic in situ hybridization analysis.

    Science.gov (United States)

    Chen, Wei; Brodsky, Sergey V; Zhao, Weiqiang; Otterson, Gregory A; Villalona-Calero, Miguel; Satoskar, Anjali A; Hasan, Ayesha; Pelletier, Ronald; Ivanov, Iouri; Ross, Patrick; Nadasdy, Tibor; Shilo, Konstantin

    2014-05-01

    Owing to the need of lifelong immunosuppression, solid-organ transplant recipients are known to have an increased risk of posttransplant malignancies including lung cancer. Posttransplant neoplastic transformation of donor-derived cells giving rise to hematopoietic malignancies, Kaposi sarcoma, and basal cell carcinoma in nongraft tissues has been reported. The goal of this study was to assess the cell origin (donor versus recipient derived) of posttransplant non-small cell lung carcinomas (NSCLCs) in kidney and heart transplant recipients. An institutional database search identified 2557 kidney and heart transplant recipients in 8 consecutive years. Among this cohort, 20 (0.8%) renal and 18 (0.7%) heart transplant recipients developed NSCLC. The study cohort comprised 6 of 38 NSCLCs arising in donor-recipient sex-mismatched transplant patients. The tumor cell origin was evaluated by chromogenic in situ hybridization with Y-chromosome probe on formalin-fixed, paraffin-embedded tissues. Y-chromosome was identified in 97% ± 1% (range from 92% to 99%) of all types of nucleated cells in male control tissues. In all 5 NSCLCs from male recipients of female donor organ, Y-chromosome was identified in 97% ± 2% (range from 92% to 100%) of tumor cells, statistically equivalent to normal control (P recipient of male kidney. These findings suggest a recipient derivation of NSCLC arising in kidney and heart transplant recipients. A combination of histologic evaluation and chromogenic in situ hybridization with Y-chromosome analysis allows reliable determination of tissue origin in sex-mismatched solid-organ transplant recipients and may aid in management of posttransplant malignancy in such cases. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. HONO and Inorganic Fine Particle Composition in Typical Monsoon Region with Intensive Anthropogenic Emission: In-situ Observations and Source Identification.

    Science.gov (United States)

    Xie, Y.; Nie, W.; Ding, A.; Huang, X.

    2015-12-01

    Yangtze River Delta (YRD) is one of the most typical monsoon area with probably the most largest population intensity in the world. With sharply economic development and the large anthropogenic emissions, fine particle pollution have been one of the major air quality problem and may further have impact on the climate system. Though a lot of control policy (sulfur emission have been decreasing from 2007) have been conducted in the region, studies showed the sulfate in fine particles still take major fraction as the nitrate from nitrogen oxides increased significantly. In this study, the role of inorganic chemical compositions in fine particles was investigated with two years in-situ observation. Sulfate and Nitrate contribute to fine particle mass equally in general, but sulfate contributes more during summer and nitrate played more important role in winter. Using lagrangian dispersion backward modeling and source contribution clustering method, the impact of airmass coming from different source region (industrial, dust, biogenic emissions, etc) on fine particle inorganic compositions were discussed. Furthermore, we found two unique cases showing in-situ implications for sulfate formation by nitrogen dioxide oxidation mechanisms. It was showed that the mixing of anthropogenic pollutants with long-range transported mineral dust and biomass burning plume would enhance the sulfate formation by different chemistry mechanisms. This study focus on the complex aspects of fine particle formation in airmasses from different source regions: . It highlights the effect of NOx in enhancing the atmospheric oxidization capacity and indicates a potentially very important impact of increasing NOx on air pollution formation and regional climate change in East Asia.

  12. Retention and subcellular distribution of 67Ga in normal organs

    International Nuclear Information System (INIS)

    Ando, A.; Ando, I.; Hiraki, T.

    1986-01-01

    Using normal rats, retention values and subcellular distribution of 67 Ga in each organ were investigated. At 10 min after administration of 67 Ga-citrate the retention value of 67 Ga in blood was 6.77% dose/g, and this value decreased with time. The values for skeletal muscle, lung, pancreas, adrenal, heart muscle, brain, small intestine, large intestine and spinal cord were the highest at 10 min after administration, and they decreased with time. Conversely this value in bone increased until 10 days after injection. But in the liver, kidney, and stomach, these values increased with time after administration and were highest 24 h or 48 h after injection. After that, they decreased with time. The value in spleen reached a plateau 48 h after administration, and hardly varied for 10 days. From the results of subcellular fractionation, it was deduced that lysosome plays quite an important role in the concentration of 67 Ga in small intestine, stomach, lung, kidney and pancreas; a lesser role in its concentration in heart muscle, and hardly any role in the 67 Ga accumulation in skeletal muscle. In spleen, the contents in nuclear, mitochrondrial, microsomal, and supernatant fractions all contributed to the accumulation of 67 Ga. (orig.) [de

  13. Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

    Science.gov (United States)

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

    2013-01-01

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

  14. Gene ontology based transfer learning for protein subcellular localization

    Directory of Open Access Journals (Sweden)

    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. Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

    Science.gov (United States)

    Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

    2005-08-01

    In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

  16. Exploiting external reflection FTIR spectroscopy for the in-situ identification of pigments and binders in illuminated manuscripts. Brochantite and posnjakite as a case study

    Science.gov (United States)

    Zaffino, Chiara; Guglielmi, Vittoria; Faraone, Silvio; Vinaccia, Alessandro; Bruni, Silvia

    2015-02-01

    In the present work, the use of portable instrumentation allowing in-situ reflection FTIR analyses is exploited to identify the coloring matters of northern-Italian illuminations dating to the XVI century. In order to build a database of spectra, reference paint samples were prepared spreading the pigments on parchment with two different binders, i.e. gum arabic and egg white, used in antiquity. Pigments for the database were chosen considering their use in the Middle Ages and in the Renaissance and their response in the mid- and near-IR region. The reflection FTIR spectra obtained resulted to be dominated by the specular reflection component, allowing the use of the Kramers-Kronig transform to convert them to the more conventional absorbance FTIR spectra. Several pigments could thus be identified in ancient illuminations, even if some green details showed a spectral pattern different with respect to the most common commercial green pigments of the database. Therefore, in addition, basic copper sulfates brochantite and posnjakite were synthesized and characterized. In three green details, posnjakite was identified, both as a pure compound and together with malachite.

  17. Utility and impact of early t(15;17) identification by Fluorescence In Situ Hybridization (FISH) in clinical decision making for patients in Acute Promyelocytic Leukemia (APL).

    Science.gov (United States)

    Kolhe, R; Mangaonkar, A; Mansour, J; Clemmons, A; Shaw, J; Dupont, B; Walczak, L; Mondal, A; Rojiani, A; Jillella, A; Kota, V

    2015-08-01

    Acute Promyelocytic Leukemia (APL) is a curable malignancy with studies showing above 90% survival. However, population-based studies looking at survival suggest that approximately 30% of patients with APL die during induction. Early demonstration of t(15;17) will lead to accurate decision making regarding treatment. The aim of this project was to validate earlier time frames for the Abbott Molecular Vysis LSI promyelocytic leukemia (PML)/ retinoic acid receptor alpha (RARA) fluorescence in situ hybridization (FISH) probe (ASR 6-16 h). Twenty patients (15 APL cases and five non-APL cases) were selected for validating various hybridization times for the FISH probe. Expected normal signal pattern was two red and two green signals (2R2G), and the most common expected abnormal signal pattern was two fusion (yellow) signals, one red and one green (2F1R1G) and/or one fusion, one red and one green (1F1R1G). The specificity of the probe ranged from 84% at 2 h, 86% at 4 h, 84% at 6 h, and 87% for overnight hybridization. The sensitivity increased from 79% at 2 h, 80% at 4 h, 81% at 6 h to 87% for overnight hybridization. Based on the validation studies, we recommend reading of FISH results at the 4-h incubation mark for a preliminary diagnosis and confirmation with overnight hybridization. © 2015 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2008-01-01

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

  19. Dosimetric characterization and identification of TL defect centres in sand for its application in sludge irradiators as an in situ dosimeter

    International Nuclear Information System (INIS)

    Benny, P.G.; Shah, M.R.; Sabharwal, S.; Bhatt, B.C.; Gundu Rao, T.K.

    2003-08-01

    The report presents investigations that have been carried out to establish a method for the routine/ periodic dosimetry for Sludge Hygienisation Research Irradiator (SHRI) facility set up at Baroda (India) for disinfection of liquid sewage sludge in bulk quantity. For this purpose, the possibility of using the sand, one of the components of inorganic matter found in sewage sludge, directly as a TL dosimeter has been explored. The report is presented in two parts. Part 1 presents dosimetric characterization of sand for its application as an in situ dosimeter. A review on various sewage sludge irradiators operating in the world and the different dosimetric techniques used for these facilities are briefly described. In the present studies, in order to investigate the thermoluminescence properties of sand, it was separated from the sewage sludge by an extensive cleaning procedure. Part 1 also describes the procedure for separation of sand from sewage sludge, study on its TL properties, dosimetric characterization of sand and application of cleaned sand collected at the outlet of the SHRI facility for estimating radiation absorbed dose imparted to the sludge during its disinfection as well as for determining distribution of dose for an irradiated sludge batch. A new method by using phototransferred thermoluminescence (PTTL) in quartz separated from sand has been explored for high-level gamma dosimetry. Part 2 of the report presents sensitization properties of TL peaks and study of TL defect centres in quartz. It describes the results on pre-dose sensitization of 220 degC and 110 degC TL peaks in the quartz samples separated from sand. From the TL and ESR studies, a mechanism for TL sensitization has been suggested, which involves the role of competing traps and E I - centres in the sensitization process. The paramagnetic radicals formed in quartz samples after gamma irradiation by using ESR technique have been briefly described. (author)

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

    International Nuclear Information System (INIS)

    Neumann, M.; Gabel, D.

    2000-01-01

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

  1. Subcellular distribution of glutathione and cysteine in cyanobacteria.

    Science.gov (United States)

    Zechmann, Bernd; Tomasić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-10-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.

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

    Directory of Open Access Journals (Sweden)

    Flavia Letícia Martins Peçanha

    2017-07-01

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

  3. 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.

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

    Science.gov (United States)

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

    2017-08-22

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

  5. Investigation of the subcellular architecture of L7 neurons of Aplysia californica using magnetic resonance microscopy (MRM) at 7.8 microns.

    Science.gov (United States)

    Lee, Choong H; Flint, Jeremy J; Hansen, Brian; Blackband, Stephen J

    2015-06-10

    Magnetic resonance microscopy (MRM) is a non-invasive diagnostic tool which is well-suited to directly resolve cellular structures in ex vivo and in vitro tissues without use of exogenous contrast agents. Recent advances in its capability to visualize mammalian cellular structure in intact tissues have reinvigorated analytical interest in aquatic cell models whose previous findings warrant up-to-date validation of subcellular components. Even if the sensitivity of MRM is less than other microscopic technologies, its strength lies in that it relies on the same image contrast mechanisms as clinical MRI which make it a unique tool for improving our ability to interpret human diagnostic imaging through high resolution studies of well-controlled biological model systems. Here, we investigate the subcellular MR signal characteristics of isolated cells of Aplysia californica at an in-plane resolution of 7.8 μm. In addition, direct correlation and positive identification of subcellular architecture in the cells is achieved through well-established histology. We hope this methodology will serve as the groundwork for studying pathophysiological changes through perturbation studies and allow for development of disease-specific cellular modeling tools. Such an approach promises to reveal the MR contrast changes underlying cellular mechanisms in various human diseases, for example in ischemic stroke.

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

    Science.gov (United States)

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

    2003-01-01

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

  7. Rational Design of Semiconductor Nanostructures for Functional Subcellular Interfaces.

    Science.gov (United States)

    Parameswaran, Ramya; Tian, Bozhi

    2018-05-15

    One of the fundamental questions guiding research in the biological sciences is how cellular systems process complex physical and environmental cues and communicate with each other across multiple length scales. Importantly, aberrant signal processing in these systems can lead to diseases that can have devastating impacts on human lives. Biophysical studies in the past several decades have demonstrated that cells can respond to not only biochemical cues but also mechanical and electrical ones. Thus, the development of new materials that can both sense and modulate all of these pathways is necessary. Semiconducting nanostructures are an emerging class of discovery platforms and tools that can push the limits of our ability to modulate and sense biological behaviors for both fundamental research and clinical applications. These materials are of particular interest for interfacing with cellular systems due to their matched dimension with subcellular components (e.g., cytoskeletal filaments), and easily tunable properties in the electrical, optical and mechanical regimes. Rational design via traditional or new approaches, such as nanocasting and mesoscale chemical lithography, can allow us to control micro- and nanoscale features in nanowires to achieve new biointerfaces. Both processes endogenous to the target cell and properties of the material surface dictate the character of these interfaces. In this Account, we focus on (1) approaches for the rational design of semiconducting nanowires that exhibit unique structures for biointerfaces, (2) recent fundamental discoveries that yield robust biointerfaces at the subcellular level, (3) intracellular electrical and mechanical sensing, and (4) modulation of cellular behaviors through material topography and remote physical stimuli. In the first section, we discuss new approaches for the synthetic control of micro- and nanoscale features of these materials. In the second section, we focus on achieving biointerfaces with

  8. Expression and subcellular localization of ORC1 in Leishmania major

    International Nuclear Information System (INIS)

    Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

    2008-01-01

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

  9. Copper and zinc contamination in oysters: subcellular distribution and detoxification.

    Science.gov (United States)

    Wang, Wen-Xiong; Yang, Yubo; Guo, Xiaoyu; He, Mei; Guo, Feng; Ke, Caihuan

    2011-08-01

    Metal pollution levels in estuarine and coastal environments have been widely reported, but few documented reports exist of severe contamination in specific environments. Here, we report on a metal-contaminated estuary in Fujian Province, China, in which blue oysters (Crassostrea hongkongensis) and green oysters (Crassostrea angulata) were discovered to be contaminated with Cu and other metals. Extraordinarily high metal concentrations were found in the oysters collected from the estuary. Comparison with historical data suggests that the estuary has recently been contaminated with Cr, Cu, Ni, and Zn. Metal concentrations in blue oysters were as high as 1.4 and 2.4% of whole-body tissue dry wt for Cu and Zn, respectively. Cellular debris was the main subcellular fraction binding the metals, but metal-rich granules were important for Cr, Ni, and Pb. With increasing Cu accumulation, its partitioning into the cytosolic proteins decreased. In contrast, metallothionein-like proteins increased their importance in binding with Zn as tissue concentrations of Zn increased. In the most severely contaminated oysters, only a negligible fraction of their Cu and Zn was bound with the metal-sensitive fraction, which may explain the survival of oysters in such contaminated environments. Copyright © 2011 SETAC.

  10. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

    KAUST Repository

    Serag, Maged F.

    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.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  12. 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.

  13. In Situ Analysis of Martian Regolith with the SAM Experiment During the First Mars Year of the MSL Mission: Identification of Organic Molecules by Gas Chromatography from Laboratory Measurements

    Science.gov (United States)

    Millan, M.; Szopa, C.; Buch, A.; Coll, P.; Glavin, D. P.; Freissinet, C.; Navarro-Gonzalez, R.; Francois, P.; Coscia, D.; Bonnet, J. Y.; hide

    2016-01-01

    The Sample Analysis at Mars (SAM) instrument onboard the Curiosity rover, is specifically designed for in situ molecular and isotopic analyses of martian surface materials and atmosphere. It contributes to the Mars Science Laboratory (MSL) missions primary scientific goal to characterize the potential past, present or future habitability of Mars. In all of the analyses of solid samples delivered to SAM so far, chlorinated organic compounds have been detected above instrument background levels and identified by gas chromatography coupled to mass spectrometry (GC-MS) (Freissinet et al., 2015; Glavin et al., 2013). While some of these may originate from reactions between oxychlorines and terrestrial organic carbon present in the instrument background (Glavin et al., 2013), others have been demonstrated to originate from indigenous organic carbon present in samples (Freissinet et al., 2015). We present here laboratory calibrations that focused on the analyses performed with the MXT-CLP GC column (SAM GC-5 channel) used for nearly all of the GC-MS analyses of the martian soil samples carried out with SAM to date. Complementary to the mass spectrometric data, gas chromatography allows us to separate and identify the species analyzable in a nominal SAM-GC run time of about 21 min. To characterize the analytical capabilities of this channel within the SAM Flight Model (FM) operating conditions on Mars, and their implications on the detection of organic matter, it is required to perform laboratory experimental tests and calibrations on spare model components. This work assesses the SAM flight GC-5 column efficiency, confirms the identification of the molecules based on their retention time, and enables a better understanding of the behavior of the SAM injection trap (IT) and its release of organic molecules. This work will enable further optimization of the SAM-GC runs for additional samples to be analyzed during the MSL mission.

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

    Directory of Open Access Journals (Sweden)

    Thomas DY

    2006-04-01

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

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

    Science.gov (United States)

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

    2015-12-07

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

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

    Science.gov (United States)

    Wang, Xiao; Li, Guo-Zheng

    2013-03-12

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

  17. Predicting protein subcellular locations using hierarchical ensemble of Bayesian classifiers based on Markov chains

    Directory of Open Access Journals (Sweden)

    Eils Roland

    2006-06-01

    Full Text Available Abstract Background The subcellular location of a protein is closely related to its function. It would be worthwhile to develop a method to predict the subcellular location for a given protein when only the amino acid sequence of the protein is known. Although many efforts have been made to predict subcellular location from sequence information only, there is the need for further research to improve the accuracy of prediction. Results A novel method called HensBC is introduced to predict protein subcellular location. HensBC is a recursive algorithm which constructs a hierarchical ensemble of classifiers. The classifiers used are Bayesian classifiers based on Markov chain models. We tested our method on six various datasets; among them are Gram-negative bacteria dataset, data for discriminating outer membrane proteins and apoptosis proteins dataset. We observed that our method can predict the subcellular location with high accuracy. Another advantage of the proposed method is that it can improve the accuracy of the prediction of some classes with few sequences in training and is therefore useful for datasets with imbalanced distribution of classes. Conclusion This study introduces an algorithm which uses only the primary sequence of a protein to predict its subcellular location. The proposed recursive scheme represents an interesting methodology for learning and combining classifiers. The method is computationally efficient and competitive with the previously reported approaches in terms of prediction accuracies as empirical results indicate. The code for the software is available upon request.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-12-15

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

  20. Sub-cellular distribution and translocation of TRP channels.

    Science.gov (United States)

    Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

    2011-01-01

    Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

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

    Directory of Open Access Journals (Sweden)

    Al Mehedi Hasan

    2017-07-01

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

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

    Science.gov (United States)

    Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2009-07-21

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

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

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2016-12-01

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

  6. The incorporation of labelled amino acids into the subcellular fractions of the rabbit brain

    International Nuclear Information System (INIS)

    Ogrodnik, W.

    1980-01-01

    Radioactive amino acids were injected into the fourth ventriculum of adult rabbits. After 3, 6 and 13 hours the animals were killed and tissue subcellular fractions were prepared from their brains. Nucleic acids were extracted and quantitatively determined from nucleic, myelin, mitochondrial, microsomal and cytoplasmic fractions. The radioactivity was determined in the protein and nucleic acid fractions. It was found out that the incorporation of radioactive amino acids increased in relation to time. In the analyzed subcellular fractions a very rapid incorporation of glutamic acid and leucine into cytoplasmic proteins was observed. The chromatographic analysis of the nucleic acids showed that radioactivity in the nucleic acid fractions depended on a radioactive protein contamination. Radioactive aminoacyl-tRNA was not found in the nucleic acid fractions, extracted from different subcellular fractions. (author)

  7. 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

  8. 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

  9. Subcellular Redox Targeting: Bridging in Vitro and in Vivo Chemical Biology.

    Science.gov (United States)

    Long, Marcus J C; Poganik, Jesse R; Ghosh, Souradyuti; Aye, Yimon

    2017-03-17

    Networks of redox sensor proteins within discrete microdomains regulate the flow of redox signaling. Yet, the inherent reactivity of redox signals complicates the study of specific redox events and pathways by traditional methods. Herein, we review designer chemistries capable of measuring flux and/or mimicking subcellular redox signaling at the cellular and organismal level. Such efforts have begun to decipher the logic underlying organelle-, site-, and target-specific redox signaling in vitro and in vivo. These data highlight chemical biology as a perfect gateway to interrogate how nature choreographs subcellular redox chemistry to drive precision redox biology.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wester, P O

    1964-12-15

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

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

    International Nuclear Information System (INIS)

    Wester, P.O.

    1964-12-01

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

  12. Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

    Science.gov (United States)

    Noe, BD; Baste, CA; Bauer, GE

    1977-01-01

    Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level. PMID:328517

  13. Femtosecond laser nanosurgery of sub-cellular structures in HeLa cells by employing Third Harmonic Generation imaging modality as diagnostic tool.

    Science.gov (United States)

    Tserevelakis, George J; Psycharakis, Stylianos; Resan, Bojan; Brunner, Felix; Gavgiotaki, Evagelia; Weingarten, Kurt; Filippidis, George

    2012-02-01

    Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relatively new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the surrounding regions. The targeted structures have to be stained in order to be clearly visualized for the nanosurgery procedure. However, the validation of the final nanosurgery result is difficult, since the targeted structure could be simply photobleached rather than selectively destroyed. This fact comprises a main drawback of this technique. In our study we employed a multimodal system which integrates non-linear imaging modalities with nanosurgery capabilities, for the selective disruption of sub-cellular structures in HeLa cancer cells. Third Harmonic Generation (THG) imaging modality was used as a tool for the identification of structures that were subjected to nanosurgery experiments. No staining of the biological samples was required, since THG is an intrinsic property of matter. Furthermore, cells' viability after nanosurgery processing was verified via Two Photon Excitation Fluorescence (TPEF) measurements. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 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

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

    NARCIS (Netherlands)

    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,

  16. 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.

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

    International Nuclear Information System (INIS)

    Winter, R.

    1980-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  19. 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.

  20. 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.

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

    Science.gov (United States)

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

    2018-01-25

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

  2. 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 w...

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

  5. Precise Photodynamic Therapy of Cancer via Subcellular Dynamic Tracing of Dual-loaded Upconversion Nanophotosensitizers

    NARCIS (Netherlands)

    Chang, Y.; Li, X.; Zhang, L.; Xia, L.; Liu, Xiaomin; Li, C.; Zhang, Y.; Tu, L.; Xue, B.; Zhao, H.; Zhang, H.; Kong, X.

    2017-01-01

    Recent advances in upconversion nanophotosensitizers (UCNPs-PS) excited by near-infrared (NIR) light have led to substantial progress in improving photodynamic therapy (PDT) of cancer. For a successful PDT, subcellular organelles are promising therapeutic targets for reaching a satisfactory

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

    International Nuclear Information System (INIS)

    Kamunde, Collins; MacPhail, Ruth

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-15

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

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

    International Nuclear Information System (INIS)

    Babahosseini, H.; Carmichael, B.; Strobl, J.S.; Mahmoodi, S.N.; Agah, M.

    2015-01-01

    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

  9. 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.

  10. 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.

  11. Identification of microstructures

    International Nuclear Information System (INIS)

    Padilha, A.F.; Ambrozio Filho, F.

    1984-01-01

    The identification of phases in a material can require the utilization of several techniques. The most used technique and discussed are: optical microscope, scanning electron microscope, transmission electron microscope, X-ray diffraction and 'in-situ' chemical analysis of the phases. The microstructures were classified, in according to the size and phase volumetric fraction, in four types. For each type the most appropriate techniques for identifying the phases are discussed. (E.G.) [pt

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

    Science.gov (United States)

    2010-01-01

    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 (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene. 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

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

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Wang Yuanchang

    1990-11-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  15. 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.

  16. Differential subcellular distribution of ion channels and the diversity of neuronal function.

    Science.gov (United States)

    Nusser, Zoltan

    2012-06-01

    Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Subhash, Chandra

    2008-01-01

    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.

  18. 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.

  19. 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

    2014-01-01

    In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator...... of sarcoplasmic reticulum Ca(2+) release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca(2+) ([Ca(2+)]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz...... in tetanic [Ca(2+)]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca(2+)]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca(2+) release critically depends on energy supply from...

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

    Directory of Open Access Journals (Sweden)

    Maritess Gay Asumen

    2010-07-01

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

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

    International Nuclear Information System (INIS)

    Golnik, N.; Zielczynski, M.

    1999-01-01

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

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

    International Nuclear Information System (INIS)

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

    1992-01-01

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

  3. Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

    OpenAIRE

    Zinser, E; Paltauf, F; Daum, G

    1993-01-01

    Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergostero...

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

    Directory of Open Access Journals (Sweden)

    King Brian R

    2012-07-01

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

  5. Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

    Science.gov (United States)

    Kostal, Vratislav; Arriaga, Edgar A.

    2011-01-01

    Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

  6. Accumulation, subcellular distribution and toxicity of inorganic mercury and methylmercury in marine phytoplankton

    Energy Technology Data Exchange (ETDEWEB)

    Wu Yun [Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2011-10-15

    We examined the accumulation, subcellular distribution, and toxicity of Hg(II) and MeHg in three marine phytoplankton (the diatom Thalassiosira pseudonana, the green alga Chlorella autotrophica, and the flagellate Isochrysis galbana). For MeHg, the inter-species toxic difference could be best interpreted by the total cellular or intracellular accumulation. For Hg(II), both I. galbana and T. pseudonana exhibited similar sensitivity, but they each accumulated a different level of Hg(II). A higher percentage of Hg(II) was bound to the cellular debris fraction in T. pseudonana than in I. galbana, implying that the cellular debris may play an important role in Hg(II) detoxification. Furthermore, heat-stable proteins were a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). Elucidating the different subcellular fates of Hg(II) and MeHg may help us understand their toxicity in marine phytoplankton at the bottom of aquatic food chains. - Highlights: > The inter-species toxic difference of methylmercury in marine phytoplankton can be explained by its total cellular or intracellular accumulation. > The inter-species toxic difference of inorganic mercury in marine phytoplankton can be explained by its subcellular distribution. > Heat-stable protein was a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). - The inter-species difference in methylmercury and inorganic mercury toxicity in phytoplankton can be explained by cellular accumulation and subcellular distribution.

  7. Accumulation, subcellular distribution and toxicity of inorganic mercury and methylmercury in marine phytoplankton

    International Nuclear Information System (INIS)

    Wu Yun; Wang Wenxiong

    2011-01-01

    We examined the accumulation, subcellular distribution, and toxicity of Hg(II) and MeHg in three marine phytoplankton (the diatom Thalassiosira pseudonana, the green alga Chlorella autotrophica, and the flagellate Isochrysis galbana). For MeHg, the inter-species toxic difference could be best interpreted by the total cellular or intracellular accumulation. For Hg(II), both I. galbana and T. pseudonana exhibited similar sensitivity, but they each accumulated a different level of Hg(II). A higher percentage of Hg(II) was bound to the cellular debris fraction in T. pseudonana than in I. galbana, implying that the cellular debris may play an important role in Hg(II) detoxification. Furthermore, heat-stable proteins were a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). Elucidating the different subcellular fates of Hg(II) and MeHg may help us understand their toxicity in marine phytoplankton at the bottom of aquatic food chains. - Highlights: → The inter-species toxic difference of methylmercury in marine phytoplankton can be explained by its total cellular or intracellular accumulation. → The inter-species toxic difference of inorganic mercury in marine phytoplankton can be explained by its subcellular distribution. → Heat-stable protein was a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). - The inter-species difference in methylmercury and inorganic mercury toxicity in phytoplankton can be explained by cellular accumulation and subcellular distribution.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  9. Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

    International Nuclear Information System (INIS)

    King, B.F.; Somani, S.M.

    1987-01-01

    The distribution of 3 H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3 H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3 H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27,474 +/- 2825 DPM/gm at 60 min (P < .05). The cytosol contains the highest RA: 223,341 +/- 21,044 DPM/gm at 30 min which declined to 53,475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of the organelle. 21 references, 1 figure, 2 tables

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-02

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

  12. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

    Science.gov (United States)

    Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F

    2014-03-01

    Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

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

    Science.gov (United States)

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    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 used 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 to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. © 2016 Neve et al.; Published by Cold Spring Harbor Laboratory Press.

  14. 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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  16. High Accumulation and Subcellular Distribution of Thallium in Green Cabbage (Brassica Oleracea L. Var. Capitata L.).

    Science.gov (United States)

    Ning, Zengping; He, Libin; Xiao, Tangfu; Márton, László

    2015-01-01

    The accumulation of thallium (Tl) in brassicaceous crops is widely known, but both the uptake extents of Tl by the individual cultivars of green cabbage and the distribution of Tl in the tissues of green cabbage are not well understood. Five commonly available cultivars of green cabbage grown in the Tl-spiked pot-culture trials were studied for the uptake extent and subcellular distribution of Tl. The results showed that all the trial cultivars mainly concentrated Tl in the leaves (101∼192 mg/kg, DW) rather than in the roots or stems, with no significant differences among cultivars (p = 0.455). Tl accumulation in the leaves revealed obvious subcellular fractionation: cell cytosol and vacuole > cell wall > cell organelles. The majority (∼ 88%) of leaf-Tl was found to be in the fraction of cytosol and vacuole, which also served as the major storage site for other major elements such as Ca and Mg. This specific subcellular fractionation of Tl appeared to enable green cabbage to avoid Tl damage to its vital organelles and to help green cabbage tolerate and detoxify Tl. This study demonstrated that all the five green cabbage cultivars show a good application potential in the phytoremediation of Tl-contaminated soils.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xia eGuo

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-01

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

  20. Identification of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in Blood Cultures: a Multicenter Performance Evaluation of a Three-Color Peptide Nucleic Acid Fluorescence In Situ Hybridization Assay▿

    Science.gov (United States)

    Della-Latta, Phyllis; Salimnia, Hossein; Painter, Theresa; Wu, Fann; Procop, Gary W.; Wilson, Deborah A.; Gillespie, Wendy; Mender, Alayna; Crystal, Benjamin S.

    2011-01-01

    A multicenter evaluation was undertaken to evaluate the performance of a new three-color peptide nucleic acid fluorescence in situ hybridization assay that identifies isolates directly from blood cultures positive for Gram-negative bacilli (GNB). The assay correctly identified 100% (186/186) of the Escherichia coli isolates, 99.1% (109/110) of the Klebsiella pneumoniae isolates, and 95.8% (46/48) of the Pseudomonas aeruginosa isolates in this study. Negative assay results were correctly obtained for 162 of 165 other GNB (specificity, 98.2%). PMID:21490185

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

    Directory of Open Access Journals (Sweden)

    Nagarajan eParamasivam

    2011-11-01

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

  2. Savannah River Site Experiences in In Situ Field Measurements of Radioactive Materials

    International Nuclear Information System (INIS)

    Moore, F.S.

    1999-01-01

    This paper discusses some of the field gamma-ray measurements made at the Savannah River Site, the equipment used for the measurements, and lessons learned during in situ identification and characterization of radioactive materials

  3. Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct.

    Science.gov (United States)

    Orihuela, Pedro A; Zuñiga, Lidia M; Rios, Mariana; Parada-Bustamante, Alexis; Sierralta, Walter D; Velásquez, Luis A; Croxatto, Horacio B

    2009-11-30

    Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Bo Fu

    2015-12-01

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

  6. Metabolic Interplay between Peroxisomes and Other Subcellular Organelles Including Mitochondria and the Endoplasmic Reticulum

    Science.gov (United States)

    Wanders, Ronald J. A.; Waterham, Hans R.; Ferdinandusse, Sacha

    2016-01-01

    Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD+, which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved. PMID:26858947

  7. Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

    Science.gov (United States)

    Khimchenko, Anna; Bikis, Christos; Schweighauser, Gabriel; Hench, Jürgen; Joita-Pacureanu, Alexandra-Teodora; Thalmann, Peter; Deyhle, Hans; Osmani, Bekim; Chicherova, Natalia; Hieber, Simone E.; Cloetens, Peter; Müller-Gerbl, Magdalena; Schulz, Georg; Müller, Bert

    2017-09-01

    Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  10. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Science.gov (United States)

    Chandra, Subhash

    2008-12-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  11. 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...

  12. Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)

    Science.gov (United States)

    Wallace, W.G.; Luoma, S.N.

    2003-01-01

    This paper examines how the subcellular partitioning of Cd and Zn in the bivalves Macoma balthica and Potamocorbula amurensis may affect the trophic transfer of metal to predators. Results show that the partitioning of metals to organelles, 'enzymes' and metallothioneins (MT) comprise a subcellular compartment containing trophically available metal (TAM; i.e. metal trophically available to predators), and that because this partitioning varies with species, animal size and metal, TAM is similarly influenced. Clams from San Francisco Bay, California, were exposed for 14 d to 3.5 ??g 1-1 Cd and 20.5 ??g 1-1 Zn, including 109Cd and 65Zn as radiotracers, and were used in feeding experiments with grass shrimp Palaemon macrodatylus, or used to investigate the subcellular partitioning of metal. Grass shrimp fed Cd-contaminated P. amurensis absorbed ???60% of ingested Cd, which was in accordance with the partitioning of Cd to the bivalve's TAM compartment (i.e. Cd associated with organelles, 'enzymes' and MT); a similar relationship was found in previous studies with grass shrimp fed Cd-contaminated oligochaetes. Thus, TAM may be used as a tool to predict the trophic transfer of at least Cd. Subcellular fractionation revealed that ???34% of both the Cd and Zn accumulated by M. balthica was associated with TAM, while partitioning to TAM in P. amurensis was metal-dependent (???60% for TAM-Cd%, ???73% for TAM-Zn%). The greater TAM-Cd% of P. amurensis than M. balthica is due to preferential binding of Cd to MT and 'enzymes', while enhanced TAM-Zn% of P. amurensis results from a greater binding of Zn to organelles. TAM for most species-metal combinations was size-dependent, decreasing with increased clam size. Based on field data, it is estimated that of the 2 bivalves, P. amurensis poses the greater threat of Cd exposure to predators because of higher tissue concentrations and greater partitioning as TAM; exposure of Zn to predators would be similar between these species.

  13. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    International Nuclear Information System (INIS)

    Chandra, Subhash

    2008-01-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2 + ) revealed local secondary ion signal enhancements correlated with the water image signals of 19 (H 3 O) + . A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  14. Subcellular SIMS imaging of gadolinium isotopes in human glioblastoma cells treated with a gadolinium containing MRI agent

    Science.gov (United States)

    Smith, Duane R.; Lorey, Daniel R.; Chandra, Subhash

    2004-06-01

    Neutron capture therapy is an experimental binary radiotherapeutic modality for the treatment of brain tumors such as glioblastoma multiforme. Recently, neutron capture therapy with gadolinium-157 has gained attention, and techniques for studying the subcellular distribution of gadolinium-157 are needed. In this preliminary study, we have been able to image the subcellular distribution of gadolinium-157, as well as the other six naturally abundant isotopes of gadolinium, with SIMS ion microscopy. T98G human glioblastoma cells were treated for 24 h with 25 mg/ml of the metal ion complex diethylenetriaminepentaacetic acid Gd(III) dihydrogen salt hydrate (Gd-DTPA). Gd-DTPA is a contrast enhancing agent used for MRI of brain tumors, blood-brain barrier impairment, diseases of the central nervous system, etc. A highly heterogeneous subcellular distribution was observed for gadolinium-157. The nuclei in each cell were distinctly lower in gadolinium-157 than in the cytoplasm. Even within the cytoplasm the gadolinium-157 was heterogeneously distributed. The other six naturally abundant isotopes of gadolinium were imaged from the same cells and exhibited a subcellular distribution consistent with that observed for gadolinium-157. These observations indicate that SIMS ion microscopy may be a viable approach for subcellular studies of gadolinium containing neutron capture therapy drugs and may even play a major role in the development and validation of new gadolinium contrast enhancing agents for diagnostic MRI applications.

  15. 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.

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

    Science.gov (United States)

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

    2017-12-01

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

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

    International Nuclear Information System (INIS)

    Campo, S.; Nicolau, G.; Pellizari, E.; Rivarola, M.A.

    1977-01-01

    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.) [de

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

    International Nuclear Information System (INIS)

    Bamunusinghe, Devinka; Hemenway, Cynthia L.; Nelson, Richard S.; Sanderfoot, Anton A.; Ye, Chang M.; Silva, Muniwarage A.T.; Payton, M.; Verchot-Lubicz, Jeanmarie

    2009-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  20. Prediction of protein subcellular locations by GO-FunD-PseAA predictor.

    Science.gov (United States)

    Chou, Kuo-Chen; Cai, Yu-Dong

    2004-08-06

    The localization of a protein in a cell is closely correlated with its biological function. With the explosion of protein sequences entering into DataBanks, it is highly desired to develop an automated method that can fast identify their subcellular location. This will expedite the annotation process, providing timely useful information for both basic research and industrial application. In view of this, a powerful predictor has been developed by hybridizing the gene ontology approach [Nat. Genet. 25 (2000) 25], functional domain composition approach [J. Biol. Chem. 277 (2002) 45765], and the pseudo-amino acid composition approach [Proteins Struct. Funct. Genet. 43 (2001) 246; Erratum: ibid. 44 (2001) 60]. As a showcase, the recently constructed dataset [Bioinformatics 19 (2003) 1656] was used for demonstration. The dataset contains 7589 proteins classified into 12 subcellular locations: chloroplast, cytoplasmic, cytoskeleton, endoplasmic reticulum, extracellular, Golgi apparatus, lysosomal, mitochondrial, nuclear, peroxisomal, plasma membrane, and vacuolar. The overall success rate of prediction obtained by the jackknife cross-validation was 92%. This is so far the highest success rate performed on this dataset by following an objective and rigorous cross-validation procedure.

  1. Uptake and disposition of mirex in hepatocytes and subcellular fractions in CD1 mouse liver

    International Nuclear Information System (INIS)

    Charles, A.K.; Rosenbaum, D.P.; Ashok, L.; Abraham, R.

    1985-01-01

    In vivo uptake and disposition of [ 14 C]mirex by CD1 mouse liver subcellular fractions and cells of different nuclear ploidy were examined following single or multiple doses of mirex injected intraperitoneally. Significant amounts of mirex were rapidly taken up by liver (21-29%), suggesting that liver is one of the primary sites of accumulation of the chemical. Among subcellular fractions, mirex was predominantly distributed in mitochondria and microsomes in the irreversibly bound form (about 20%), although its levels fluctuated considerably with time. Mirex was completely dissociated with trichloroacetic acid treatment from both nuclear and plasma membrane fractions, although the total uptake by these fractions was markedly high. The time course of uptake and concentration-dependent disposition of mirex revealed that polyploid hepatocytes selectively accumulated higher amounts of the chemical (two to three times) compared to diploid hepatocytes. The increased affinity of polyploid cells to mirex may indicate a greater susceptibility of this cell type to the chemical insult and also may suggest a possible early involvement of polyploids in the tumorigenic process in rodent livers

  2. Mapping the subcellular distribution of biomolecules at the ultrastructural level by ion microscopy.

    Science.gov (United States)

    Galle, P; Escaig, F; Dantin, F; Zhang, L

    1996-05-01

    Analytical ion microscopy, a method proposed and developed in 1960 by Casting and Slodzian at the Orsay University (France), makes it possible to obtain easily and rapidly analytical images representing the distribution in a tissue section of elements or isotopes (beginning from the three isotopes of hydrogen until to transuranic elements), even when these elements or isotopes are at a trace concentration of 1 ppm or less. This method has been applied to study the subcellular distribution of different varieties of biomolecules. The subcellular location of these molecules can be easily determined when the molecules contain in their structures a specific atom such as fluorine, iodine, bromine or platinum, what is the case of many pharmaceutical drugs. In this situation, the distribution of these specific atoms can be considered as representative of the distribution of the corresponding molecule. In other cases, the molecules must be labelled with an isotope which may be either radioactive or stable. Recent developments in ion microscopy allow the obtention of their chemical images at ultra structural level. In this paper we present the results obtained with the prototype of a new Scanning Ion Microscope used for the study of the intracellular distribution of different varieties of molecules: glucocorticoids, estrogens, pharmaceutical drugs and pyrimidine analogues.

  3. Multi-label learning with fuzzy hypergraph regularization for protein subcellular location prediction.

    Science.gov (United States)

    Chen, Jing; Tang, Yuan Yan; Chen, C L Philip; Fang, Bin; Lin, Yuewei; Shang, Zhaowei

    2014-12-01

    Protein subcellular location prediction aims to predict the location where a protein resides within a cell using computational methods. Considering the main limitations of the existing methods, we propose a hierarchical multi-label learning model FHML for both single-location proteins and multi-location proteins. The latent concepts are extracted through feature space decomposition and label space decomposition under the nonnegative data factorization framework. The extracted latent concepts are used as the codebook to indirectly connect the protein features to their annotations. We construct dual fuzzy hypergraphs to capture the intrinsic high-order relations embedded in not only feature space, but also label space. Finally, the subcellular location annotation information is propagated from the labeled proteins to the unlabeled proteins by performing dual fuzzy hypergraph Laplacian regularization. The experimental results on the six protein benchmark datasets demonstrate the superiority of our proposed method by comparing it with the state-of-the-art methods, and illustrate the benefit of exploiting both feature correlations and label correlations.

  4. HPSLPred: An Ensemble Multi-Label Classifier for Human Protein Subcellular Location Prediction with Imbalanced Source.

    Science.gov (United States)

    Wan, Shixiang; Duan, Yucong; Zou, Quan

    2017-09-01

    Predicting the subcellular localization of proteins is an important and challenging problem. Traditional experimental approaches are often expensive and time-consuming. Consequently, a growing number of research efforts employ a series of machine learning approaches to predict the subcellular location of proteins. There are two main challenges among the state-of-the-art prediction methods. First, most of the existing techniques are designed to deal with multi-class rather than multi-label classification, which ignores connections between multiple labels. In reality, multiple locations of particular proteins imply that there are vital and unique biological significances that deserve special focus and cannot be ignored. Second, techniques for handling imbalanced data in multi-label classification problems are necessary, but never employed. For solving these two issues, we have developed an ensemble multi-label classifier called HPSLPred, which can be applied for multi-label classification with an imbalanced protein source. For convenience, a user-friendly webserver has been established at http://server.malab.cn/HPSLPred. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fu Xiaoping; Dou Changming [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China); Chen Yingxu, E-mail: yingxu_chen@hotmail.com [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China); Chen Xincai; Shi Jiyan; Yu Mingge; Xu Jie [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China)

    2011-02-15

    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-H{sub 2}O 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. Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins

    Directory of Open Access Journals (Sweden)

    Marie-Hélène Avelange-Macherel

    2018-05-01

    Full Text Available Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement, we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.

  7. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

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

    Science.gov (United States)

    2012-01-01

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

  9. 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.

  10. Incoordination among Subcellular Compartments Is Associated with Depression-Like Behavior Induced by Chronic Mild Stress

    Science.gov (United States)

    Xu, Aiping; Cui, Shan

    2016-01-01

    Background: Major depressive disorder is characterized as persistent low mood. A chronically stressful life in genetically susceptible individuals is presumably the major etiology that leads to dysfunctions of monoamine and hypothalamus-pituitary-adrenal axis. These pathogenic factors cause neuron atrophy in the limbic system for major depressive disorder. Cell-specific pathophysiology is unclear, so we investigated prelimbic cortical GABAergic neurons and their interaction with glutamatergic neurons in depression-like mice. Methods: Mice were treated with chronic unpredictable mild stress for 3 weeks until they expressed depression-like behaviors confirmed by sucrose preference, Y-maze, and forced swimming tests. The structures and functions of GABAergic and glutamatergic units in prelimbic cortices were studied by cell imaging and electrophysiology in chronic unpredictable mild stress-induced depression mice vs controls. Results: In depression-like mice, prelimbic cortical GABAergic neurons show incoordination among the subcellular compartments, such as decreased excitability and synaptic outputs as well as increased reception from excitatory inputs. GABAergic synapses on glutamatergic cells demonstrate decreased presynaptic innervation and increased postsynaptic responsiveness. Conclusions: Chronic unpredictable mild stress-induced incoordination in prelimbic cortical GABAergic and glutamatergic neurons dysregulates their target neurons, which may be the pathological basis for depressive mood. The rebalance of compatibility among subcellular compartments would be an ideal strategy to treat neural disorders. PMID:26506857

  11. 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.

  12. The role of water flow into subcellular organella in cell death

    International Nuclear Information System (INIS)

    Chiba-Kamoshida, Kaori

    2008-01-01

    Mitochondrion is a subcellular organella producing most of the energy necessary for living cells. The structure consisting of double membrane, inner and outer membranes, has a close relationship with activity and diseases. Its accurate regulation of the membrane permeability plays an important role in the homeostatic energy production. Abnormal membrane permeability has a potential to lead to cell depth. Although, even transportation of water molecule is regulated by a specific membrane protein, aquapoline, there has not been reported any method to monitor the water flow through the membrane. Neutron small-angle scattering allows us to perform measurements with biological materials and subcellular organella such as mitochondria in solution under the experimental condition maintaining the activity of the biological samples. Outstanding advantage of neutron spectroscopy is its ability to distinguish hydrogen spread over biomolecules from deuterium. In order to explore a new method to monitor conformational change inside mitochondria, wide-range neutron small angle scattering data introducing two neutron spectrometers in JAEA JRR-3, SANS-J and PNO covering not only the size for the thickness of the double membrane but also that for isolated whole mitochondria particle, ∼1 μm was employed. Utilizing the excess protein content, 70%, in the inner membrane of mitochondria, a new attempt was began to figure out the structure change in inner membrane caused by the change such as in oxygen and in the substrate concentration, and to examine the relationship between the structure change and water flow through the mitochondria membrane. (author)

  13. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Exposure to cadmium

    International Nuclear Information System (INIS)

    Wang Mengjiao; Wang Wenxiong

    2011-01-01

    The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd 2+ ], 0.01-84 nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd 2+ ] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd 2+ ] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC 2 being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs' detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd 2+ ] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.

  14. Cadmium Disrupts Subcellular Organelles, Including Chloroplasts, Resulting in Melatonin Induction in Plants

    Directory of Open Access Journals (Sweden)

    Hyoung-Yool Lee

    2017-10-01

    Full Text Available Cadmium is a well-known elicitor of melatonin synthesis in plants, including rice. However, the mechanisms by which cadmium induces melatonin induction remain elusive. To investigate whether cadmium influences physical integrities in subcellular organelles, we treated tobacco leaves with either CdCl2 or AlCl3 and monitored the structures of subcellular organelles—such as chloroplasts, mitochondria, and the endoplasmic reticulum (ER—using confocal microscopic analysis. Unlike AlCl3 treatment, CdCl2 (0.5 mM treatment significantly disrupted chloroplasts, mitochondria, and ER. In theory, the disruption of chloroplasts enabled chloroplast-expressed serotonin N-acetyltransferase (SNAT to encounter serotonin in the cytoplasm, leading to the synthesis of N-acetylserotonin followed by melatonin synthesis. In fact, the disruption of chloroplasts by cadmium, not by aluminum, gave rise to a huge induction of melatonin in rice leaves, which suggests that cadmium-treated chloroplast disruption plays an important role in inducing melatonin in plants by removing physical barriers, such as chloroplast double membranes, allowing SNAT to gain access to the serotonin substrate enriched in the cytoplasm.

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

    Science.gov (United States)

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

    2017-12-15

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

  16. Organ accumulation and subcellular location of Cicer arietinum ST1 protein.

    Science.gov (United States)

    Albornos, Lucía; Cabrera, Javier; Hernández-Nistal, Josefina; Martín, Ignacio; Labrador, Emilia; Dopico, Berta

    2014-07-01

    The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. STUDY OF SUBCELLULAR DISTRIBUTION OF CRYSTALLINE MESO-TETRA(3-PYRIDYLBACTERIOCHLORIN NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Yu. S. Maklygina

    2016-01-01

    Full Text Available The results of the study of subcellular distribution of molecular meso-tetra(3-pyridylbacteriochlorin nanocrystals proposed as therapeutic agents for photodynamic therapy are represented in the article. Investigations and measurement of spectroscopic properties of molecular crystals of near-infrared photosensitizer were conducted using special device complex based on fiber-optic spectrometer. Investigation and analysis of the pattern of subcellular accumulation of meso-tetra(3-pyridylbacteriochlorin in molecular (dimethyl sulfoxide (DMSO as solvent and nanocrystalline forms on different cell lines: human monocytes (THP-1, human cervical cancer cells (HeLa and mouse malignant brain tumor cells (glioma C6. The dynamics of subcellylar accumulation of the agent at concentration of 5 and 10 mg/l was assessed with laser microscope-spectrum analyzer and by confocal microscopy. The study showed that in the course of interaction with cell lines molecular nanocrystals of the agent developed ability to fluorescence. Hence, in the cellular environment meso-tetra(3-pyridyl bacteriochlorin nanoparticles became phototoxic giving opportunities for their use for fluorescence diagnosis and photodynamic therapy. Specific role of meso-tetra(3-pyridylbacteriochlorin in the range of photosensitizers is determined by its spectral characteristics, i.e. absorption and fluorescence in near-infrared band, which allows measuring and affecting on deeper layers of biotissue. Thus, the use of meso-tetra(3-pyridylbacteriochlorin nanoparticles as nanophotosensitizers may improve the efficacy of diagnosis and treatment of deep-seated tumors.

  18. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: Subcellular distribution, chemical forms and thiol pools

    International Nuclear Information System (INIS)

    Weng Bosen; Xie Xiangyu; Weiss, Dominik J.; Liu Jingchun; Lu Haoliang; Yan Chongling

    2012-01-01

    Highlights: ► Cadmium tolerance mechanisms of Kandelia obovata was investigated systematacially. ► Thiol pool can play roles in cadmium detoxification mechanisms. ► Increasing cadmium treatment strength caused proportional increase of cadmium uptake. ► More than half of cadmium was localized in cell walls, and lowest in membranes. ► Sodium chloride and acetic acid extractable fractions were dominant. - Abstract: In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  19. Subcellular distribution of zinc in the benign and malignant human prostate

    International Nuclear Information System (INIS)

    Leake, A.; Chrisholm, G.D.; Busuttil, A.; Habib, F.K

    1984-01-01

    The subcellular distribution of zinc and its interaction with androgens has been examined in the benign and malignant human prostate. Endogenously, most of the zinc was associated with the nuclear fraction but signigicant concentrations were also found in the cytosol. Furthermore, the epithelium contained more zinc than that found in either the stroma or the intact gland. Zinc concentrations were lower in the subcellular fractions of the cancerous tissue when compared to hyperplastic specimens. In vitro uptake of zinc into prostatic homogenates was rapid and at equilibrium the binding was stable for both the 4degC and the 37degC incubations. At low zinc concentrations (<5mM) the uptake was higher in the nucleus, whereas at higher concentraions, the cancerous tissue exhibited a greater capacity for the metal which was predominantly retained by the cytosol. Our data suggest the presence of a saturable zinc retention mechanism in the nucleus. The zinc uptake was found to be independent of any added androgen. In contrast, the total androgen uptake by the prostate was significantly enhanced by the addition of zinc. This effect was not due to increases in the nuclear and cytosolic receptor binding since zinc inhibited the binding of the androgen to these receptors. (author)

  20. In-Situ Simulation

    DEFF Research Database (Denmark)

    Bjerregaard, Anders Thais; Slot, Susanne; Paltved, Charlotte

    2015-01-01

    , and organisational characteristic. Therefore, it might fail to fully mimic real clinical team processes. Though research on in situ simulation in healthcare is in its infancy, literature is abundant on patient safety and team training1. Patient safety reporting systems that identify risks to patients can improve......Introduction: In situ simulation offers on-site training to healthcare professionals. It refers to a training strategy where simulation technology is integrated into the clinical encounter. Training in the simulation laboratory does not easily tap into situational resources, e.g. individual, team...... patient safety if coupled with training and organisational support. This study explored the use of critical incidents and adverse events reports for in situ simulation and short-term observations were used to create learning objectives and training scenarios. Method: This study used an interventional case...

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

    International Nuclear Information System (INIS)

    Sappal, Ravinder; Burka, John; Dawson, Susan; Kamunde, Collins

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sappal, Ravinder; Burka, John; Dawson, Susan [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Kamunde, Collins [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada)], E-mail: ckamunde@upei.ca

    2009-03-09

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-07

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

  5. In situ groundwater bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Hazen, Terry C.

    2009-02-01

    In situ groundwater bioremediation of hydrocarbons has been used for more than 40 years. Most strategies involve biostimulation; however, recently bioaugmentation have been used for dehalorespiration. Aquifer and contaminant profiles are critical to determining the feasibility and strategy for in situ groundwater bioremediation. Hydraulic conductivity and redox conditions, including concentrations of terminal electron acceptors are critical to determine the feasibility and strategy for potential bioremediation applications. Conceptual models followed by characterization and subsequent numerical models are critical for efficient and cost effective bioremediation. Critical research needs in this area include better modeling and integration of remediation strategies with natural attenuation.

  6. Sex in situ

    DEFF Research Database (Denmark)

    Krøgholt, Ida

    2017-01-01

    Sex er en del af vores sociale praksis og centralt for det, vi hver især er. Men bortset fra pornoindustrien, har vi ikke mange muligheder for at få adgang til billeder af sex. Teater Nordkrafts forestilling Sex in situ vil gøre seksuelle billeder til noget, der kan deles, udveksles og tales om, og...

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

    Science.gov (United States)

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

    2009-06-01

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

  8. Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

    Directory of Open Access Journals (Sweden)

    Sierralta Walter

    2009-01-01

    Full Text Available Abstract Background Mating changes the mode of action of 17beta-estradiol (E2 to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER are required. This change was designated as intracellular path shifting (IPS. Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta in oviductal epithelial cells of rats on day 1 of cycle (C1 or pregnancy (P1 using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb and calbindin 9 kDa (s100g in rats on C1 or P1 treated with selective agonists for ESR1 (PPT or ESR2 (DPN. The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

  9. 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.

  10. Changes in subcellular elemental distributions accompanying the acrosome reaction in sea urchin sperm

    International Nuclear Information System (INIS)

    Cantino, M.E.; Schackmann, R.W.; Johnson, D.E.

    1983-01-01

    Energy-dispersive x-ray microanalysis was used to analyze changes in the subcellular distributions of Na, Mg, P, S, Cl, K, and Ca associated with the acrosome reaction of sea urchin sperm. Within 5 sec after induction of the acrosome reaction, nuclear Na and mitochondrial Ca increased and nuclear and mitochondrial K decreased. Uptake of mitochondrial P was detected after several minutes, and increases in nuclear Mg were detected only after 5-10 min of incubation following induction of the reaction. The results suggest that sudden permeability changes in the sperm plasma membrane are associated with the acrosome reaction, but that complete breakdown of membrane and cell function does not occur for several minutes

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    International Nuclear Information System (INIS)

    Pasantes-Morales, H.; Ademe, R.M.; Lopez-Colome, A.M.

    1979-01-01

    The effect of taurine on 45 Ca 2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45 Ca 2+ 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 45 Ca 2+ uptake; in the crude synaptosomal fraction, taurine reduced 45 Ca 2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45 Ca 2+ 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.)

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Ørtenblad, Niels

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

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

  18. CellMap visualizes protein-protein interactions and subcellular localization

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

    Pärnamaa, Tanel; Parts, Leopold

    2017-05-05

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  1. 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.

  2. Subcellular redistribution of trimeric G-proteins – potential mechanism of desensitization of hormone response: internalisation, solubilization, down-regulation

    Czech Academy of Sciences Publication Activity Database

    Drastichová, Zdeňka; Bouřová, Lenka; Lisý, Václav; Hejnová, L.; Rudajev, Vladimír; Stöhr, Jiří; Durchánková, Dana; Ostašov, Pavel; Teisinger, Jan; Soukup, Tomáš; Novotný, Jiří; Svoboda, Petr

    2008-01-01

    Roč. 57, Suppl.3 (2008), S1-S10 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LC554; GA ČR(CZ) GA309/06/0121 Institutional research plan: CEZ:AV0Z50110509 Keywords : brain * subcellular fractionation * trimeric G-proteins Subject RIV: CE - Biochemistry Impact factor: 1.653, year: 2008

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthe...

  5. Evaluation on subcellular partitioning and biodynamics of pulse copper toxicity in tilapia reveals impacts of a major environmental disturbance.

    Science.gov (United States)

    Ju, Yun-Ru; Yang, Ying-Fei; Tsai, Jeng-Wei; Cheng, Yi-Hsien; Chen, Wei-Yu; Liao, Chung-Min

    2017-07-01

    Fluctuation exposure of trace metal copper (Cu) is ubiquitous in aquatic environments. The purpose of this study was to investigate the impacts of chronically pulsed exposure on biodynamics and subcellular partitioning of Cu in freshwater tilapia (Oreochromis mossambicus). Long-term 28-day pulsed Cu exposure experiments were performed to explore subcellular partitioning and toxicokinetics/toxicodynamics of Cu in tilapia. Subcellular partitioning linking with a metal influx scheme was used to estimate detoxification and elimination rates. A biotic ligand model-based damage assessment model was used to take into account environmental effects and biological mechanisms of Cu toxicity. We demonstrated that the probability causing 50% of susceptibility risk in response to pulse Cu exposure in generic Taiwan aquaculture ponds was ~33% of Cu in adverse physiologically associated, metabolically active pool, implicating no significant susceptibility risk for tilapia. We suggest that our integrated ecotoxicological models linking chronic exposure measurements with subcellular partitioning can facilitate a risk assessment framework that provides a predictive tool for preventive susceptibility reduction strategies for freshwater fish exposed to pulse metal stressors.

  6. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  7. Development of a Charged Particle Microbeam for Single-Particle Subcellular Irradiations at the MIT Laboratory for Accelerator Beam Application

    International Nuclear Information System (INIS)

    Yanch, Jacquelyn C.

    2004-01-01

    The development of a charged particle microbeam for single particle, subcellular irradiations at the Massachusetts Institute of Technology Laboratory for Accelerator Beam Applications (MIT LABA) was initiated under this NEER aeard. The Microbeam apparatus makes use of a pre-existing electrostatic accelerator with a horizontal beam tube

  8. Zn subcellular distribution in liver of goldfish (carassius auratus with exposure to zinc oxide nanoparticles and mechanism of hepatic detoxification.

    Directory of Open Access Journals (Sweden)

    Wenhong Fan

    Full Text Available Zinc Oxide Nanoparticles (ZnO NPs have attracted increasing concerns because of their widespread use and toxic potential. In this study, Zn accumulations in different tissues (gills, liver, muscle, and gut of goldfish (Carassius auratus after exposure to ZnO NPs were studied in comparison with bulk ZnO and Zn(2+. And the technique of subcellular partitioning was firstly used on the liver of goldfish to study the hepatic accumulation of ZnO NPs. The results showed that at sublethal Zn concentration (2 mg/L, bioaccumulation in goldfish was tissue-specific and dependent on the exposure materials. Compared with Zn(2+, the particles of bulk ZnO and the ZnO NPs appeared to aggregate in the environmentally contacted tissues (gills and gut, rather than transport to the internal tissues (liver and muscle. The subcellular distributions of liver differed for the three exposure treatments. After ZnO NPs exposure, Zn percentage in metal-rich granule (MRG increased significantly, and after Zn(2+ exposure, it increased significantly in the organelles. Metallothionein-like proteins (MTLP were the main target for Zn(2+, while MRG played dominant role for ZnO NPs. The different results of subcellular distributions revealed that metal detoxification mechanisms of liver for ZnO NPs, bulk ZnO, and Zn(2+ were different. Overall, subcellular partitioning provided an interesting start to better understanding of the toxicity of nano- and conventional materials.

  9. Subcellular Location of PKA Controls Striatal Plasticity: Stochastic Simulations in Spiny Dendrites

    Science.gov (United States)

    Oliveira, Rodrigo F.; Kim, MyungSook; Blackwell, Kim T.

    2012-01-01

    Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity. PMID:22346744

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

    International Nuclear Information System (INIS)

    Tursunkhodjayeva, F.M.

    2004-01-01

    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 J 125 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Floriani, M.; Grasset, G.; Simon, O.; Morlon, H.; Laroche, L. [CEA Cadarache (DEI/SECRE/LRE), Laboratory of Radioecology and Ecotoxicology, Institute for Radioprotection and Nuclear Safety, 13 - Saint-Paul-lez-Durance (France)

    2004-07-01

    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)

  12. 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

  13. Visualizing Escherichia coli sub-cellular structure using sparse deconvolution Spatial Light Interference Tomography.

    Directory of Open Access Journals (Sweden)

    Mustafa Mir

    Full Text Available Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM. In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT, to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.

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

    Directory of Open Access Journals (Sweden)

    A.M. Vargas

    2004-07-01

    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.

  15. 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.

  16. Optical monitoring of spinal cord subcellular damage after acute spinal cord injury

    Science.gov (United States)

    Shadgan, Babak; Manouchehri, Neda; So, Kitty; Shortt, Katelyn; Fong, Allan; Streijger, Femke; Macnab, Andrew; Kwon, Brian K.

    2018-02-01

    Introduction: Sudden physical trauma to the spinal cord results in acute spinal cord injury (SCI), leading to spinal cord (SC) tissue destruction, acute inflammation, increased SC intraparenchymal pressure, and tissue ischemia, hypoxia, and cellular necrosis. The ability to monitor SC tissue viability at subcellular level, using a real-time noninvasive method, would be extremely valuable to clinicians for estimating acute SCI damage, and adjusting and monitoring treatment in the intensive care setting. This study examined the feasibility and sensitivity of a custommade near infrared spectroscopy (NIRS) sensor to monitor the oxidation state of SC mitochondrial cytochrome aa3 (CCO), which reflects the subcellular damage of SC tissue in an animal model of SCI. Methods: Six anesthetized Yorkshire pigs were studied using a custom-made multi-wavelength NIRS system with a miniaturized optical sensor applied directly on the surgically exposed SC at T9. The oxidation states of SC tissue hemoglobin and CCO were monitored before, during and after acute SCI, and during mean arterial pressure alterations. Results: Non-invasive NIRS monitoring reflected changes in SC tissue CCO, simultaneous but independent of changes in hemoglobin saturation following acute SCI. A consistent decrease in SC tissue CCO chromophore concentration (-1.98 +/- 2.1 ab, pElevation of mean arterial pressure can reduce SC tissue damage as suggested by different researchers and observed by significant increase in SC tissue CCO concentration (1.51 +/- 1.7 ab, p<0.05) in this study. Conclusions: This pilot study indicates that a novel miniaturized multi-wave NIRS sensor has the potential to monitor post-SCI changes of SC cytochrome aa3 oxygenation state in real time. Further development of this method may offer new options for improved SCI care.

  17. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    International Nuclear Information System (INIS)

    Chandra, Subhash

    2004-01-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13 C and 15 N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13 C 15 N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39 K, 23 Na and 40 Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors

  18. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash

    2004-06-15

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

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

    International Nuclear Information System (INIS)

    Floriani, M.; Grasset, G.; Simon, O.; Morlon, H.; Laroche, L.

    2004-01-01

    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)

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-20

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  6. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

    Science.gov (United States)

    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-12-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  7. In situ reactor

    Science.gov (United States)

    Radtke, Corey William; Blackwelder, David Bradley

    2004-01-27

    An in situ reactor for use in a geological strata, is described and which includes a liner defining a centrally disposed passageway and which is placed in a borehole formed in the geological strata; and a sampling conduit is received within the passageway defined by the liner and which receives a geological specimen which is derived from the geological strata, and wherein the sampling conduit is in fluid communication with the passageway defined by the liner.

  8. Environmental monitoring with in-situ gamma spectrometer; Umweltueberwachung mit in-situ-Gamma-Spektrometer

    Energy Technology Data Exchange (ETDEWEB)

    Reinhardt, S. [ENVINET GmbH, Haar (Germany)

    2014-01-20

    The in-situ gamma spectroscopy allows large area and continuous monitoring of the radio nuclides and there composition in the environment. In comparison to the gamma dose rate measurement the additional spectral information gives the possibility for a quick and effective action in the case of a man-made radiation exposition in the environment. The knowledge respectively localization of the possible nuclides, which a responsible for the increased dose rate, supports responsible organization in the quick identification of the situation, definition of the actions and tracking of the temporal and local process of the radiation exposition. Due to dedicate actions the risk for people and environment is reduced.

  9. Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure

    Energy Technology Data Exchange (ETDEWEB)

    Thit, Amalie, E-mail: athitj@ruc.dk [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Ramskov, Tina, E-mail: tramskov@hotmail.com [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Croteau, Marie-Noële, E-mail: mcroteau@usgs.gov [Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Selck, Henriette [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark)

    2016-11-15

    Highlights: • L. variegatus was exposed to sediment spiked with either aqueous Cu or nanoparticulate CuO. • Both aqueous and nanoparticulate Cu were marginally accumulated by L. variegatus. • Elimination of Cu accumulated from both forms was limited. • The subcellular distribution of accumulated Cu varied between Cu forms. • The use of a tracer, greater exposure concentration and duration are recommended. - Abstract: The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched {sup 65}Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus

  10. Malignant mesothelioma in situ.

    Science.gov (United States)

    Churg, Andrew; Hwang, Harry; Tan, Larry; Qing, Gefei; Taher, Altaf; Tong, Amy; Bilawich, Ana M; Dacic, Sanja

    2018-05-01

    The existence of malignant mesothelioma in situ (MIS) is often postulated, but there are no accepted morphological criteria for making such a diagnosis. Here we report two cases that appear to be true MIS on the basis of in-situ genomic analysis. In one case the patient had repeated unexplained pleural unilateral effusions. Two thoracoscopies 9 months apart revealed only visually normal pleura. Biopsies from both thoracoscopies showed only a single layer of mildly reactive mesothelial cells. However, these cells had lost BRCA1-associated protein 1 (BAP1) and showed loss of cyclin-dependent kinase inhibitor 2 (CDKN2A) (p16) by fluorescence in-situ hybridisation (FISH). NF2 was not deleted by FISH but 28% of the mesothelial cells showed hyperploidy. Six months after the second biopsy the patient has persisting effusions but no evidence of pleural malignancy on imaging. The second patient presented with ascites and minimal omental thickening on imaging, but no visual evidence of tumour at laparoscopy. Omental biopsy showed a single layer of minimally atypical mesothelial cells with rare tiny foci of superficial invasion of fat. BAP1 immunostain showed loss of nuclear BAP1 in all the surface mesothelial cells and the invasive cells. There was CDKN2A deletion, but no deletion of NF2 by FISH. These cases show that morphologically bland single-layered surface mesothelial proliferations with molecular alterations seen previously only in invasive malignant mesotheliomas exist, and presumably represent malignant MIS. More cases are need to understand the frequency of such changes and the time-course over which invasive tumour develops. © 2018 John Wiley & Sons Ltd.

  11. 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

  12. Uptake and subcellular distribution of triclosan in typical hydrophytes under hydroponic conditions.

    Science.gov (United States)

    He, Yupeng; Nie, Enguang; Li, Chengming; Ye, Qingfu; Wang, Haiyan

    2017-01-01

    The increasing discharge of pharmaceuticals and personal care products (PPCPs) into the environment has generated serious public concern. The recent awareness of the environmental impact of this emerging class of pollutants and their potential adverse effects on human health have been documented in many reports. However, information regarding uptake and intracellular distribution of PPCPs in hydrophytes under hydroponic conditions, and potential human exposure is very limited. A laboratory experiment was conducted using 14 C-labeled triclosan (TCS) to investigate uptake and distribution of TCS in six aquatic plants (water spinach, purple perilla, cress, penny grass, cane shoot, and rice), and the subcellular distribution of 14 C-TCS was determined in these plants. The results showed that the uptake and removal rate of TCS from nutrient solution by hydrophytes followed the order of cress (96%) > water spinach (94%) > penny grass (87%) > cane shoot (84%) > purple perilla (78%) > rice (63%) at the end of incubation period (192 h). The range of 14 C-TCS content in the roots was 94.3%-99.0% of the added 14 C-TCS, and the concentrations in roots were 2-3 orders of magnitude greater than those in shoots. Furthermore, the subcellular fraction-concentration factor (3.6 × 10 2 -2.6 × 10 3  mL g -1 ), concentration (0.58-4.47 μg g -1 ), and percentage (30%-61%) of 14 C-TCS in organelles were found predominantly greater than those in cell walls and/or cytoplasm. These results indicate that for these plants, the roots are the primary storage for TCS, and within plant cells organelles are the major domains for TCS accumulation. These findings provide a better understanding of translocation and accumulation of TCS in aquatic plants at the cellular level, which is valuable for environmental and human health assessments of TCS. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    Science.gov (United States)

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

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

    International Nuclear Information System (INIS)

    Seebaugh, David R.; Wallace, William G.

    2009-01-01

    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 109 Cd- or 203 Hg-labeled amphipods or 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- 109 Cd% and TAM- 203 Hg% in radiolabeled amphipods were ∼64% and ∼73%, respectively. Gradients in AE- 109 Cd% (∼54% to ∼75%) and AE- 203 Hg% (∼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- 14 C% were not observed. Assimilated 109 Cd% partitioned to the TAM compartment in grass shrimp varied between ∼67% and ∼75%. 109 Cd bound to HSP in shrimp varied between ∼15% and ∼47%, while 109 Cd associated with metal-sensitive HDP was ∼17% to ∼44%. Percentages of assimilated 109 Cd bound to ORG were constant at ∼10%. Assimilated 203 Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated 203 Hg bound to HDP (∼47%) and ORG (∼11%) did not vary among populations and partitioning of 203 Hg 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

  15. In situ breast cancer

    International Nuclear Information System (INIS)

    Pacheco, Luis

    2004-01-01

    In situ breast cancer, particularly the ductal type, is increasing in frequency in the developed countries as well as in Ecuador, most probably. These lesions carry a higher risk of developing a subsequent invasive cancer. Treatment has changed recently due to results of randomized studies, from classical mastectomy to conservative surgery associated to radiotherapy. The Van Nuys Prognostic Index is currently the most usual instrument to guide diagnosis and treatment. Tamoxifen seems to decrease significantly the risk of tumor recurrence after initial treatment. (The author)

  16. 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.

  17. Quantifying the Sub-Cellular Distributions of Gold Nanospheres Uptaken by Cells through Stepwise, Site-Selective Etching.

    Science.gov (United States)

    Xia, Younan; Huo, Da

    2018-04-10

    A quantitative understanding of the sub-cellular distributions of nanoparticles uptaken by cells is important to the development of nanomedicine. With Au nanospheres as a model system, here we demonstrate, for the first time, how to quantify the numbers of nanoparticles bound to plasma membrane, accumulated in cytosol, and entrapped in lysosomes, respectively, through stepwise, site-selective etching. Our results indicate that the chance for nanoparticles to escape from lysosomes is insensitive to the presence of targeting ligand although ligand-receptor binding has been documented as a critical factor in triggering internalization. Furthermore, the presence of serum proteins is shown to facilitate the binding of nanoparticles to plasma membrane lacking the specific receptor. Collectively, these findings confirm the potential of stepwise etching in quantitatively analyzing the sub-cellular distributions of nanoparticles uptaken by cells in an effort to optimize the therapeutic effect. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Endoplasmic Reticulum Export, Subcellular Distribution, and Fibril Formation by Pmel17 Require an Intact N-terminal Domain Junction*

    Science.gov (United States)

    Leonhardt, Ralf M.; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J.; Cresswell, Peter

    2010-01-01

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes. PMID:20231267

  19. Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2

    Directory of Open Access Journals (Sweden)

    Samuel K. Campos

    2017-12-01

    Full Text Available Since 2012, our understanding of human papillomavirus (HPV subcellular trafficking has undergone a drastic paradigm shift. Work from multiple laboratories has revealed that HPV has evolved a unique means to deliver its viral genome (vDNA to the cell nucleus, relying on myriad host cell proteins and processes. The major breakthrough finding from these recent endeavors has been the realization of L2-dependent utilization of cellular sorting factors for the retrograde transport of vDNA away from degradative endo/lysosomal compartments to the Golgi, prior to mitosis-dependent nuclear accumulation of L2/vDNA. An overview of current models of HPV entry, subcellular trafficking, and the role of L2 during initial infection is provided below, highlighting unresolved questions and gaps in knowledge.

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

    International Nuclear Information System (INIS)

    Dubois, Maitee; Hare, Landis

    2009-01-01

    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. - Selenium assimilation by a predatory aquatic insect depends on Se availability in the cells of its prey

  1. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells

    Science.gov (United States)

    Sampathkumar, Arun; Krupinski, Pawel; Wightman, Raymond; Milani, Pascale; Berquand, Alexandre; Boudaoud, Arezki; Hamant, Olivier; Jönsson, Henrik; Meyerowitz, Elliot M

    2014-01-01

    Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001 PMID:24740969

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

    Science.gov (United States)

    Zheng, Wu; Blake, Catherine

    2015-10-01

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

  3. Subcellular distribution and mitogenic effect of basic fibroblast growth factor in mesenchymal uncommitted stem cells.

    Science.gov (United States)

    Benavente, Claudia A; Sierralta, Walter D; Conget, Paulette A; Minguell, José J

    2003-06-01

    Uncommitted mesenchymal stem cells (MSC), upon commitment and differentiation give rise to several mature mesenchymal lineages. Although the involvement of specific growth factors, including FGF2, in the development of committed MSC is known, the effect of FGF2 on uncommitted progenitors remains unclear. We have analyzed on a comparative basis, the subcellular distribution and mitogenic effect of FGF2 in committed and uncommitted MSC prepared from human bone marrow. Indirect immunofluorescence studies showed strong nuclear FGF2 staining in both progenitors; however, cytoplasmic staining was only detected in committed cells. Western blot analysis revealed the presence of 22.5 and 21-22 kDa forms of FGF2 in the nucleus of both progenitors; however, their relative content was higher in uncommitted than in committed cells. Exogenous FGF2 stimulated proliferation and sustained quiescence in committed and uncommitted cells, respectively. These results show that both type of progenitors, apart from morphological and proliferative differences, display specific patterns of response to FGF2.

  4. Subcellular analysis of interaction between breast cancer cells and drug by digital holography

    Science.gov (United States)

    Zhao, Jie; Lin, Qiaowen; Wang, Dayong; Wang, Yunxin; Ouyang, Liting; Guo, Sha; Yao, Qian

    2017-10-01

    Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don't need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.

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

    Science.gov (United States)

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

    2017-03-01

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

  6. Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

    Science.gov (United States)

    de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

    2018-02-01

    Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

  7. Subcellular distribution of apolipoprotein E along the lipoprotein synthetic pathway of rat liver

    International Nuclear Information System (INIS)

    Cole, T.G.; Stockhausen, D.C.

    1986-01-01

    Apolipoprotein E (apoE) is synthesized by the liver and is secreted as a component of VLDL. To define the intracellular locations of apoE, liver from 10 nonfasted male rats were removed and subcellular organelles prepared by differential pelleting through sucrose gradients. Mass of apoE was measured by radioimmunoassay. Approximately 10% of total hepatic apoE was recovered in rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER) and Golgi fractions. Concentrations of apoE (ng/mg protein) were: homogenate, 302 +/- 59; RER, 653 +/- 251; SER, 1250 +/- 471; Golgi, 11,044 +/- 4291. Total apoE content of each reaction (μg/organelle) was: homogenate (whole liver), 517 +/- 103; RER, 15 +/- 3; SER, 9 +/- 3; Golgi, 28 +/- 8. These data indicate that along the putative pathway of lipoprotein synthesis (RER->SER->Golgi), apoE concentration increases in each successive organelle and that flux of apoE is apparently most rapid through SER. Furthermore, the majority of apoE in the rat liver is apparently not directly associated with the lipoprotein synthetic pathway and may be associated with internalized lipoproteins or may be involved in non-lipoprotein related functions

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

    Science.gov (United States)

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

    2013-01-01

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

  9. Absorption and subcellular distribution of cadmium in tea plant (Camellia sinensis cv. "Shuchazao").

    Science.gov (United States)

    Cao, De-Ju; Yang, Xun; Geng, Geng; Wan, Xiao-Chun; Ma, Ru-Xiao; Zhang, Qian; Liang, Yue-Gan

    2018-03-21

    A hydroponic experiment was performed to investigate the Cd absorption and subcellular distribution in tea plant, Camellia sinensis. Increased Cd accumulation potential was observed in the tea plant in a Cd-enriched environment, but most of the Cd was absorbed by the roots of C. sinensis. The Cd in all the root fractions was mostly distributed in the soluble fraction, followed by the cell wall fraction. By contrast, the Cd was least distributed in the organelle fraction. The adsorption of Cd onto the C. sinensis roots was described well by the Langmuir isotherm model than the Freundlich isotherm. Most of the Cd (38.6 to 59.4%) was integrated with pectates and proteins in the roots and leaves. Fourier transform infrared spectroscopy (FTIR) analysis showed that small molecular organic substances, such as amino acids, organic acids, and carbohydrates with N-H, C=O, C-N, and O-H functional groups in the roots, bonded with Cd(II). The Cd accumulation in the C. sinensis leaves occurred in the cell wall and organelle fractions. C. sinensis has great capability to transport Cd, thereby indicating pollution risk. The metal homeostasis of Fe, Mn, Ca, and Mg in C. sinensis was affected when the Cd concentration was 1.0-15.0 mg/L.

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

    Science.gov (United States)

    Dienemann, Axel; Sprenger, Frank

    2004-06-22

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

  11. Perspective: On the importance of hydrodynamic interactions in the subcellular dynamics of macromolecules

    Science.gov (United States)

    Skolnick, Jeffrey

    2016-09-01

    An outstanding challenge in computational biophysics is the simulation of a living cell at molecular detail. Over the past several years, using Stokesian dynamics, progress has been made in simulating coarse grained molecular models of the cytoplasm. Since macromolecules comprise 20%-40% of the volume of a cell, one would expect that steric interactions dominate macromolecular diffusion. However, the reduction in cellular diffusion rates relative to infinite dilution is due, roughly equally, to steric and hydrodynamic interactions, HI, with nonspecific attractive interactions likely playing rather a minor role. HI not only serve to slow down long time diffusion rates but also cause a considerable reduction in the magnitude of the short time diffusion coefficient relative to that at infinite dilution. More importantly, the long range contribution of the Rotne-Prager-Yamakawa diffusion tensor results in temporal and spatial correlations that persist up to microseconds and for intermolecular distances on the order of protein radii. While HI slow down the bimolecular association rate in the early stages of lipid bilayer formation, they accelerate the rate of large scale assembly of lipid aggregates. This is suggestive of an important role for HI in the self-assembly kinetics of large macromolecular complexes such as tubulin. Since HI are important, questions as to whether continuum models of HI are adequate as well as improved simulation methodologies that will make simulations of more complex cellular processes practical need to be addressed. Nevertheless, the stage is set for the molecular simulations of ever more complex subcellular processes.

  12. Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites.

    Science.gov (United States)

    Znamensky, Vladimir; Akama, Keith T; McEwen, Bruce S; Milner, Teresa A

    2003-03-15

    In addition to genomic pathways, estrogens may regulate gene expression by activating specific signal transduction pathways, such as that involving phosphatidylinositol 3-kinase (PI3-K) and the subsequent phosphorylation of Akt (protein kinase B). The Akt pathway regulates various cellular events, including the initiation of protein synthesis. Our previous studies showed that synaptogenesis in hippocampal CA1 pyramidal cell dendritic spines is highest when brain estrogen levels are highest. To address the role of Akt in this process, the subcellular distribution of phosphorylated Akt immunoreactivity (pAkt-I) in the hippocampus of female rats across the estrous cycle and male rats was analyzed by light microscopy (LM) and electron microscopy (EM). By LM, the density of pAkt-I in stratum radiatum of CA1 was significantly higher in proestrus rats (or in estrogen-supplemented ovariectomized females) compared with diestrus, estrus, or male rats. By EM, pAkt-I was found throughout the shafts and in select spines of stratum radiatum dendrites. Quantitative ultrastructural analysis identifying pAkt-I with immunogold particles revealed that proestrus rats compared with diestrus, estrus, and male rats contained significantly higher pAkt-I associated with (1) dendritic spines (both cytoplasm and plasmalemma), (2) spine apparati located within 0.1 microm of dendritic spine bases, (3) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts, and (4) the plasmalemma of dendritic shafts. These findings suggest that estrogens may regulate spine formation in CA1 pyramidal neurons via Akt-mediated signaling events.

  13. Uniquely high turnover of nickel in contaminated oysters Crassostrea hongkongensis: Biokinetics and subcellular distribution.

    Science.gov (United States)

    Yin, Qijun; Wang, Wen-Xiong

    2018-01-01

    Despite the environmental concerns regarding nickel (Ni) especially in China, it has received little attention in aquatic animals due to its comparatively weak toxicity. In the present study, we explored the bioaccumulation, biokinetics, and subcellular distribution of Ni in an estuarine oyster Crassostrea hongkongensis. We demonstrated that Ni represented a new pattern of bioaccumulation in oysters characterized by rapid elimination and low dissolved uptake. The waterborne uptake rate constant and dietary assimilation efficiency were 0.036L/g/h and 28%, respectively, and dissolved uptake was the predominant exposure route. The efflux rate constant was positively related to tissue Ni concentration, with the highest efflux of 0.155d -1 . Such high elimination resulted in a high Ni turnover and steady-state condition reached rapidly, as shown with a 4-week waterborne exposure experiment at different Ni concentrations. Ni in oysters was mainly sequestered in metallothionein-like protein (MTLP), metal-rich granule, and cellular debris. MTLP was the most important binding fraction during accumulation and depuration, and played a dynamic role leading to rapid Ni elimination. Pre-exposure to Ni significantly reduced the dissolved uptake, probably accompanied by depressed filtration activity. Overall, the high turnover and regulation of Ni in oysters were achieved by enhanced efflux, suppressed uptake, and sequestration of most Ni into the detoxified pool. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Shrestha, Bindesh; Sripadi, Prabhakar; Reschke, Brent R; Henderson, Holly D; Powell, Matthew J; Moody, Sally A; Vertes, Akos

    2014-01-01

    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.

  15. Subcellular distribution of folate and folate binding protein in renal proximal tubules

    International Nuclear Information System (INIS)

    Sharkey, C.; Hjelle, J.T.; Selhub, J.

    1986-01-01

    High affinity folate binding protein (FBP) found in brush border membranes derived from renal cortices is thought to be involved in the renal conservation of folate. To examine the mechanisms of folate recovery, the subcellular distribution of FBP and 3 H-folate in rabbit renal proximal tubules (PT) was examined using analytical cell fractionation techniques. Tubules contain 3.41 +/- 0.32 picomoles FBP/mg protein (X +/- S.D.; n = 5). Postnuclear supernates (PNS) of PT were layered atop Percoll-sucrose gradients, centrifuged, fractions collected and assayed for various marker enzymes and FBP. Pooled fractions from such gradients were subsequently treated with digitonin and centrifuged in a stoichiometric manner with the activity of the microvillar enzyme, alanylaminopeptidase (AAP); excess FBP distributed with more buoyant particles. Infusion of 3 H-folate into rabbit kidneys followed by tubule isolation and fractionation revealed a time dependent shift in distribution of radiolabel from the AAP-rich gradient fractions to a region containing more buoyant particles; radiolevel was not associated with lysosomal markers. EM-radioautography revealed grains over intracellular vesicles. These results are consistent with the hypothesis that folate is recovered by a process involving receptor-mediated endocytosis or transcytosis

  16. Subcellular distribution of 111In and 169Yb in tumor and liver

    International Nuclear Information System (INIS)

    Ando, A.; Ando, I.; Takeshita, M.; Hiraki, T.; Hisada, K.

    1981-01-01

    Subcellular distribution of 111 In and 169 Yb was quantitatively determined to evaluate the role of the lysosome in accumulation of these nuclides in malignant tumor tissue and in the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of these nuclides was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of these nuclides and reached approximately 50% of the total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30% of the total radioactivity was concentrated in this fraction after 24 h. It is concluded that the lysosome does not play a major role in the tumor concentration of these nuclides, although it may play an important role in their liver concentration. In the case of hepatoma AH109A, it is pressumed that lysosome plays a considerably important role in the tumor concentration of these nuclides, hepatoma AH109A possessing some residual features of the liver. (orig.)

  17. Subcellular distribution and chemical forms of cadmium in the edible seaweed, Porphyra yezoensis.

    Science.gov (United States)

    Zhao, Yanfang; Wu, Jifa; Shang, Derong; Ning, Jinsong; Zhai, Yuxiu; Sheng, Xiaofeng; Ding, Haiyan

    2015-02-01

    The subcellular distribution and chemical forms of Cd were investigated in the edible seaweed, Porphyra yezoensis. The seaweed was exposed to different Cd concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 5.0mgl(-1)) for up to 96h. In both the controls (no Cd added) and treatment groups, 41.2-79.2% of Cd was localised in the cell wall, and the proportion of Cd in the cell wall increased with increasing concentrations of Cd and exposure time. In the control groups, 74.8% of Cd was extracted by 1M NaCl, followed by 2% acetic acid, HAC (18.9%). In the treatment groups, most Cd was extracted by 2% HAC. The proportion of Cd extracted by 2% HAC increased with exposure to increasing concentrations of Cd and over time. Cell wall deposition and forming of precipitates with phosphate may be a key strategy to reduce Cd toxicity in P. yezoensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  19. 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.

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

    Science.gov (United States)

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

    2002-04-12

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

  1. Subcellular distribution of /sup 111/In and /sup 169/Yb in tumor and liver

    Energy Technology Data Exchange (ETDEWEB)

    Ando, A; Ando, I; Takeshita, M; Hiraki, T; Hisada, K

    1981-05-01

    Subcellular distribution of /sup 111/In and /sup 169/Yb was quantitatively determined to evaluate the role of the lysosome in accumulation of these nuclides in malignant tumor tissue and in the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of these nuclides was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of these nuclides and reached approximately 50% of the total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30% of the total radioactivity was concentrated in this fraction after 24 h. It is concluded that the lysosome does not play a major role in the tumor concentration of these nuclides, although it may play an important role in their liver concentration. In the case of hepatoma AH109A, it is pressumed that lysosome plays a considerably important role in the tumor concentration of these nuclides, hepatoma AH109A possessing some residual features of the liver.

  2. 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.

  3. Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein.

    Science.gov (United States)

    Tien, Nguyen T; Karaca, Ilker; Tamboli, Irfan Y; Walter, Jochen

    2016-05-13

    The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    International Nuclear Information System (INIS)

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

    1976-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  6. Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

    Science.gov (United States)

    Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

    1999-04-01

    4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

  7. Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

    Science.gov (United States)

    Peterson, Soren J; Krasnow, Mark A

    2015-01-15

    To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Subcellular distribution and chemical forms of thorium in Brassica juncea var. foliosa.

    Science.gov (United States)

    Zhou, Sai; Kai, Hailu; Zha, Zhongyong; Fang, Zhendong; Wang, Dingna; Du, Liang; Zhang, Dong; Feng, Xiaojie; Jin, Yongdong; Xia, Chuanqin

    2016-06-01

    Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress.

    Science.gov (United States)

    Meneghel, Julie; Passot, Stéphanie; Cenard, Stéphanie; Réfrégiers, Matthieu; Jamme, Frédéric; Fonseca, Fernanda

    2017-09-01

    Cryopreservation of lactic acid bacteria may lead to undesirable cell death and functionality losses. The membrane is the first target for cell injury and plays a key role in bacterial cryotolerance. This work aimed at investigating at a subcellular resolution the membrane fluidity of two populations of Lactobacillus delbrueckii subsp. bulgaricus when subjected to cold and osmotic stresses associated to freezing. Cells were cultivated at 42 °C in mild whey medium, and they were exposed to sucrose solutions of different osmolarities (300 and 1800 mOsm L -1 ) after harvest. Synchrotron fluorescence microscopy was used to measure membrane fluidity of cells labeled with the cytoplasmic membrane probe 1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Images were acquired at 25 and 0 °C, and more than a thousand cells were individually analyzed. Results revealed that a bacterial population characterized by high membrane fluidity and a homogeneous distribution of fluidity values appeared to be positively related to freeze-thaw resistance. Furthermore, rigid domains with different anisotropy values were observed and the occurrence of these domains was more important in the freeze-sensitive bacterial population. The freeze-sensitive cells exhibited a broadening of existing highly rigid lipid domains with osmotic stress. The enlargement of domains might be ascribed to the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.

  10. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

    Science.gov (United States)

    Chamberland, Simon; Yang, Helen H; Pan, Michael M; Evans, Stephen W; Guan, Sihui; Chavarha, Mariya; Yang, Ying; Salesse, Charleen; Wu, Haodi; Wu, Joseph C; Clandinin, Thomas R; Toth, Katalin; Lin, Michael Z; St-Pierre, François

    2017-07-27

    Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila . These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

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

    Directory of Open Access Journals (Sweden)

    Miguel A. Ibeas

    2017-12-01

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

  12. Sub-cellular damage by copper in the cnidarian Zoanthus robustus.

    Science.gov (United States)

    Grant, A; Trompf, K; Seung, D; Nivison-Smith, L; Bowcock, H; Kresse, H; Holmes, S; Radford, J; Morrow, P

    2010-09-01

    Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110 microg Cu L(-1)). A decrease in net photosynthesis was observed only at the highest copper concentration (156 microg Cu L(-1)). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae. Copyright (c) 2010 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-02-01

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

  14. 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.

  15. Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

    OpenAIRE

    Nishi, Yoshihiro; Mifune, Hiroharu; Yabuki, Akira; Tajiri, Yuji; Hirata, Rumiko; Tanaka, Eiichiro; Hosoda, Hiroshi; Kangawa, Kenji; Kojima, Masayasu

    2013-01-01

    Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the sub-cellular processes governing the acylation of ghrelin remain to be elucidated.Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-pro...

  16. Analysis of sublethal arsenic toxicity to Ceratophyllum demersum: subcellular distribution of arsenic and inhibition of chlorophyll biosynthesis

    Czech Academy of Sciences Publication Activity Database

    Mishra, S.; Alfred, M.; Sobotka, Roman; Andresen, E.; Falkenberg, G.; Küpper, Hendrik

    2016-01-01

    Roč. 67, č. 15 (2016), s. 4639-4646 ISSN 0022-0957 R&D Projects: GA ČR GBP501/12/G055; GA MŠk(CZ) LO1416 Institutional support: RVO:61388971 ; RVO:60077344 Keywords : arsenic toxicity * chlorophyll biosynthesis * subcellular distribution of arsenic * synchrotron micro-X-ray fluorescence Subject RIV: EE - Microbiology, Virology; CE - Biochemistry (BC-A) Impact factor: 5.830, year: 2016

  17. Isotope Identification

    Energy Technology Data Exchange (ETDEWEB)

    Karpius, Peter Joseph [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-09-18

    The objective of this training modules is to examine the process of using gamma spectroscopy for radionuclide identification; apply pattern recognition to gamma spectra; identify methods of verifying energy calibration; and discuss potential causes of isotope misidentification.

  18. Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture.

    Science.gov (United States)

    de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Van Gorp, Hanne; Parthoens, Eef; Lamkanfi, Mohamed

    2018-04-17

    Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.

  19. Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

    Science.gov (United States)

    Avaritt, Brittany R; Swaan, Peter W

    2015-06-01

    Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

  20. Imaging cells and sub-cellular structures with ultrahigh resolution full-field X-ray microscopy.

    Science.gov (United States)

    Chien, C C; Tseng, P Y; Chen, H H; Hua, T E; Chen, S T; Chen, Y Y; Leng, W H; Wang, C H; Hwu, Y; Yin, G C; Liang, K S; Chen, F R; Chu, Y S; Yeh, H I; Yang, Y C; Yang, C S; Zhang, G L; Je, J H; Margaritondo, G

    2013-01-01

    Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refractive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The concluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-08-01

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

  2. 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.

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

    Science.gov (United States)

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

    2010-05-15

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

  4. Identification of Microbial Gene Biomarkers for in situ RDX Biodegradation

    Science.gov (United States)

    2012-12-01

    synthetase activity. The expected product, glutamate, could not be detected by thin layer chromatography in the reaction mixture (solvent: butanol: acetic... urease ) gamma subunit 5.344 KTR9_0529 Urea amidohydrolase ( urease ) alpha subunit 4.671 KTR9_34231 Aspartyl aminopeptidase 4.028 KTR9_0611

  5. Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.

    Science.gov (United States)

    Bleckmann, Andrea; Dresselhaus, Thomas

    2016-04-01

    First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in space and time, it is necessary to visualize mRNAs quantitatively with a subcellular resolution, without sectioning the tissues. We have adapted and optimized a protocol for colorimetric whole-mount RNA in situ hybridization (WISH) using egg cell-specific digoxigenin (DIG) labeled probes (Hejátko et al., 2006) [1] on ovules and early seeds of Arabidopsis. Furthermore, we established a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol, which allows mRNA visualization on a subcellular level. The polar localized mRNA of SBT4.13, encoding a subtilase, was identified using this protocol. Both methods are described and discussed in detail. Additionally a (F)-WISH flow-chart is provided along with a troubleshooting table. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. In situ zymography.

    Science.gov (United States)

    George, Sarah J; Johnson, Jason L

    2010-01-01

    In situ zymography is a unique laboratory technique that enables the localisation of matrix-degrading metalloproteinase (MMP) activity in histological sections. Frozen sections are placed on glass slides coated with fluorescently labelled matrix proteins. After incubation MMP activity can be observed as black holes in the fluorescent background due to proteolysis of the matrix protein. Alternatively frozen sections can be incubated with matrix proteins conjugated to quenched fluorescein. Proteolysis of the substrate by MMPs leads to the release of fluorescence. This technique can be combined with immunohistochemistry to enable co-location of proteins such as cell type markers or other proteins of interest. Additionally, this technique can be adapted for use with cell cultures, permitting precise location of MMP activity within cells, time-lapse analysis of MMP activity and analysis of MMP activity in migrating cells.

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

    Science.gov (United States)

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

    2017-01-01

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

  8. Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL).

    Science.gov (United States)

    Hooper, Cornelia M; Castleden, Ian R; Aryamanesh, Nader; Jacoby, Richard P; Millar, A Harvey

    2016-01-01

    Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

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

  10. Dosimetric characterization of radionuclides for systemic tumor therapy: Influence of particle range, photon emission, and subcellular distribution

    International Nuclear Information System (INIS)

    Uusijaervi, Helena; Bernhardt, Peter; Ericsson, Thomas; Forssell-Aronsson, Eva

    2006-01-01

    Various radionuclides have been proposed for systemic tumor therapy. However, in most dosimetric analysis of proposed radionuclides the charged particles are taken into consideration while the potential photons are ignored. The photons will cause undesirable irradiation of normal tissue, and increase the probability of toxicity in, e.g., the bone marrow. The aim of this study was to investigate the dosimetric properties according to particle range, photon emission, and subcellular radionuclide distribution, of a selection of radionuclides used or proposed for radionuclide therapy, and to investigate the possibility of dividing radionuclides into groups according to their dosimetric properties. The absorbed dose rate to the tumors divided by the absorbed dose rate to the normal tissue (TND) was estimated for different tumor sizes in a mathematical model of the human body. The body was simulated as a 70-kg ellipsoid and the tumors as spheres of different sizes (1 ng-100 g). The radionuclides were either assumed to be uniformly distributed throughout the entire tumor and normal tissue, or located in the nucleus or the cytoplasm of the tumor cells and on the cell membrane of the normal cells. Fifty-nine radionuclides were studied together with monoenergetic electrons, positrons, and alpha particles. The tumor and normal tissue were assumed to be of water density. The activity concentration ratio between the tumor and normal tissue was assumed to be 25. The radionuclides emitting low-energy electrons combined with a low photon contribution, and the alpha emitters showed high TND values for most tumor sizes. Electrons with higher energy gave reduced TND values for small tumors, while a higher photon contribution reduced the TND values for large tumors. Radionuclides with high photon contributions showed low TND value for all tumor sizes studied. The radionuclides studied could be divided into four main groups according to their TND values: beta emitters, Auger electron

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zaosheng, E-mail: zswang@iue.ac.cn [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Boulevard, Xiamen 361021 (China); State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012 (China); Feng, Chenglian; Ye, Chun [State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012 (China); Wang, Youshao [State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301 (China); Yan, Changzhou, E-mail: czyan@iue.ac.cn [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Boulevard, Xiamen 361021 (China); Li, Rui; Yan, Yijun; Chi, Qiaoqiao [Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Boulevard, Xiamen 361021 (China)

    2016-07-15

    Highlights: • Subcellular partitioning profile of metals were investigated in biomonitor organism. • Cu, Zn and Cd levels in main fraction of HSP increase along accumulation gradients. • Despite MTs as the major binding pool, detoxification of Cd and Pb was incomplete. • Induced MTs were sequentially correlated with Cu, Zn and Cd levels in HSP fraction. • Intracellular metal fates highlighted the metabolic availability within organism. - Abstract: 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

  12. PERFORMANCE CONFIRMATION IN-SITU INSTRUMENTATION

    International Nuclear Information System (INIS)

    N.T. Raczka

    2000-01-01

    The purpose of this document is to identify and analyze the types of in-situ instruments and methods that could be used in support of the data acquisition portion of the Performance Confirmation (PC) program at the potential nuclear waste repository at Yucca Mountain. The PC program will require geomechanical , geophysical, thermal, and hydrologic instrumentation of several kinds. This analysis is being prepared to document the technical issues associated with each type of measurement during the PC period. This analysis utilizes the ''Performance Confirmation Input Criteria'' (CRWMS M andO 1999a) as its starting point. The scope of this analysis is primarily on the period after the start of waste package emplacement and before permanent closure of the repository, a period lasting between 15 and 300 years after last package emplacement (Stroupe 2000, Attachment 1, p. 1). The primary objectives of this analysis are to: (1) Review the design criteria as presented in the ''Performance Confirmation Input Criteria'' (CRWMS M andO 1999a). The scope of this analysis will be limited to the instrumentation related to parameters that require continuous monitoring of the conditions underground. (2) Preliminary identification and listing of the data requirements and parameters as related to the current repository layout in support of PC monitoring. (3) Preliminary identification of methods and instrumentation for the acquisition of the required data. Although the ''Performance Confirmation Input Criteria'' (CRWMS M andO 1999a) defines a broad range of data that must be obtained from a variety of methods, the focus of this analysis is on instrumentation related to the performance of the rock mass and the formation of water in the repository environment, that is obtainable from in-situ observation, testing, and monitoring

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

    International Nuclear Information System (INIS)

    Kosior, Ewelina

    2013-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Xiaoqing Peng

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

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

    Science.gov (United States)

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

    2017-07-21

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

  16. Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

    Science.gov (United States)

    Kolb, Dagmar; Müller, Maria; Zellnig, Günther; Zechmann, Bernd

    2010-07-01

    Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca GREB: . Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 microM cadmium chloride (CdCl(2)) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.

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

    Science.gov (United States)

    Scherer, G F

    1984-03-01

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

  18. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

  19. 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.

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

    Science.gov (United States)

    Stubbs, M; Aiston, S; Agius, L

    2000-12-01

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

  1. Subcellular topological effect of particle monolayers on cell shapes and functions.

    Science.gov (United States)

    Miura, Manabu; Fujimoto, Keiji

    2006-12-01

    We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene-co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell-cell adhesion became stronger than cell-substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm(-2), some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and

  2. Agent-Based Modeling of Mitochondria Links Sub-Cellular Dynamics to Cellular Homeostasis and Heterogeneity.

    Directory of Open Access Journals (Sweden)

    Giovanni Dalmasso

    amplifying, cell-to-cell variability of mitochondrial morphology and energetic stress states. Overall, our modeling approach integrates biochemical and imaging knowledge, and presents a novel open-modeling approach to investigate how spatial and temporal mitochondrial dynamics contribute to functional homeostasis, and how subcellular organelle heterogeneity contributes to the emergence of cell heterogeneity.

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

    Science.gov (United States)

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

    2015-10-01

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

  4. 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.

  5. Subcellular distribution and chemical forms of thorium in Brassica juncea var. foliosa

    International Nuclear Information System (INIS)

    Zhou, Sai; Kai, Hailu; Zha, Zhongyong; Fang, Zhendong; Wang, Dingna; Du, Liang; Zhang, Dong; Feng, Xiaojie; Jin, Yongdong; Xia, Chuanqin

    2016-01-01

    Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th. - Highlights: • Brassica juncea var. foliosa can adapt to the stress of Th(<200 μM) under hydroponic condition. • Th was selectively distributed on cell wall, membranes and soluble fraction. • Th mainly existed in low-toxicity forms which were benefit for Th tolerance.

  6. Study of subcellular distribution of /sup 67/Ga in tumor and liver

    Energy Technology Data Exchange (ETDEWEB)

    Ando, A; Takeshita, M; Hiraki, T [Kanazawa Univ. (Japan). School of Paramedicine; Ando, T; Hisada, K

    1977-02-01

    The following animals and transplanted tumors were used: rats implanted with Yoshida sarcoma and hepatoma AH109A, and mice implanted with Ehrlich tumor. /sup 67/Ga-citrate was injected into the rats intravenously and into the mice intraperitoneally. Ten minutes to 48 hours after the administration of /sup 67/Ga-citrate, the animals were sacrificed, and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and livers was carried out according to the method of Hogeboom and Schneider. Radioactivity of each fraction was counted with a well type scintillation counter, and the protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and a small amount of radioactivity was localized in the mitochondrial fraction (lysosome contains in this fraction). But in the liver, most of the radioactivity was concentrated in the mitochondrial fraction, and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of the total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction was increased with the passage of time after administration, and about 30% of total activity was concentrated in this fraction at 24 hours after administration. From these results it is concluded that the lysosome does not play an important role in the concentration of /sup 67/Ga in the tumor, but that the lysosome plays an important role in the concentration of /sup 67/Ga in the liver. In the case of hepatoma AH109A, it is presumed that the lysosome plays a very important role in the concentration of /sup 67/Ga in the tumor, hepatoma AH109A having some nature of liver.

  7. Study of subcellular distribution of /sup 169/Yb and /sup 111/In in tumor and liver

    Energy Technology Data Exchange (ETDEWEB)

    Ando, A; Takeshita, M; Hiraki, T [Kanazawa Univ. (Japan). School of Paramedicine; Ando, Itsuko; Hisada, Kinichi

    1977-03-01

    Rats were implanted with Yoshida sarcoma and hepatoma AH109A; and mice were implanted with Ehrlich tumor. /sup 169/Yb-citrate and /sup 111/In-citrate were injected into the rats intravenously and into the mice intraperitoneally. Ten minutes to 48 hours after the administration of /sup 169/Yb-citrate and /sup 111/In-citrate, the animals were sacrificed and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and liver was carried out according to the method of Hogeboom and Schneider. The /sup 169/Yb and /sup 111/In of each fraction were counted by a well type scintillation counter, and the protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and a small amount of radioactivity was accumulated in the mitochondrial fraction (lysosome is contained in this fraction). But, in the liver, most of the radioactivity was concentrated in the mitochondrial fraction, and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of the total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction was increased with time after administration, and about 30% of total radioactivity was concentrated in this fraction 24 hours after administration. From these results it is concluded that the lysosome does not play an important role in the concentration of /sup 169/Yb and /sup 111/In in the tumor, and that the lysosome plays an important role in the concentration of /sup 169/Yb and /sup 111/In in the liver. In the case of hepatoma AH109A it is presumed that the lysosome plays a very important role in the concentration of /sup 169/Yb and /sup 111/In, in the tumor as hepatoma AH109A retains some nature of liver.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mengjiao [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2011-01-25

    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 [Cd{sup 2+}] ([Cd{sup 2+}]-based-IC{sub 50}, 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.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  12. Trophic transfer of trace metals: Subcellular compartmentalization in a polychaete and assimilation by a decapod crustacean

    Science.gov (United States)

    Rainbow, P.S.; Poirier, L.; Smith, B.D.; Brix, K.V.; Luoma, S.N.

    2006-01-01

    The chemical form of accumulated trace metal in prey is important in controlling the bioavailataility of dietary metal to a predator. This study investigated the trophic transfer of radiolabelled Ag, Cd and Zn from the polychaete worm Nereis diversicolor to the decapod crustacean Palaemonetes varians. We used 2 populations of worms with different proportions of accumulated metals in different subcellular fractions as prey, and loaded the worms with radiolabelled metals either from sediment or from solution. Accumulated radiolabelled metals were fractionated into 5 components : metal-rich granules (MRG), cellular debris, organelles, metallothionein-like proteins (MTLP), and other (heat-sensitive) proteins (HSP). Assimilation efficiencies (AE) of the metals by P. varians were measured from the 4 categories of prey (i.e. 2 populations, radiolabelled from sediment or solution). There were significant differences for each metal between the AEs from the different prey categories, confirming that origin of prey and route of uptake of accumulated trace metal will cause intraspecific differences in subsequent metal assimilation. Correlations were sought between AEs and selected fractions or combinations of fractions of metals in the prey-MRG, Trophically Available Metal (TAM = MTLP + HSP + organelles) and total protein (MTLP + HSP). TAM explained 28% of the variance in AEs for Ag, but no consistent relationships emerged between AEs and TAM or total protein when the metals were considered separately. AEs did, however, show significant positive regressions with both TAM and total protein when the 3 metals were considered together, explaining only about 21 % of the variance in each case. A significant negative relationship was observed between MRG and AE for all metals combined. The predator (P. varians) can assimilate dietary metal from a range of the fractions binding metals in the prey (N. diversicolor), with different assimilation efficiencies summated across these

  13. Fluorescence resonance energy transfer (FRET-based subcellular visualization of pathogen-induced host receptor signaling

    Directory of Open Access Journals (Sweden)

    Zimmermann Timo

    2009-11-01

    Full Text Available Abstract Background Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET to visualize pathogen-initiated signaling events in human cells. Results Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3. In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2 domain of Hck (Hck-SH2 was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet-Hck-SH2 and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. Conclusion These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

  14. 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.

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

    OpenAIRE

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

    2009-01-01

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

  16. Optimized Whole-Mount In Situ Immunolocalization for Arabidopsis thaliana Root Meristems and Lateral Root Primordia.

    Science.gov (United States)

    Karampelias, Michael; Tejos, Ricardo; Friml, Jiří; Vanneste, Steffen

    2018-01-01

    Immunolocalization is a valuable tool for cell biology research that allows to rapidly determine the localization and expression levels of endogenous proteins. In plants, whole-mount in situ immunolocalization remains a challenging method, especially in tissues protected by waxy layers and complex cell wall carbohydrates. Here, we present a robust method for whole-mount in situ immunolocalization in primary root meristems and lateral root primordia in Arabidopsis thaliana. For good epitope preservation, fixation is done in an alkaline paraformaldehyde/glutaraldehyde mixture. This fixative is suitable for detecting a wide range of proteins, including integral transmembrane proteins and proteins peripherally attached to the plasma membrane. From initiation until emergence from the primary root, lateral root primordia are surrounded by several layers of differentiated tissues with a complex cell wall composition that interferes with the efficient penetration of all buffers. Therefore, immunolocalization in early lateral root primordia requires a modified method, including a strong solvent treatment for removal of hydrophobic barriers and a specific cocktail of cell wall-degrading enzymes. The presented method allows for easy, reliable, and high-quality in situ detection of the subcellular localization of endogenous proteins in primary and lateral root meristems without the need of time-consuming crosses or making translational fusions to fluorescent proteins.

  17. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas

    2010-01-01

    target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal-to-noise ratio but low detection frequency (up to 15% for single-copy genes and up to 43% for the multi-copy 16S rRNA gene...... as Candidatus Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells.......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

  18. Modeling in situ vitrification

    International Nuclear Information System (INIS)

    Mecham, D.C.; MacKinnon, R.J.; Murray, P.E.; Johnson, R.W.

    1990-01-01

    In Situ Vitrification (ISV) process is being assessed by the Idaho National Engineering Laboratory (INEL) to determine its applicability to transuranic and mixed wastes buried at INEL'S Subsurface Disposal Area (SDA). This process uses electrical resistance heating to melt waste and contaminated soil in place to produce a durable glasslike material that encapsulates and immobilizes buried wastes. This paper outlines the requirements for the model being developed at the INEL which will provide analytical support for the ISV technology assessment program. The model includes representations of the electric potential field, thermal transport with melting, gas and particulate release, vapor migration, off-gas combustion and process chemistry. The modeling objectives are to help determine the safety of the process by assessing the air and surrounding soil radionuclides and chemical pollution hazards, the nuclear criticality hazard, and the explosion and fire hazards, help determine the suitability of the ISV process for stabilizing the buried wastes involved, and help design laboratory and field tests and interpret results. 3 refs., 2 figs., 1 tab

  19. In-situ uranium leaching

    International Nuclear Information System (INIS)

    Dotson, B.J.

    1986-01-01

    This invention provides a method for improving the recovery of mineral values from ore bodies subjected to in-situ leaching by controlling the flow behaviour of the leaching solution. In particular, the invention relates to an in-situ leaching operation employing a foam for mobility control of the leaching solution. A foam bank is either introduced into the ore bed or developed in-situ in the ore bed. The foam then becomes a diverting agent forcing the leaching fluid through the previously non-contacted regions of the deposit

  20. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. 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.

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

    Science.gov (United States)

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber’s oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  4. In situ leaching of uranium

    International Nuclear Information System (INIS)

    Martin, B.

    1980-01-01

    A process is described for the in-situ leaching of uranium-containing ores employing an acidic leach liquor containing peroxymonosulphuric acid. Preferably, additionally, sulphuric acid is present in the leach liquor. (author)

  5. CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels.

    Science.gov (United States)

    Kartasalo, Kimmo; Pölönen, Risto-Pekka; Ojala, Marisa; Rasku, Jyrki; Lekkala, Jukka; Aalto-Setälä, Katriina; Kallio, Pasi

    2015-10-26

    Orientation and the degree of isotropy are important in many biological systems such as the sarcomeres of cardiomyocytes and other fibrillar structures of the cytoskeleton. Image based analysis of such structures is often limited to qualitative evaluation by human experts, hampering the throughput, repeatability and reliability of the analyses. Software tools are not readily available for this purpose and the existing methods typically rely at least partly on manual operation. We developed CytoSpectre, an automated tool based on spectral analysis, allowing the quantification of orientation and also size distributions of structures in microscopy images. CytoSpectre utilizes the Fourier transform to estimate the power spectrum of an image and based on the spectrum, computes parameter values describing, among others, the mean orientation, isotropy and size of target structures. The analysis can be further tuned to focus on targets of particular size at cellular or subcellular scales. The software can be operated via a graphical user interface without any programming expertise. We analyzed the performance of CytoSpectre by extensive simulations using artificial images, by benchmarking against FibrilTool and by comparisons with manual measurements performed for real images by a panel of human experts. The software was found to be tolerant against noise and blurring and superior to FibrilTool when analyzing realistic targets with degraded image quality. The analysis of real images indicated general good agreement between computational and manual results while also revealing notable expert-to-expert variation. Moreover, the experiment showed that CytoSpectre can handle images obtained of different cell types using different microscopy techniques. Finally, we studied the effect of mechanical stretching on cardiomyocytes to demonstrate the software in an actual experiment and observed changes in cellular orientation in response to stretching. CytoSpectre, a versatile, easy

  6. 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

  7. Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells

    Directory of Open Access Journals (Sweden)

    Grotewold Erich

    2003-12-01

    Full Text Available Abstract Background Little is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively. Results We investigated the accumulation and sub-cellular localization of the green and yellow auto-fluorescent compounds in maize BMS cells expressing the P1 transcription factor from an estradiol inducible promoter. We established that the yellow fluorescent compounds accumulate inside the vacuole in YFBs that resemble AVIs. The green fluorescent compounds accumulate initially in the cytoplasm in large spherical GFBs. Cells accumulating GFBs also contain electron-dense structures that accumulate initially in the ER and which later appear to fuse with the plasma membrane. Structures resembling the GFBs were also observed in the periplasmic space of plasmolized cells. Ultimately, the green fluorescence accumulates in the cell wall, in a process that is insensitive to the Golgi-disturbing agents BFA and monensin. Conclusions Our results suggest the presence of at least two distinct trafficking pathways, one to the cell wall and the other to the vacuole, for different auto-fluorescent compounds induced by the same transcription factor in maize BMS cells. These compartments represent two of the major sites of accumulation of phenolic compounds characteristic of maize cells. The secretion of the green auto-fluorescent compounds occurs by a pathway that does not involve the TGN, suggesting that it is different from the secretion of most proteins, polysaccharides or epicuticular waxes. The

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

    International Nuclear Information System (INIS)

    Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

    1976-01-01

    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- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  9. 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

  10. The in vitro synthesis of β-galactosidase induced in a subcellular structure of Escherichia coli (1961)

    International Nuclear Information System (INIS)

    Nisman, B.; Kayser, A.; Demailly, J.; Genin, C.

    1961-01-01

    Isopropyl-thio-galactoside (IPTG), an inducer of 3-galactosidase, makes it possible to synthesise this enzyme in vitro with the subcellular structure (P 1 ). The enzyme is isolated from the bacteria Escherichia coli K 12 which are inductive but not induced. The incorporation of radioactive amino-acids, which is stimulated by the presence of an inducer, was studied during the course of the enzyme synthesis. Saccharose suppresses the induction of β-galactosidase. The presence of a specific inhibitor in the structure studied is considered. (authors) [fr

  11. Extreme Spectroscopy: In situ nuclear materials behavior from optical data

    Energy Technology Data Exchange (ETDEWEB)

    Guimbretiere, G.; Canizares, A.; Raimboux, N.; Omnee, R.; Duval, F.; Ammar, M.R.; Simon, P. [CNRS - UPR3079 CEMHTI, Universite d' Orleans, 45071Orleans cedex 2 (France); Desgranges, L.; Mohun, R. [CEA, DEN, DEC, F-13108 Saint-Paul-Lez-Durance (France); Jegou, C.; Magnin, M. [CEA/DTCD/SECM/LMPA, Marcoule 30207 Bagnols Sur Ceze (France); Clavier, N.; Dacheux, N. [ICSM-UMR5257 CEA/CNRS/UM2/ENSCM, Marcoule, BP17171, 30207 Bagnols sur Ceze (France)

    2015-07-01

    In the nuclear industry, materials are regularly exposed to high temperature or/and irradiation and a better knowledge and understanding of their behavior under such extreme conditions is a key-point for improvements and further developments. Nowadays, Raman spectroscopy begins to be well known as a promising technique in the post mortem and remote characterization of nuclear materials exposed to extreme conditions. On this topic, at ANIMMA 2013 conference, we have presented some results about its implementation in the study of model or real nuclear fuel. However, the strength of Raman spectroscopy as in situ characterization tool is mainly its ability to be implemented remotely through optical fibers. Aware of this, implementation of other optical techniques can be considered in order to gain information not only on the structural dynamics of materials but also on the electronic charge carrier populations. In this paper, we propose to present our last advances in Raman characterization of nuclear materials and enlarge to the in situ use of complementary optical spectroscopies. Emphasis will be made on the information that can be gained to the behavior of the model fuel depleted UO{sub 2} under extreme conditions of high temperature and ionic irradiation: - In Situ Raman identification of the radiolysis alteration products of UO{sub 2} in contact with water under ionic irradiation. - In Situ Raman recording of the damaged dynamic of UO{sub 2} under inert atmosphere. - In Situ Raman and photo-luminescence study of virgin and damaged UO2 at high temperature. - In Situ study of electronic charge carriers' behavior in U{sub x}Th{sub 1-x}O{sub 2} solid solutions by mean of Iono- and Thermo- luminescence under and post- ionic irradiation. (authors)

  12. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA

    Directory of Open Access Journals (Sweden)

    N. Charles Harata

    2014-09-01

    Full Text Available Background: An in‐frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE‐torsinA results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown.Methods: We evaluated the literature regarding the subcellular localization of torsinA.Results: Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild‐type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A into host cells and overexpress the protein therein. In both neurons and non‐neuronal cells, exogenous wild‐type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE‐torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate.Discussion: As patients’ cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non‐overexpressed vs. exogenously introduced (overexpressed proteins.

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

    Science.gov (United States)

    Salvatore, M; Shu, N; Elofsson, A

    2018-01-01

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

  14. Prenatal alcohol exposure modifies glucocorticoid receptor subcellular distribution in the medial prefrontal cortex and impairs frontal cortex-dependent learning.

    Directory of Open Access Journals (Sweden)

    Andrea M Allan

    Full Text Available Prenatal alcohol exposure (PAE has been shown to impair learning, memory and executive functioning in children. Perseveration, or the failure to respond adaptively to changing contingencies, is a hallmark on neurobehavioral assessment tasks for human fetal alcohol spectrum disorder (FASD. Adaptive responding is predominantly a product of the medial prefrontal cortex (mPFC and is regulated by corticosteroids. In our mouse model of PAE we recently reported deficits in hippocampal formation-dependent learning and memory and a dysregulation of hippocampal formation glucocorticoid receptor (GR subcellular distribution. Here, we examined the effect of PAE on frontal cortical-dependent behavior, as well as mPFC GR subcellular distribution and the levels of regulators of intracellular GR transport. PAE mice displayed significantly reduced response flexibility in a Y-maze reversal learning task. While the levels of total nuclear GR were reduced in PAE mPFC, levels of GR phosphorylated at serines 203, 211 and 226 were not significantly changed. Cytosolic, but not nuclear, MR levels were elevated in the PAE mPFC. The levels of critical GR trafficking proteins, FKBP51, Hsp90, cyclophilin 40, dynamitin and dynein intermediate chain, were altered in PAE mice, in favor of the exclusion of GR from the nucleus, indicating dysregulation of GR trafficking. Our findings suggest that there may be a link between a deficit in GR nuclear localization and frontal cortical learning deficits in prenatal alcohol-exposed mice.

  15. 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.

  16. Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

    Science.gov (United States)

    Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

    2006-07-01

    Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

  17. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA.

    Science.gov (United States)

    Harata, N Charles

    2014-01-01

    An in-frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE-torsinA) results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown. We evaluated the literature regarding the subcellular localization of torsinA. Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild-type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A) into host cells and overexpress the protein therein. In both neurons and non-neuronal cells, exogenous wild-type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE-torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate. As patients' cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non-overexpressed) vs. exogenously introduced (overexpressed) proteins.

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Science.gov (United States)

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

    2009-08-01

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

  20. Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

    Science.gov (United States)

    Panieri, Emiliano; Millia, Carlo; Santoro, Massimo M

    2017-08-01

    Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H 2 O 2 ) levels and glutathione redox potential (E GSH ), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. A New Technique for Deep in situ Measurements of the Soil Water Retention Behaviour

    DEFF Research Database (Denmark)

    Rocchi, Irene; Gragnano, Carmine Gerardo; Govoni, Laura

    2018-01-01

    In situ measurements of soil suction and water content in deep soil layers still represent an experimental challenge. Mostly developed within agriculture related disciplines, field techniques for the identification of soil retention behaviour have been so far employed in the geotechnical context ...

  2. Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

    NARCIS (Netherlands)

    Moers, M.H.P.; Moers, M.H.P.; Kalle, W.H.J.; Kalle, W.H.J.; Ruiter, A.G.T.; Wiegant, J.C.A.G.; Raap, A.K.; Greve, Jan; de Grooth, B.G.; van Hulst, N.F.

    1996-01-01

    Fluorescence in situ hybridization o­n human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments o­n a single

  3. Preliminary study of selenium and mercury distribution in some porcine tissues and their subcellular fractions by NAA and HG-AFS

    International Nuclear Information System (INIS)

    Jiujiang Zhao; Chunying Chen; Peiqun Zhang; Zhifang Chai

    2004-01-01

    Selenium and mercury distribution in porcine tissues and their subcellular fractions from a mercury-polluted area of Guizhou Province and from a not mercury-exposed area of Beijing in China have been studied with neutron activation analysis and hydride generation-atomic fluorescence spectrometry. Both the selenium and mercury levels are higher in Guizhou porcine tissues and their subcellular fractions than those in Beijing. These two elements are highly enriched in kidney and liver of Guizhou pig, while selenium is only enriched in the kidney of Beijing pig. Exposure of mercury may result in redistribution of Se and Hg in vivo. The Hg/Se molar ratio of the subcellular fractions is very low in the case of relatively low mercury level and gradually reaches to a high constant value with increasing level of mercury, which implies that selenium and mercury may form some special complexes in the organisms. (author)

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

    Science.gov (United States)

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

    2014-10-01

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

  5. Subcellular partitioning kinetics, metallothionein response and oxidative damage in the marine mussel Mytilus galloprovincialis exposed to cadmium-based quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, Thiago Lopes [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Gomes, Tânia [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo (Norway); Durigon, Emerson Giuliani [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Bebianno, Maria João, E-mail: mbebian@ualg.pt [CIMA, Faculty of Science and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)

    2016-06-01

    The environmental health impact of metal-based nanomaterials is of emerging concern, but their metabolism and detoxification pathways in marine bioindicator species remain unclear. This study investigated the role of subcellular partitioning kinetics, metallothioneins (MTs) response and oxidative damage (lipid peroxidation – LPO) in the marine mussel Mytilus galloprovincialis exposed to CdTe quantum dots (QDs) in comparison with its dissolved counterpart. Mussels were exposed to QDs and dissolved Cd for 21 days at 10 μg Cd L{sup −1} followed by a 50 days depuration. Higher Cd concentrations were detected in fractions containing mitochondria, nucleus and lysosomes, suggesting potential subcellular targets of QDs toxicity in mussel tissues. Tissue specific metabolism patterns were observed in mussels exposed to both Cd forms. Although MT levels were directly associated with Cd in both forms, QDs subcellular partitioning is linked to biologically active metal (BAM), but no increase in LPO occurred, while in the case of dissolved Cd levels are in the biologically detoxified metal (BDM) form, indicating nano-specific effects. Mussel gills showed lower detoxification capability of QDs, while the digestive gland is the major tissue for storage and detoxification of both Cd forms. Both mussel tissues were unable to completely eliminate the Cd accumulated in the QDs form (estimated half-life time > 50 days), highlighting the potential source of Cd and QDs toxicity for human and environmental health. Results indicate tissue specific metabolism patterns and nano-specific effects in marine mussel exposed to QDs. - Highlights: • Subcellular partitioning and MT response are Cd form, tissue and time dependent. • Tissue specific metabolism of Cd-based quantum dots (QDs) in marine mussels. • QDs are slower biologically detoxified when compared to dissolved Cd. • Subcellular partitioning and biomarker responses indicate nano-specific effects. • Subcellular

  6. Multivariate profiling of neurodegeneration-associated changes in a subcellular compartment of neurons via image processing

    Directory of Open Access Journals (Sweden)

    Kumarasamy Saravana K

    2008-11-01

    Full Text Available Abstract Background Dysfunction in the endolysosome, a late endosomal to lysosomal degradative intracellular compartment, is an early hallmark of some neurodegenerative diseases, in particular Alzheimer's disease. However, the subtle morphological changes in compartments of affected neurons are difficult to quantify quickly and reliably, making this phenotype inaccessible as either an early diagnostic marker, or as a read-out for drug screening. Methods We present a method for automatic detection of fluorescently labeled endolysosomes in degenerative neurons in situ. The Drosophila blue cheese (bchs mutant was taken as a genetic neurodegenerative model for direct in situ visualization and quantification of endolysosomal compartments in affected neurons. Endolysosomal compartments were first detected automatically from 2-D image sections using a combination of point-wise multi-scale correlation and normalized correlation operations. This detection algorithm performed well at recognizing fluorescent endolysosomes, unlike conventional convolution methods, which are confounded by variable intensity levels and background noise. Morphological feature differences between endolysosomes from wild type vs. degenerative neurons were then quantified by multivariate profiling and support vector machine (SVM classification based on compartment density, size and contrast distribution. Finally, we ranked these distributions according to their profiling accuracy, based on the backward elimination method. Results This analysis revealed a statistically significant difference between the neurodegenerative phenotype and the wild type up to a 99.9% confidence interval. Differences between the wild type and phenotypes resulting from overexpression of the Bchs protein are detectable by contrast variations, whereas both size and contrast variations distinguish the wild type from either of the loss of function alleles bchs1 or bchs58. In contrast, the density measurement

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

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Krustrup, Peter; Nybo, Lars

    2012-01-01

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

  8. Whale Identification

    Science.gov (United States)

    1991-01-01

    R:BASE for DOS, a computer program developed under NASA contract, has been adapted by the National Marine Mammal Laboratory and the College of the Atlantic to provide and advanced computerized photo matching technique for identification of humpback whales. The program compares photos with stored digitized descriptions, enabling researchers to track and determine distribution and migration patterns. R:BASE is a spinoff of RIM (Relational Information Manager), which was used to store data for analyzing heat shielding tiles on the Space Shuttle Orbiter. It is now the world's second largest selling line of microcomputer database management software.

  9. In Situ TEM Electrical Measurements

    DEFF Research Database (Denmark)

    Canepa, Silvia; Alam, Sardar Bilal; Ngo, Duc-The

    2016-01-01

    understanding of complex physical and chemical interactions in the pursuit to optimize nanostructure function and device performance. Recent developments of sample holder technology for TEM have enabled a new field of research in the study of functional nanomaterials and devices via electrical stimulation...... influence the sample by external stimuli, e.g. through electrical connections, the TEM becomes a powerful laboratory for performing quantitative real time in situ experiments. Such TEM setups enable the characterization of nanostructures and nanodevices under working conditions, thereby providing a deeper...... and measurement of the specimen. Recognizing the benefits of electrical measurements for in situ TEM, many research groups have focused their effort in this field and some of these methods have transferred to ETEM. This chapter will describe recent advances in the in situ TEM investigation of nanostructured...

  10. Triplex in-situ hybridization

    Science.gov (United States)

    Fresco, Jacques R.; Johnson, Marion D.

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  11. Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

    International Nuclear Information System (INIS)

    Wu, G; Wei, J; Zheng, Z; Ye, J; Zeng, S

    2014-01-01

    The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM. (letters)

  12. Subcellular partitioning of cadmium and zinc in mealworm beetle (Tenebrio molitor) larvae exposed to metal-contaminated flour.

    Science.gov (United States)

    Bednarska, Agnieszka J; Świątek, Zuzanna

    2016-11-01

    By studying the internal compartmentalization of metals in different subcellular fractions we are able to better understand the mechanisms of metal accumulation in organisms and the transfer of metals through trophic chains. We investigated the internal compartmentalization of cadmium (Cd) and zinc (Zn) in mealworm beetle (Tenebrio molitor) larvae by breeding them in flour contaminated with either Cd at 100, 300 and 600mgkg(-1), or Zn at 1000 and 2000mgkg(-1). We separated the cellular components of the larvae into 3 fractions: the S1 or cytosolic fraction containing organelles, heat-sensitive and heat-stable proteins, the S2 or cellular debris fraction and the G or metal-rich granule fraction. The concentration of Cd and Zn in each fraction was measured at 0, 7, 14 and 21 days of being fed the flour. The concentration of Cd in the flour affected the concentration of Cd measured in each larval subcellular fraction (p≤0.0001), while the concentration of Zn in the flour only affected the Zn concentration in the S2 and G fractions (p≤0.02). Both Cd and Zn concentrations in mealworms remained relatively constant during the exposure (days 7, 14 and 21) in all three fractions, but the Cd concentrations were much higher than those found in larvae before the exposure (day 0). The concentration of Cd in the flour, however, did not affect the percentage of Cd in the S1 fraction. The contribution of Cd in the G fraction to the total Cd amount was similar (30-40%) in all Cd treatments. The percentage of Zn in all three fractions was not affected by the concentration of Zn in the flour and the relative contributions of each subcellular fraction to the total burden of Zn remained generally constant for both control and treated larvae. In general, larvae sequestered approximately 30% of Cd and Zn in the S1 fraction, which is important for the transport of metals to higher trophic levels in a food web. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. 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)

    Eyckmans, Marleen; Blust, Ronny; De Boeck, Gudrun

    2012-01-01

    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

  14. 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

  15. Impacts of BDE209 addition on Pb uptake, subcellular partitioning and gene toxicity in earthworm (Eisenia fetida)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei, E-mail: wzhang@ecust.edu.cn [State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, Shanghai 200237 (China); School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China); Liu, Kou; Li, Jing; Liang, Jun; Lin, Kuangfei [State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, Shanghai 200237 (China); School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2015-12-30

    Highlights: • 10 or 100 μg g{sup −1} BDE209 addition caused histological changes in Pb-exposed earthworms’ body wall. • Strong histopathological effects with BDE209 addition confirmed the enhanced Pb bioavailability. • The presence of higher levels of BDE209 altered subcellular partitioning of Pb in earthworm. • Co-exposure to Pb and BDE209 declined SOD and CAT gene transcripts synergistically. • BDE209 addition elicited up-regulation of Hsp90 gene expression compared to Pb exposure alone. - Abstract: Lead (Pb) and decabromodiphenyl ether (BDE209) are the mainly co-existed contaminants at e-waste recycling sites. The potential toxicity of Pb (250 μg g{sup −1}) to earthworm Eisenia fetida in the presence of BDE209 (1, 10 and 100 μg g{sup −1}) was determined during 14-d incubation period. Compared to Pb treatment alone, the co-exposure with 1 μg g{sup −1} BDE209 barely affected Pb uptake, subcellular partitioning and gene expression; however, histopathological changes in earthworms’ body wall (epidermal, circular and longitudinal muscles) demonstrated that 10 and 100 μg g{sup −1} BDE209 additions enhanced Pb uptake and altered its subcellular partitioning, indicating that Pb redistributed from fractions E (cell debris) and D (metal-rich granules) to fraction C (cytosols); Additionally, BDE209 supply significantly inhibited (p < 0.05) the induction of SOD (superoxide dismutase) and CAT (catalase) gene expressions (maximum down-regulation 59% for SOD gene at Pb + 100 μg g{sup −1} BDE209 and 89% for CAT gene at Pb + 10 μg g{sup −1} BDE209), while facilitated (p < 0.05) Hsp90 (heat shock protein 90) gene expression with maximum induction rate of 120% after exposure to Pb + 10 μg g{sup −1} BDE209. These findings illustrate the importance of considering environmental BDE209 co-exposure when assessing Pb bioaccumulation and toxicity in multi-contaminated soil ecosystems.

  16. Impacts of BDE209 addition on Pb uptake, subcellular partitioning and gene toxicity in earthworm (Eisenia fetida)

    International Nuclear Information System (INIS)

    Zhang, Wei; Liu, Kou; Li, Jing; Liang, Jun; Lin, Kuangfei

    2015-01-01

    Highlights: • 10 or 100 μg g −1 BDE209 addition caused histological changes in Pb-exposed earthworms’ body wall. • Strong histopathological effects with BDE209 addition confirmed the enhanced Pb bioavailability. • The presence of higher levels of BDE209 altered subcellular partitioning of Pb in earthworm. • Co-exposure to Pb and BDE209 declined SOD and CAT gene transcripts synergistically. • BDE209 addition elicited up-regulation of Hsp90 gene expression compared to Pb exposure alone. - Abstract: Lead (Pb) and decabromodiphenyl ether (BDE209) are the mainly co-existed contaminants at e-waste recycling sites. The potential toxicity of Pb (250 μg g −1 ) to earthworm Eisenia fetida in the presence of BDE209 (1, 10 and 100 μg g −1 ) was determined during 14-d incubation period. Compared to Pb treatment alone, the co-exposure with 1 μg g −1 BDE209 barely affected Pb uptake, subcellular partitioning and gene expression; however, histopathological changes in earthworms’ body wall (epidermal, circular and longitudinal muscles) demonstrated that 10 and 100 μg g −1 BDE209 additions enhanced Pb uptake and altered its subcellular partitioning, indicating that Pb redistributed from fractions E (cell debris) and D (metal-rich granules) to fraction C (cytosols); Additionally, BDE209 supply significantly inhibited (p < 0.05) the induction of SOD (superoxide dismutase) and CAT (catalase) gene expressions (maximum down-regulation 59% for SOD gene at Pb + 100 μg g −1 BDE209 and 89% for CAT gene at Pb + 10 μg g −1 BDE209), while facilitated (p < 0.05) Hsp90 (heat shock protein 90) gene expression with maximum induction rate of 120% after exposure to Pb + 10 μg g −1 BDE209. These findings illustrate the importance of considering environmental BDE209 co-exposure when assessing Pb bioaccumulation and toxicity in multi-contaminated soil ecosystems.

  17. Identification of proteins in the postsynaptic density fraction by mass spectrometry

    DEFF Research Database (Denmark)

    Walikonis, R S; Jensen, Ole Nørregaard; Mann, M

    2000-01-01

    Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed...... not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog...

  18. Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.

    Science.gov (United States)

    Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J H; Risseeuw, Martijn D P; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

    2014-12-01

    Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. In situ solution mining technique

    International Nuclear Information System (INIS)

    Learmont, R.P.

    1978-01-01

    A method of in situ solution mining is disclosed in which a primary leaching process employing an array of 5-spot leaching patterns of production and injection wells is converted to a different pattern by converting to injection wells all the production wells in alternate rows

  20. 'In situ' expanded graphite extinguishant

    International Nuclear Information System (INIS)

    Cao Qixin; Shou Yuemei; He Bangrong

    1987-01-01

    This report is concerning the development of the extinguishant for sodium fire and the investigation of its extinguishing property. The experiment result shows that 'in situ' expanded graphite developed by the authors is a kind of extinguishant which extinguishes sodium fire quickly and effectively and has no environment pollution during use and the amount of usage is little

  1. In Situ Cardiovascular Tissue Engineering

    NARCIS (Netherlands)

    Talacua, H

    2016-01-01

    In this thesis, the feasibility of in situ TE for vascular and valvular purposes were tested with the use of different materials, and animal models. First, the feasibility of a decellularized biological scaffold (pSIS-ECM) as pulmonary heart valve prosthesis is examined in sheep (Chapter 2). Next,

  2. Diamond identification

    International Nuclear Information System (INIS)

    Lang, A.R.

    1979-01-01

    Methods of producing sets of records of the internal defects of diamonds as a means of identification of the gems by x-ray topography are described. To obtain the records one can either use (a) monochromatic x-radiation reflected at the Bragg angle from crystallographically equivalent planes of the diamond lattice structure, Bragg reflections from each such plane being recorded from a number of directions of view, or (b) white x-radiation incident upon the diamond in directions having a constant angular relationship to each equivalent axis of symmetry of the diamond lattice structure, Bragg reflections being recorded for each direction of the incident x-radiation. By either method an overall point-to-point three dimensional representation of the diamond is produced. (U.K.)

  3. Subcellular localization of secondary lipid metabolites including fragrance volatiles in carnation petals

    International Nuclear Information System (INIS)

    Hudak, K.A.; Thompson, J.E.

    1997-01-01

    Pulse-chase labeling of carnation (Dianthus caryophyllus L. cv Improved White Sim) petals with [14C]acetate has provided evidence for a hydrophobic subcompartment of lipid-protein particles within the cytosol that resemble oil bodies, are formed by blebbing from membranes, and are enriched in lipid metabolites (including fragrance volatiles) derived from membrane fatty acids. Fractionation of the petals during pulse-chase labeling revealed that radiolabeled fatty acids appear first in microsomal membranes and subsequently in cytosolic lipid-protein particles, indicating that the particles originate from membranes. This interpretation is supported by the finding that the cytosolic lipid-protein particles contain phospholipid as well as the same fatty acids found in microsomal membranes. Radiolabeled polar lipid metabolites (methanol/ water-soluble) were detectable in both in situ lipid-protein particles isolated from the cytosol and those generated in vitro from isolated radiolabeled microsomal membranes. The lipid-protein particles were also enriched in hexanal, trans-2-hexenal, 1-hexanol, 3-hexen-1-ol, and 2-hexanol, volatiles of carnation flower fragrance that are derived from membrane fatty acids through the lipoxygenase pathway. Therefore, secondary lipid metabolites, including components of fragrance, appear to be formed within membranes of petal tissue and are subsequently released from the membrane bilayers into the cytosol by blebbing of lipid-protein particles

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-09-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Temporal variations in metallothionein concentration and subcellular distribution of metals in gills and digestive glands of the oyster Crassostrea angulata

    Directory of Open Access Journals (Sweden)

    Chiara Trombini

    2010-11-01

    Full Text Available The metallothionein levels and metal concentrations in whole body, digestive gland and gills of Crassostrea angulata were analyzed in field samples collected from the River Guadalquivir estuary over several years following a mining waste spill upstream. The subcellular distribution of metals was analyzed to determine the mechanisms involved in the detoxification process. The highest metallothionein levels were reported in the digestive gland shortly after the mining contamination event. In this organ, metals are stored preferentially in the non-cytosolic fraction when increased bioaccumulation takes place. In the cytosol of the gills, metals are associated with metallothionein, whereas in the digestive gland, the distribution of metals between metallothioneins and high molecular weight proteins is similar. Metallothionein variation cannot be explained by metals alone; other abiotic factors must be taken into account. In order to use metallothionein as a metal exposure biomarker in field studies, natural variability needs to be taken into account for the correct interpretation of results.

  8. High resolution alpha-autoradiography for measurement of 10B distribution in subcellular scale using CR-39 and AFM

    International Nuclear Information System (INIS)

    Amemiya, K.; Takahashi, H.; Yasuda, N.

    2000-01-01

    In order to measure 10 B distribution in tumor tissues for BNCT at subcellular scale, we have developed a new method for high resolution alpha-autoradiography using contact X-ray microscopy technique with CR-39 plastic track detectors. Sliced sections of boron-injected brain tumors in rats were mounted on CR-39 and irradiated with thermal neutrons at KUR. Then the samples were exposed to soft X-rays from a laser plasma source. After etching the CR-39 in NaOH solution for a short time (1-5 min.), transmission X-ray image of tumor cells appeared as relief on CR-39 surface, and could be observed with the atomic force microscopy (AFM). Very small etch pits of about 100 nm in diameter corresponding to particle tracks from 10 B(n, α) 7 Li reactions were also observed in the image simultaneously. This method provides an accurate distribution of 10 B inside the cell. (author)

  9. Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

    Science.gov (United States)

    Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

    2009-01-01

    Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

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

    Science.gov (United States)

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

    1987-09-01

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

  11. A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout.

    Science.gov (United States)

    McConnell, Gail; Trägårdh, Johanna; Amor, Rumelo; Dempster, John; Reid, Es; Amos, William Bradshaw

    2016-09-23

    Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.

  12. Ex situ Flora of China

    Directory of Open Access Journals (Sweden)

    Hongwen Huang

    2017-12-01

    Full Text Available The role of living collections-based research and discovery has been a prominent feature throughout the history of evolution and advance of botanical science: such research is the core and soul of the botanical gardens. Currently, there are c. 162 Chinese botanical gardens, harboring c. 20,000 species in China. As an example of initiatives to utilize the garden cultivated flora to address plant diversity conservation and germplasm discovery for sustainable agriculture and the bio-industries, the Ex situ Flora of China project aims to catalog and document this mega-diversity of plants that are cultivated in the Chinese botanical gardens. The concept of Ex situ Flora of China is a complete new formulation of species, based on garden cultivated individuals and populations, to obtain better morphological descriptions, provide multi-purpose applicability and a fundamental data service that will support national bio-strategies and bio-industries. It emphasises integrative information, accurately collected from living collections across different Chinese botanical gardens, on biology, phenology, cultivation requirements and uses of plant resources, which are normally not available from traditional Floras based on herbarium specimens. The ex situ flora should provide better information coverage for taxonomy, biological and introduction and collection data and color photos of stems, leaves, flowers, fruits and seed, as well as useful information of cultivation key points and main use of each plant. In general, the Ex situ Flora of China provides more useful information than the traditional Flora Reipublicae Popularis Sinicae. The project of Ex situ Flora of China is planned to be one of the most important initiatives of the plant diversity research platform for sustainable economic and social development in China.

  13. Pronounced limb and fibre type differences in subcellular lipid droplet content and distribution in elite skiers before and after exhaustive exercise

    DEFF Research Database (Denmark)

    Koh, Han-Chow E; Nielsen, Joachim; Saltin, Bengt

    2017-01-01

    Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Science.gov (United States)

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

  16. High-resolution sub-cellular imaging by correlative NanoSIMS and electron microscopy of amiodarone internalisation by lung macrophages as evidence for drug-induced phospholipidosis.

    Science.gov (United States)

    Jiang, Haibo; Passarelli, Melissa K; Munro, Peter M G; Kilburn, Matt R; West, Andrew; Dollery, Colin T; Gilmore, Ian S; Rakowska, Paulina D

    2017-01-26

    Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation of the drug at a sub-cellular level and reveals its accumulation within the lysosomes, providing direct evidence for amiodarone-induced phospholipidosis. Chemical fixation using tannic acid effectively seals cellular membranes aiding intracellular retention of diffusible drugs.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2012-03-09

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

  2. Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution.

    Science.gov (United States)

    Porras, Pablo; McDonagh, Brian; Pedrajas, Jose Rafael; Bárcena, J Antonio; Padilla, C Alicia

    2010-04-01

    We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide. Copyright 2009 Elsevier B.V. All rights reserved.

  3. 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)

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

    Science.gov (United States)

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

    2017-11-15

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

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

    Science.gov (United States)

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

    2013-04-05

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

  6. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    Directory of Open Access Journals (Sweden)

    Takemoto Daigo

    2008-06-01

    Full Text Available Abstract Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the

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

    Science.gov (United States)

    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 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 mg·kg−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. PMID:23139811

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

    Directory of Open Access Journals (Sweden)

    Angela C Cone

    2013-02-01

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

  9. Effects of cooking and subcellular distribution on the bioaccessibility of trace elements in two marine fish species.

    Science.gov (United States)

    He, Mei; Ke, Cai-Huan; Wang, Wen-Xiong

    2010-03-24

    In current human health risk assessment, the maximum acceptable concentrations of contaminants in food are mostly based on the total concentrations. However, the total concentration of contaminants may not always reflect the available amount. Bioaccessibility determination is thus required to improve the risk assessment of contaminants. This study used an in vitro digestion model to assess the bioaccessibility of several trace elements (As, Cd, Cu, Fe, Se, and Zn) in the muscles of two farmed marine fish species (seabass Lateolabrax japonicus and red seabream Pagrosomus major ) of different body sizes. The total concentrations and subcellular distributions of these trace elements in fish muscles were also determined. Bioaccessibility of these trace elements was generally high (>45%), and the lowest bioaccessibility was observed for Fe. Cooking processes, including boiling, steaming, frying, and grilling, generally decreased the bioaccessibility of these trace elements, especially for Cu and Zn. The influences of frying and grilling were greater than those of boiling and steaming. The relationship of bioaccessibility and total concentration varied with the elements. A positive correlation was found for As and Cu and a negative correlation for Fe, whereas no correlation was found for Cd, Se, and Zn. A significant positive relationship was demonstrated between the bioaccessibility and the elemental partitioning in the heat stable protein fraction and in the trophically available fraction, and a negative correlation was observed between the bioaccessibility and the elemental partitioning in metal-rich granule fraction. Subcellular distribution may thus affect the bioaccessibility of metals and should be considered in the risk assessment for seafood safety.

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

    Science.gov (United States)

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

    2013-12-20

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  12. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

    DEFF Research Database (Denmark)

    Pauli, Andrea; Valen, Eivind; Lin, Michael F.

    2012-01-01

    Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in human and mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during...... of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression...... and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms...

  13. Noise canceling in-situ detection

    Science.gov (United States)

    Walsh, David O.

    2014-08-26

    Technologies applicable to noise canceling in-situ NMR detection and imaging are disclosed. An example noise canceling in-situ NMR detection apparatus may comprise one or more of a static magnetic field generator, an alternating magnetic field generator, an in-situ NMR detection device, an auxiliary noise detection device, and a computer.

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

    Science.gov (United States)

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

    2008-09-05

    Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell