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Sample records for accumulation subcellular fate

  1. 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.

  2. [Influence of NTA on accumulation and subcellular distribution of copper and zinc in corn (Zea mays)].

    Science.gov (United States)

    Zhou, Jian-min; Dang, Zhi; Tao, Xue-qin; Zhou, Yong-zhang

    2005-11-01

    The differential centrifugation method was used to study the subcellular distribution of Cu and Zn in the roots, stems and leaves of corn (zea mays) growing on multi-metal contaminated soil with the addition of chelator Nitrilotriacetic Acid (NTA). The results show that the subcellular distributions of Cu and Zn have significant relationship with the ability of metal uptake and accumulation in corn. NTA could evidently promote the uptake and accumulation of Cu and Zn in corn and affect on their distribution in cell wall and vacuole. Most of Cu was bound to the cell wall fraction and secondly cytoplasm fraction and only a small quantity of Cu bound to organelle fractions. For Zn, however, most of Zn was bound to cytoplasm fraction and there was more Zn bound to organelles than Cu. Under the inducement of NTA, there were increasing Cu and Zn bound to cytoplasm fraction, which mostly came from cell wall fraction and partly came from organelle fractions.

  3. [Cadmium accumulation, subcellular distribution, and chemical forms in Vitis vinifera cv. chardonnay grapevine].

    Science.gov (United States)

    Du, Yuan-Peng; Li, Hong-Jing; Yin, Ke-Lin; Zhai, Heng

    2012-06-01

    A pot culture experiment was conducted to study the Cd absorption, Cd subcellular distribution, and Cd chemical forms in one-year old self-rooted Chardonnay and SO4 rootstock-grafted Chardonnay grapevine after root-irrigating with different concentration CdCl and CaCl2. In the grapevine, the absorbed Cd was mostly distributed in underground organs (root and rhizome). In treatment 4 mmol x L(-1) of CdCl2, 77.1% and 1.4% of the absorbed Cd in self-rooted Chardonnay were accumulated in underground organs and leaves, respectively, while 93.9% and 0.1% of the absorbed Cd in grafted Chardonnay were accumulated in the organs below graft position and in leaves, respectively. 5 mmol L(-1) of CaCl2 decreased the plant Cd absorption and accumulation, while 10 mmol x L(-1) of CaCl2 increased the plant Cd absorption and accumulation significantly. The Cd subcellular distribution in roots and leaves was in the order of cell wall > soluble fraction > organelle, and more than 50% of the Cd was accumulated in cell wall. In the roots, NaCl-extractable Cd had a major proportion, followed by HAc-extractable Cd, and water-extractable Cd. The contents of all the Cd chemical forms varied with the increasing concentration of Cd in the treatments.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

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    Ting eZhao

    2016-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    YANG; ZHENGSHAOJIAN; 等

    1999-01-01

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

  7. Integration of Cadmium Accumulation, Subcellular Distribution, and Physiological Responses to Understand Cadmium Tolerance in Apple Rootstocks

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    Jiangtao Zhou

    2017-06-01

    Full Text Available Cadmium (Cd is a nonessential and highly toxic element causing agricultural problems. However, little information is available about the variation in Cd tolerance among apple rootstocks and its underlying physiological regulation mechanisms. This study investigated Cd accumulation, subcellular distribution, and chemical forms as well as physiological changes among four apple rootstocks exposed to either 0 or 300 μM CdCl2. The results showed that variations in Cd tolerance existed among these rootstocks. Cd exposure caused decline in photosynthesis, chlorophyll and biomass in four apple rootstocks, which was less pronounced in M. baccata, indicating its higher Cd tolerance. This finding was corroborated with higher Cd tolerance indexes (TIs of the whole plant in M. baccata than those in the other three apple rootstocks. Among the four apple rootstocks, M. baccata displayed the lowest Cd concentrations in roots, wood, and leaves, the smallest total Cd amounts as well as the lowest BCF. In apple rootstocks, it was found that to immobilize Cd in cell wall and soluble fraction (most likely in vacuole and to convert it into pectate- or protein- integrated forms and undissolved Cd phosphate forms may be the primary strategies to reduce Cd mobility and toxicity. The physiological changes including ROS, carbohydrates and antioxidants were in line with the variations of Cd tolerance among four apple rootstocks. In comparison with the other three apple rootstocks, M. baccata had lower concentrations of ROS in roots and bark, H2O2 in roots and leaves and MDA in roots, wood and bark, but higher concentrations of soluble sugars in bark and starch in roots and leaves, and enhanced antioxidants. These results indicate that M. baccata are more tolerant to Cd stress than the other three apple rootstocks under the current experiment conditions, which is probably related to Cd accumulation, subcellular partitioning and chemical forms of Cd and well

  8. Nickel tolerance, accumulation and subcellular distribution in the halophytes Sesuvium portulacastrum and Cakile maritima.

    Science.gov (United States)

    Fourati, Emna; Wali, Mariem; Vogel-Mikuš, Katarina; Abdelly, Chedly; Ghnaya, Tahar

    2016-11-01

    It has been shown that halophytes are able to successfully cope with heavy metal toxicity, suggesting their possible use for remediation of metal contaminated soils. In this work, Ni tolerance and accumulation in two halophytes, Sesuvium portulacastrum (L.) L. and Cakile maritima Scop. was investigated. Seedlings of both species were subjected hydroponically during 21 days to 0, 25, 50, and 100 μM of NiCl2. The growth and photosynthesis parameters revealed that S. portulacastrum tolerates Ni better than C. maritima. The photosynthesis activity, chlorophyll content and photosystem II integrity were less impacted in Ni-treated S. portulacastrum as compared to C. maritima, although, Ni accumulated in higher concentrations in the shoots of S. portulacastrum (1050 μg g(-1) DW) than in those of C. maritima (550 μg g(-1) DW). The subcellular fractionation of Ni in the shoots of both species showed that C. maritima accumulated about 65% of Ni in the soluble fraction, while 28% was associated with the cell walls. In S. portulacastrum 44% of the total cellular Ni was seen in the soluble fraction and 43% was bound to the cell walls. It can be concluded that S. portulacastrum tolerates Ni better than C. maritima, most probably due to a better ability to sequester Ni in the cell walls, restricting its accumulation in the soluble fraction. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Cadmium uptake, chemical forms, subcellular distribution, and accumulation in Echinodorus osiris Rataj.

    Science.gov (United States)

    Zhang, Chaolan; Zhang, Peng; Mo, Chuangrong; Yang, Weiwei; Li, Qinfeng; Pan, Liping; Lee, D K

    2013-07-01

    Phytoremediation is a technology for extracting or inactivating pollutants in soil. Echinodorus osiris (E. osiris) is a fast growing perennial wetland plant that is common in tropical and subtropical areas and has a high tolerance to cadmium (Cd). However, the absorption dynamics, subcellular distribution and accumulation of Cd by E. osiris had not been investigated. In this paper, hydroponic experiments with different levels of Cd(2+) (0, 5.0, 15.0 mg L(-1)) were carried out to determine these characteristics of E. osiris. The results indicated that the Cd absorption rate of Echinodorus osiris decreased over time, and the absorption rate within 0.5-1.0 h was faster than after 1.0 h. In a 6.0 hour time period, the rate of Cd uptake fit a quadratic polynomial curve when E. osiris was grown under the 5 mg L(-1) Cd treatment. However, the rate of Cd uptake by E. osiris fit a cubic polynomial model with the 15 mg L(-1) Cd treatment. In the roots, the ethanol-extractable Cd, water-extractable Cd, and NaCl-extractable Cd were the largest proportions of the total Cd. The HAc-extractable Cd, HCl-extractable Cd, and residual-Cd represented a larger proportion of the total Cd in the leaves which was combined with phosphate including CdHPO4, Cd3 (PO4)2, and oxalic acid. When analyzing the subcellular distribution of Cd in the plant, the soluble fraction containing Cd accounted for the largest part (69.49-88.39%) followed by the Cd bound to the cell wall (8.44-25.62%). Both the lower and the higher Cd treatments demonstrated that compartmentation by the vacuole and cell wall binding were two effective defense mechanisms of the plant. However, the vacuole became the main site for Cd accumulation in the leaves under the 15 mg L(-1) Cd treatment. E. osiris was able to accumulate high concentrations of Cd in both the roots and the leaves. The Cd concentration reached 502.97 mg kg(-1) and 2742.95 mg kg(-1) in the shoots and roots, respectively, after 27 days of cultivation. It was

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  11. Pb uptake, accumulation, subcellular distribution in a Pb-accumulating ecotype of Sedum alfredii (Hance)

    Institute of Scientific and Technical Information of China (English)

    何冰; 杨肖娥; 倪吾钟; 魏幼璋; 叶海波

    2003-01-01

    Lead concentrations in roots, stems and leaves of accumulating and non-accumulating ecotypes of Sedum alfredii (Hance) were studied through a hydroponic experiment with different Pb concentrations sup-plied as Pb( NO3 )2 .Lead concentrations in leaves and stems of the accumulating ecotype were 4-9 times and 3-5 times those of the non-accumulating ecotype, and Pb-accumulated amounts in stems and leaves of theaccumulating ecotype were 4-9 times and 8-1l times higher than those of the non-accumulating ecotype,respectively. The results indicated that the accumulating ecotype had better ability to transport Pb from roots toshoots. The subeellular distributions of Pb in the root, stem and leaf tissues were studied using sucrose differ-ential centrifugation. Approximately 50% of Pb contents was found to be associated with the cell wall fractionin stems of the accumulating ecotype and the percentage increased to 80% both in roots and leaves, no matter when plants were grown with different levels of Pb. The results indicated that the distribution of Pb on cell walls of the accumulating ecotype could mainly account for the high tolerance to Pb.

  12. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii.

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    Qiu, Rong-Liang; Thangavel, Palaniswamy; Hu, Peng-Jie; Senthilkumar, Palaninaicker; Ying, Rong-Rong; Tang, Ye-Tao

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii

    Energy Technology Data Exchange (ETDEWEB)

    Qiu Rongliang, E-mail: eesqrl@mail.sysu.edu.cn [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Guangdong Provincial Key Lab of Environmental Pollution Control and Remediation Technology, Guangzhou 510275 (China); Thangavel, Palaniswamy; Hu Pengjie; Senthilkumar, Palaninaicker; Ying Rongrong [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Tang Yetao [School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275 (China); Guangdong Provincial Key Lab of Environmental Pollution Control and Remediation Technology, Guangzhou 510275 (China)

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii.

  14. Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

    Energy Technology Data Exchange (ETDEWEB)

    Sokolova, I.M. [Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States)]. E-mail: insokolo@uncc.edu; Ringwood, A.H. [Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States); Johnson, C. [Johnson C. Smith University, 100 Beatties Ford Road, Charlotte, NC 28216 (United States)

    2005-09-10

    Cadmium distribution was studied in different subcellular fractions of gill and hepatopancreas tissues of eastern oysters Crassostrea virginica. Oysters were exposed for up to 21 days to low sublethal Cd concentrations (25 {mu}g L{sup -1}). Gill and hepatopancreas tissues were sampled and divided into organelle fractions and cytosol by differential centrifugation. Organelle content of different fractions was verified by activities of marker enzymes, citrate synthase and acid phosphatase for mitochondria and lysosomes, respectively. In both tissue types, there was a significant accumulation of cadmium in cytosol reaching 230-350 ng mg{sup -1} protein. Among organelles, mitochondria were the main target for Cd bioaccumulation in gills (250-300 ng mg{sup -1} protein), whereas in hepatopancreas tissues, the highest cadmium accumulation occurred in lysosomes (90-94 ng mg{sup -1} protein). Although 75-83% of total cadmium burden was associated with the cytosol reflecting high volume fraction of this compartment, Cd concentrations in organelle fractions reached levels that could cause dysfunction of mitochondria and lysosomes. Organ- and organelle-specific patterns of cadmium bioaccumulation support our previous in vivo studies, which showed adverse effects of cadmium exposures on mitochondrial oxidation in gills and on the lysosomal system of hepatopancreas. This may have important implications for the development of biomarkers of effect for heavy metals and for understanding the mechanisms of toxic effects of metals.

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

    Science.gov (United States)

    Araújo, Olinda; Pereira, Patrícia; Cesário, Rute; Pacheco, Mário; Raimundo, Joana

    2015-06-15

    Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas - contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys+Mic). At LAR, Lys+Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions.

  16. The accumulation and subcellular distribution of arsenic and antimony in four fern plants.

    Science.gov (United States)

    Feng, R; Wang, X; Wei, C; Tu, S

    2015-01-01

    In the present study, Pteris cretica 'Albo-Lineata' (PC), Pteris fauriei (PF), Humata tyermanii Moore (HT), and Pteris ensiformis Burm (PE), were selected to explore additional plant materials for the phytoremediation of As and Sb co-contamination. To some extent, the addition of As and Sb enhanced the growth of HT, PE, and PF. Conversely, the addition of As and Sb negatively affected the growth of PC and was accompanied with the accumulation of high levels of As and Sb in the roots. The highest concentration of Sb was recorded as 6405 mg kg(-1) in the roots of PC, and that for As was 337 mg kg(-1) in the rhizome of PF. To some degree, As and Sb stimulated the uptake of each other in these ferns. Arsenic was mainly stored in the cytoplasmic supernatant (CS) fraction, followed by the cell wall (CW) fraction. In contrast, Sb was mainly found in the CW fraction and, to a lesser extent, in the CS fraction, suggesting that the cell wall and cytosol play different roles in As and Sb accumulation by fern plants. This study demonstrated that these fern plants show a good application potential in the phytoremediation of As and Sb co-contaminated environments.

  17. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    Science.gov (United States)

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  18. Subcellular cadmium distribution and antioxidant enzymatic activities in the leaves of two castor (Ricinus communis L.) cultivars exhibit differences in Cd accumulation.

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    Zhang, Hanzhi; Guo, Qingjun; Yang, Junxing; Shen, Jianxiu; Chen, Tongbin; Zhu, Guangxu; Chen, Hui; Shao, Chunyan

    2015-10-01

    The aims of this study were: (1) the study of cadmium (Cd) accumulation and toxicity in different castor cultivars (Ricinus communis L.); (2) to investigate changes in antioxidant enzymatic activities and the subcellular distribution of Cd in young and old leaves from two different castor cultivars, after exposure to two different Cd concentrations, and explore the underlying mechanism of Cd detoxification focusing on antioxidant enzymes and subcellular compartmentalization. The Cd concentration, toxicity, and subcellular distribution, as well as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were measured in Zibo-3 and Zibo-9 cultivars after exposure to two different concentrations of Cd (2mg/L and 5mg/L) for 10 days. This research revealed Cd accumulation characteristics in castor are root>stem>young leaf>old leaf. Castor tolerance was Cd dose exposure and the cultivars themselves dependent. Investigation of subcellular Cd partitioning showed that Cd accumulated mainly in the heat stable protein (HSP) and cellular debris fractions, followed by the Cd rich granule (MRG), heat denatured protein (HDP), and organelle fractions. With increasing Cd concentration in nutrient solution, the decreased detoxified fractions (BDM) and the increased Cd-sensitive fractions (MSF) in young leaves may indicate the increased Cd toxicity in castor cultivars. The BDM-Cd fractions or MSF-Cd in old leaves may be linked with Cd tolerance of different cultivars of castor. The antioxidant enzymes that govern Cd detoxification were not found to be active in leaves. Taken together, these results indicate Cd tolerance and toxicity in castor can be explained by subcellular partitioning.

  19. The fate of fertilizer nitrogen in a high nitrate accumulated agricultural soil.

    Science.gov (United States)

    Quan, Zhi; Huang, Bin; Lu, Caiyan; Shi, Yi; Chen, Xin; Zhang, Haiyang; Fang, Yunting

    2016-02-12

    Well-acclimatized nitrifiers in high-nitrate agricultural soils can quickly nitrify NH4(+) into NO3(-) subject to leaching and denitrifying loss. A 120-day incubation experiment was conducted using a greenhouse soil to explore the fates of applied fertilizer N entering into seven soil N pools and to examine if green manure (as ryegrass) co-application can increase immobilization of the applied N into relatively stable N pools and thereby reduce NO3(-) accumulation and loss. We found that 87-92% of the applied (15)N-labelled NH4(+) was rapidly recovered as NO3(-) since day 3 and only 2-4% as microbial biomass and soil organic matter (SOM), while ryegrass co-application significantly decreased its recovery as NO3(-) but enhanced its recovery as SOM (17%) at the end of incubation. The trade-off relationship between (15)N recoveries in microbial biomass and SOM indicated that ryegrass co-application stabilized newly immobilized N via initial microbial uptake and later breakdown. Nevertheless, ryegrass application didn't decrease soil total NO3(-) accumulation due to its own decay. Our results suggest that green manure co-application can increase immobilization of applied N into stable organic N via microbial turnover, but the quantity and quality of green manure should be well considered to reduce N release from itself.

  20. When nanoparticles meet biofilms - Interactions guiding the environmental fate and accumulation of nanoparticles

    Directory of Open Access Journals (Sweden)

    Kaoru eIkuma

    2015-06-01

    Full Text Available Bacteria are essential components of all natural and many engineered systems. The most active fractions of bacteria are now recognized to occur as ‘biofilms’, where cells are attached and surrounded by a secreted matrix of sticky extracellular polymeric substances (EPS. Recent investigations have established that significant accumulation of nanoparticles (NPs occurs in aquatic biofilms. These studies point to the emerging roles of biofilms for influencing partitioning and possibly transformations of NPs in both natural and engineered systems. While attached biofilms are efficient sponges for NPs, efforts to elucidate the fundamental mechanisms guiding interactions between NPs and biofilms have just begun. In this mini review, special attention is focused on NP-biofilm interactions within the aquatic environment. We highlight key physical, chemical and biological processes that affect interactions and accumulation of NPs by bacterial biofilms. We posit that these biofilm processes present the likely possibility for unique biological and chemical transformations of NPs. Ultimately, the environmental fate of NPs is influenced by biofilms, and therefore requires a more-in depth understanding of their fundamental properties.

  1. Engineering metal-binding sites of bacterial CusF to enhance Zn/Cd accumulation and resistance by subcellular targeting

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Pengli; Yuan, Jinhong [Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China); Zhang, Hui [Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China); Deng, Xin [Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637 (United States); Ma, Mi [Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China); Zhang, Haiyan, E-mail: hyz@ibcas.ac.cn [Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China)

    2016-01-25

    Highlights: • mCusF is specifically targeted to different subcellular compartments in Arabidopsis. • Plants expressing vacuole-targeted mCusF exhibit strongest Zn resistance. • All transgenic lines accumulate more Zn under Zn exposure. • All transgenic lines enhance root-to-shoot translocation of Cd. • Metal homeostasis is improved in mCusF plants under Cd exposure. - Abstract: The periplasmic protein CusF acts as a metallochaperone to mediate Cu resistance in Escherichia coli. CusF does not contain cysteine residues and barely binds to divalent cations. Here, we addressed effects of cysteine-substitution mutant (named as mCusF) of CusF on zinc/cadmium (Zn/Cd) accumulation and resistance. We targeted mCusF to different subcellular compartments in Arabidopsis. We found that plants expressing vacuole-targeted mCusF were more resistant to excess Zn than WT and plants with cell wall-targeted or cytoplasmic mCusF. Under long-term exposure to excess Zn, all transgenic lines accumulated more Zn (up to 2.3-fold) in shoots than the untransformed plants. Importantly, plants with cytoplasmic mCusF showed higher efficiency of Zn translocation from root to shoot than plants with secretory pathway-targeted-mCusF. Furthermore, the transgenic lines exhibited enhanced resistance to Cd and significant increase in root-to-shoot Cd translocation. We also found all transgenic plants greatly improved manganese (Mn) and iron (Fe) homeostasis under Cd exposure. Our results demonstrate heterologous expression of mCusF could be used to engineer a new phytoremediation strategy for Zn/Cd and our finding also deepen our insights into mechanistic basis for relieving Cd toxicity in plants through proper root/shoot partitioning mechanism and homeostatic accumulation of Mn and Fe.

  2. 茭白对铬的吸收积累及其亚细胞分布%Cr Accumulation and Sub-cellular Distribution in Zizania latifolia

    Institute of Scientific and Technical Information of China (English)

    朱四喜; 王凤友; 刘文; 史云勋; 杨秀琴; 吴云杰

    2014-01-01

    为茭白在水体铬污染修复的运用提供科学依据,在模拟小型湿地试验系统中,用不同浓度(0,5 mg/L,10 mg/L,20 mg/L,40 mg/L,60 mg/L)的含铬(Ⅵ)废水进行污染胁迫,研究在不同 Cr(Ⅵ)浓度处理下,茭白的生长状况、各器官中铬的含量与积累量及其亚细胞的分布特征。结果表明,不同铬浓度处理下,茭白各器官生物量表现为根>叶>茎,且在铬胁迫浓度为20 mg/L时,株高、根长、生物量均较大;相同 Cr浓度胁迫下,不同器官中铬含量不同,总趋势为茎>根>叶;茭白对铬的耐性指数与转运系数均较高,且在20 mg/L时最高;茭白各器官亚细胞中铬分布总趋势为胞液>细胞壁>细胞器。结论:茭白对低浓度(0~20 mg/L)水体铬污染具有较强的积累力、转运力和耐受力,在水体铬污染植物修复中具有一定的应用价值。%Z.latifolia plants were cultivated in waste water with different Cr concentrations to study their growth status,Cr accumulation in different organs and sub-cellular distribution characteristics under different Cr stress and to provide the scientific basis for phytoremediation of;Z.latifolia in Cr pollution of water.The biomass of different organs was root leaf stem under different Cr concentrations and Z. latifolia had higher height,root length and biomass when Cr concentration was 20mg/L.The general trend of Cr content and Cr accumulation in different organs was stem root leaf and root stem leaf under the same Cr concentration.Z.latifolia had higher Cr tolerance index and transfer coefficient and the Cr tolerance index and transfer coefficient was up to the highest when Cr concentration was 20mg/L.Cr was distributed in cell wall and cytosol mainly and the general trend of Cr distribution in sub-cellular level was cytosol cell wall organelle.In conclusion,Z.latifolia with the strong accumulation ability,transfer ability and tolerance

  3. Nanomaterial translocation - the biokinetics, tissue accumulation, toxicity and fate of materials in secondary organs

    DEFF Research Database (Denmark)

    Kermanizadeh, Ali; Balharry, Dominique; Wallin, Håkan

    2015-01-01

    into the toxicity posed by the NMs in these secondary organs is expanding due to the realisation that some materials may reach and accumulate in these target sites. The translocation to secondary organs includes, but is not limited to, the hepatic, central nervous, cardiovascular and renal systems. Current data......Engineered nanomaterials (NMs) offer great technological advantages but their risks to human health are still not fully understood. An increasing body of evidence suggests that some NMs are capable of distributing from the site of exposure to a number of secondary organs. The research...... the gastrointestinal tract seems to follow the same pattern as inhalation translocation, whereas the dermal uptake of NMs is generally reported to be low. The toxicological effects in secondary organs include oxidative stress, inflammation, cytotoxicity and dysfunction of cellular and physiological processes...

  4. Subcellular Size

    Science.gov (United States)

    Marshall, Wallace F.

    2015-01-01

    All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all of its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something? PMID:25957302

  5. Influence of a step-change in metal exposure (Cd, Cu, Zn) on metal accumulation and subcellular partitioning in a freshwater bivalve, Pyganodon grandis: A long-term transplantation experiment between lakes with contrasting ambient metal levels

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Sophie [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada); Bonneris, Emmanuelle [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada) and Bayer S.A.S., Bayer CropScience, 16 Rue Jean-Marie Leclair, CP 90106, F 69266 Lyon Cedex 09 (France); Michaud, Annick [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada) and Direction des Évaluations environnementales, Ministère du Développement durable, de l’Environnement et des Parcs, 675, boul. René-Lévesque Est, 6e étage, Québec, QC G1R 5V7 (Canada); Pinel-Alloul, Bernadette [Groupe de Recherche Interuniversitaire en Limnologie et Environnement Aquatique (GRIL), Département de Sciences Biologiques, Université de Montréal, C.P. 6128, Montréal, QC H3C 3J7 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [INRS-Eau, Terre et Environnement, Université du Québec, 490 de la Couronne, Québec, QC G1K 9A9 (Canada)

    2013-05-15

    Highlights: ? We transferred freshwater bivalves from a reference lake to a Cd and Zn contaminated lake. ? Changes in metal accumulation and subcellular partitioning were followed over time (up to 860 d). ? Metal detoxification strategies differed between target organs (gills vs. digestive gland). ? The ability to handle Cd is inherent in P. grandis, not a trait acquired after long-term adaptation. -- Abstract: The objective of the present field experiment was to identify detoxification responses in the gills and digestive gland of a freshwater unionid bivalve, Pyganodon grandis, subjected to a step-change in metal exposure. Adult bivalves were transferred from a reference site (Lake Opasatica) and a metal-contaminated lake (Lake Héva) to a second contaminated lake (Lake Vaudray) in northwestern Quebec, Canada. Changes in organ metal concentrations, in the subcellular distribution of metals and in metallothionein concentrations were followed over time (t = 0, 132, (400) and 860 days). At each collection time and for each bivalve, the gills and digestive gland were excised and gently homogenized; six sub-cellular fractions were separated by differential centrifugation and analyzed for their Cd, Cu and Zn content, and metallothionein was quantified independently. Metal detoxification strategies were shown to differ between target organs: in the gills, incoming metals were sequestered largely in the granules, whereas in the digestive gland the same metals primarily accumulated in the cytosol, in the metallothionein-like protein fraction. These metal-handling strategies, as employed by the metal-naïve bivalves originating in the reference lake, closely resemble those identified in free-living P. grandis chronically exposed in the metal-contaminated lake, suggesting that the ability to handle incoming metals (Cd in particular) is inherent in P. grandis and is not a trait acquired after long-term adaptation of the bivalve to metal-contaminated environments. The

  6. Silver nanoparticle accumulation by aquatic organisms – neutron activation as a tool for the environmental fate of nanoparticles tracing

    Directory of Open Access Journals (Sweden)

    Asztemborska Monika

    2014-12-01

    Full Text Available Water environments are noted as being some of the most exposed to the influence of toxic nanoparticles (NPs. Therefore, there is a growing need for the investigation of the accumulation and toxicity of NPs to aquatic organisms. In our studies neutron activation followed by gamma spectrometry and liquid scintillation counting were used for studying the accumulation of silver nanoparticles (AgNPs by freshwater larvae of Chironomus and fish Danio rerio. The influence of exposition time, concentration and the source of nanoparticles on the efficiency of AgNP accumulation were studied. It was found that AgNPs are efficiently accumulated by Chironomid larvae for the first 30 hours of exposition; then, the amount of silver nanoparticles decreases. The silver content in larvae increases together with the NP concentration in water. Larvae which have accumulated AgNPs can be a source of nanoparticles for fish and certainly higher levels of Ag in the trophic chain. In comparison with water contamination, silver nanoparticles are more efficiently accumulated if fish are fed with AgNP-contaminated food. Finally, it was concluded that the applied study strategy, including neutron activation of nanoparticles, is very useful technique for tracing the uptake and accumulation of NPs in organisms

  7. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-01

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  8. Photothermal-triggered control of sub-cellular drug accumulation using doxorubicin-loaded single-walled carbon nanotubes for the effective killing of human breast cancer cells

    Science.gov (United States)

    Oh, Yunok; Jin, Jun-O.; Oh, Junghwan

    2017-03-01

    Single-walled carbon nanotubes (SWNTs) are often the subject of investigation as effective photothermal therapy (PTT) agents owing to their unique strong optical absorption. Doxorubicin (DOX)-loaded SWNTs (SWNTs-DOX) can be used as an efficient therapeutic agent for combined near infrared (NIR) cancer photothermal and chemotherapy. However, SWNTs-DOX-mediated induction of cancer cell death has not been fully investigated, particularly the reaction of DOX inside cancer cells by PTT. In this study, we examined how the SWNTs-DOX promoted effective MDA-MB-231 cell death compared to DOX and PTT alone. We successfully synthesized the SWNTs-DOX. The SWNTs-DOX exhibited a slow DOX release, which was accelerated by NIR irradiation. Furthermore, DOX released from the SWNTs-DOX accumulated inside the cells at high concentration and effectively localized into the MDA-MB-231 cell nucleus. A combination of SWNTs-DOX and PTT promoted an effective MDA-MB-231 cell death by mitochondrial disruption and ROS generation. Thus, SWNTs-DOX can be utilized as an excellent anticancer agent for early breast cancer treatment.

  9. 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

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

    Science.gov (United States)

    Thit, Amalie; Ramskov, Tina; Croteau, Marie-Noele; Selck, Henriette

    2016-01-01

    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 65Cu 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 handle the two Cu forms differently. However, longer-term exposures are suggested in order to clearly highlight differences in the subcellular distribution of these two Cu forms.

  11. Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1.

    Science.gov (United States)

    Tian, Qing; Olsen, Lene; Sun, Beimeng; Lid, Stein Erik; Brown, Roy C; Lemmon, Betty E; Fosnes, Kjetil; Gruis, Darren Fred; Opsahl-Sorteberg, Hilde-Gunn; Otegui, Marisa S; Olsen, Odd-Arne

    2007-10-01

    DEFECTIVE KERNEL1 (DEK1), which consists of a membrane-spanning region (DEK1-MEM) and a calpain-like Cys proteinase region (DEK1-CALP), is essential for aleurone cell formation at the surface of maize (Zea mays) endosperm. Immunolocalization and FM4-64 dye incubation experiments showed that DEK1 and CRINKLY4 (CR4), a receptor kinase implicated in aleurone cell fate specification, colocalized to plasma membrane and endosomes. SUPERNUMERARY ALEURONE LAYER1 (SAL1), a negative regulator of aleurone cell fate encoding a class E vacuolar sorting protein, colocalized with DEK1 and CR4 in endosomes. Immunogold localization, dual-axis electron tomography, and diffusion of fluorescent dye tracers showed that young aleurone cells established symplastic subdomains through plasmodesmata of larger dimensions than those connecting starchy endosperm cells and that CR4 preferentially associated with plasmodesmata between aleurone cells. Genetic complementation experiments showed that DEK1-CALP failed to restore wild-type phenotypes in maize and Arabidopsis thaliana dek1 mutants, and DEK1-MEM also failed to restore wild-type phenotypes in Arabidopsis dek1-1 mutants. Instead, ectopic expression of DEK1-MEM under the control of the cauliflower mosaic virus 35S promoter gave a dominant negative phenotype. These data suggest a model for aleurone cell fate specification in which DEK1 perceives and/or transmits a positional signal, CR4 promotes the lateral movement of aleurone signaling molecules between aleurone cells, and SAL1 maintains the proper plasma membrane concentration of DEK1 and CR4 proteins via endosome-mediated recycling/degradation.

  12. Subcellular site and nature of intracellular cadmium in plants

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, George J.

    1979-01-01

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

  13. ACCUMULATION AND FATE OF PERCHLORATE IN PLANTS

    Science.gov (United States)

    Perchlorate, a component of solid rocket fuels, has emerged as a potential threat to surface water and groundwater at several locations in the U.S. Perchlorate levels up to 16 ug/L were detected in Lake Mead and 5-9 ug/L in the lower Colorado River. The water from the Colorado Ri...

  14. Decision making at a subcellular level determines the outcome of bacteriophage infection.

    Science.gov (United States)

    Zeng, Lanying; Skinner, Samuel O; Zong, Chenghang; Sippy, Jean; Feiss, Michael; Golding, Ido

    2010-05-14

    When the process of cell-fate determination is examined at single-cell resolution, it is often observed that individual cells undergo different fates even when subject to identical conditions. This "noisy" phenotype is usually attributed to the inherent stochasticity of chemical reactions in the cell. Here we demonstrate how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the subcellular level. We follow the postinfection decision in bacteriophage lambda at single-virus resolution, and show that a choice between lysis and lysogeny is first made at the level of the individual virus. The decisions by all viruses infecting a single cell are then integrated in a precise (noise-free) way, such that only a unanimous vote by all viruses leads to the establishment of lysogeny. By detecting and integrating over the subcellular "hidden variables," we are able to predict the level of noise measured at the single-cell level.

  15. Sequence conserved for subcellular localization

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2002-01-01

    The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes. PMID:12441382

  16. Effects of Cd-spiking Treatment on Cd Accumulation, Subcellular Distribution and Content of Nonprotein Thiols in Rice%外加镉对水稻镉吸收、亚细胞分布及非蛋白巯基含量的影响

    Institute of Scientific and Technical Information of China (English)

    史静; 潘根兴

    2015-01-01

    staple crops, rice (Oryza sativa L.) is the particular food crop with high Cd uptake and accumulation in grains. There had been much evidences of differences in Cd accumulation in plants and plant tolerance to between rice species and genotypes. Meanwhile, Cd tolerance and low Cd accumulation in grains may be used as a strategy for low Cd rice production. However, the mechanisms involved in Cd tolerance and grain accumulation has been not clearly demonstrated. Studying cadmium absorption characteristics under different rice varieties with Cd contaminated soil, the detoxification mechanism of PCs (mainly Non-protein thiol) and from micro level explore the distribution of cadmium subcellular. Its has huge significance to understand the resistance mechanism of Cd in different rice genotypes. In an effort to understand the biological process of Cd bioaccumulation, a pot experiments with subcelluar fractionation were carried out to investigate the subcelluar distribution of Cd in roots and leaves of rice. Two rice cultivars, Zhongzheyou-1, a high grain Cd accumulation cultivar, and J196, a low Cd accumulating cultivar, were grown in red soils. The experiment was conducted to study the effect of Cd-spiking treatment on Cd content, nonprotein thiols (NPT) production and its subcellular distribution in rice. The results showed that Cd stress had significant inhibitory effects on NPT overproduction. The Cd uptake of rice was affected by the Cd-spiking treatment and the genotypes. Furthermore, the Cd content was enhanced with increasing Cd supply, coinciding with the enhancement of NPT level in root. Compared the two varieties, when soil without Cd. The ratio of J196 aboveground Cd content/root Cd content is higher than Zhong-Zhe Optimal number 1;when the soil Cd content was 5 mg·kg-1, the amount of root A transform Cd to aboveground is far more than C, respectively 25.7% and 7.4%; but when the soil Cd content was 25 mg·kg-1, there was no significant difference between the two

  17. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

    KAUST Repository

    Bayoumi, Maged Fouad

    2012-12-12

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

  18. 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.

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

    NARCIS (Netherlands)

    Oyoo-Okoth, E.; Admiraal, W.; Osano, O.; Kraak, M.H.S.; Gichuki, J.; Ogwai, C.

    2012-01-01

    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

  20. Of switches and hourglasses: regulation of subcellular traffic in circadian clocks by phosphorylation

    OpenAIRE

    Tataroğlu, Özgür; Schafmeier, Tobias

    2011-01-01

    A major aspect of molecular timekeeping is the daytime-specific nuclear accumulation of circadian clock proteins. The authors discuss recent insights into the regulation of subcellular shuttling and consider the importance of these cycles in regulating circadian period in different organisms.

  1. Subcellular effects of drought stress in Rosmarinus officinalis.

    Science.gov (United States)

    Olmos, E; Sánchez-Blanco, M J; Ferrández, T; Alarcón, J J

    2007-01-01

    The use of Rosmarinus officinalis, and other wild plant species, in the Mediterranean area is an interesting solution in order to avoid the desertification and rapid soil erosion, because of their good resistance to environmental conditions. Previous articles have described experiments designed to determine the impact of water stress at the plant level in this species, but more knowledge is required at the subcellular and ultrastructural levels. An anatomic and ultrastructural study of the leaves was conducted on Rosmarinus officinalis plants growing under different water treatments. In the leaves of water-stressed plants, the leaf water potential and turgor decreased, and leaf osmotic potential became more negative with respect to control plants. The anatomic investigations showed that both the mesophyll intercellular spaces and the epidermal cell size were reduced significantly under the more intense drought stress conditions. At the subcellular level, chloroplasts accumulated plastoglobuli and lipid bodies, and cuticle thickness was increased under water stress. In our experiment, the anatomic and ultrastructural modifications of Rosmarinus officinalis could be considered an additional adaptation to drought stress together with physiological and biochemical modifications as antioxidant accumulation.

  2. Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

    Science.gov (United States)

    Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

    2011-01-01

    Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

  3. 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

  4. 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.

  5. Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity.

    Directory of Open Access Journals (Sweden)

    Jun Wu

    2004-07-01

    Full Text Available The Frizzled (Fz; called here Fz1 and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.

  6. 外源一氧化氮介导铜胁迫下番茄幼苗中铁、锌、锰的累积及亚细胞分布%Exogenous nitric oxide involved in the accumulation and subcellular distribution of Fe, Zn and Mn in tomato seedlings under copper stress

    Institute of Scientific and Technical Information of China (English)

    张敏; 梁国鹏; 姜春辉; 崔秀敏

    2014-01-01

    A tomato cultivar, Gailiang Maofen 802F1, was selected as the planting material to investigate effects of NO on the absorption and distribution of Fe , Zn and Mn in tomato seedlings under copper stress using nutrient solution cultivation in greenhouse .The results show that the biomass and plant height of tomato seedlings are decreased by 33.7% and 23.1% respectively under the 50 μmol/L copper stress , while the addition of 100μmol/L SNP could remarkably alleviate this inhibition effect . For mineral elements , SNP could remarkably increase the Fe and Mn concentrations in roots and stems , and Fe and Zn concentrations in petioles and leaves , and the Zn concentration in stems and the Mn concentrations in petioles and leaves are reduced under the copper stress . Meanwhile the exogenous NO could improve Fe and Zn accumulation amounts in roots , stems, petioles and leaves and Mn accumulation amounts in roots and stems , while the exogenous NO reduces the transformation of the absorbed Fe , Zn and Mn red from roots to shoots .Under the Cu stress , the exogenous NO could remarkably increase the Fe and Zn concentrations in vacuole and organelle of all organs and decrease Fe , Zn and Mn concentrations in cell wall of roots and leaves .Mn mainly concentrates on the cell walls of stems and petioles that function as transport organizations , while its concentrations in vacuole and organelle of petioles and leaves are also increased .These results suggest that the exogenous NO could effectively regulate Fe , Zn and Mn rational distributions in organs and subcellular microzone under the Cu stress , so that the seedlings could keep a better balance between mineral nutrition elements and intracellular ion homeostasis to maintain normal physiological metabolism of tomato.%采用营养液培养方法,以“改良毛粉802F1”番茄为材料,研究外源一氧化氮( NO,SNP为供体)对铜( Cu)胁迫下番茄幼苗铁( Fe)、锌( Zn)、锰( Mn)吸收

  7. Subcellular characterization of glucose uptake in coronary endothelial cells.

    Science.gov (United States)

    Gaudreault, N; Scriven, D R L; Laher, I; Moore, E D W

    2008-01-01

    Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

  8. FY08 LDRD Final Report Stem Cell Fate Decisions

    Energy Technology Data Exchange (ETDEWEB)

    Hiddessen, A

    2009-03-02

    A detailed understanding of the biological control of fate decisions of stem and progenitor cells is needed to harness their full power for tissue repair and/or regeneration. Currently, internal and external factors that regulate stem cell fate are not fully understood. We aim to engineer biocompatible tools to facilitate the measurement and comparison of the roles and significance of immobilized factors such as extracellular matrix and signaling peptides, synergistic and opposing soluble factors and signals, and cell-to-cell communication, in stem cell fate decisions. Our approach is based on the development of cell microarrays to capture viable stem/progenitor cells individually or in small clusters onto substrate-bound signals (e.g. proteins), combined with conventional antibody and customized subcellular markers made in-house, to facilitate tracking of cell behavior during exposure to relevant signals. Below we describe our efforts, including methods to manipulate a model epithelial stem cell system using a custom subcellular reporter to track and measure cell signaling, arrays with surface chemistry that support viable cells and enable controlled presentation of immobilized signals to cells on the array and fluorescence-based measurement of cell response, and successful on-array tests via conventional immunofluorescence assays that indicate correct cell polarity, localization of junctional proteins, and phenotype, properties which are essential to measuring true cell responses.

  9. Birth and rapid subcellular adaptation of a hominoid-specific CDC14 protein.

    Directory of Open Access Journals (Sweden)

    Lia Rosso

    2008-06-01

    Full Text Available Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18-25 million years ago (Mya. CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7-12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function.

  10. CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Dosemeci, Ayse, E-mail: dosemeca@mail.nih.gov [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Thein, Soe; Yang, Yijung; Reese, Thomas S. [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Tao-Cheng, Jung-Hwa [EM Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. Black-Right-Pointing-Pointer Presence of CYLD in PSDs is established by biochemistry and immunoEM. Black-Right-Pointing-Pointer CYLD accumulates on PSDs upon depolarization of neurons. Black-Right-Pointing-Pointer Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

  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. Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation.

    Science.gov (United States)

    Dölle, Christian; Niere, Marc; Lohndal, Emilia; Ziegler, Mathias

    2010-02-01

    Poly-ADP-ribose polymerases (PARPs) use NAD(+) as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD(+) detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD(+) pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD(+) and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.

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

    Science.gov (United States)

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

    2016-03-22

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

  14. Subcellular compartmentation of glutathione in dicotyledonous plants

    Science.gov (United States)

    Müller, Maria

    2010-01-01

    This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method. PMID:20186447

  15. Subcellular distribution and translocation of radionuclides in plants

    Energy Technology Data Exchange (ETDEWEB)

    Gouthu, S.; Weginwar, R.; Arie, Tsutomu; Ambe, Shizuko; Ozaki, Takuo; Enomoto, Shuichi; Ambe, Fumitoshi; Yamaguchi, Isamu

    1999-09-01

    The subcellular distribution of radionuclides in Glycine max Merr. (soybean) and Cucumis sativus L. (cucumber) and translocation of plant absorbed radionuclides with growth in soybean were studied. More than 60% of cellular incorporated Rb{sup {minus}83}, Sr{sup {minus}85}, Mn{sup {minus}54}, Nb{sup {minus}95}, and Se{sup {minus}75} remained in the supernatant fraction; 55% and 20% of Cr{sup {minus}51} was bound to soybean and cucumber cell wall fractions, respectively; 70% or more of Be{sup {minus}7}, Y{sup {minus}88}, and Fe{sup {minus}59} was fixed in the chloroplast fraction; and approx. 10% of Sc{sup {minus}46}, Fe{sup {minus}59}, V{sup {minus}48}, and As were fixed in the mitochondrial fraction. Translocation of nuclides within the soybean plant at different stages of growth has been determined. Vanadium, Y{sup {minus}88}, Be{sup {minus}7}, Se{sup {minus}75}, Nb{sup {minus}95}, Sc{sup {minus}46}, Cr{sup {minus}51}, and Zr{sup {minus}88} were predominantly accumulated in the root. Although the total percentage of plant uptake of Sc{sup {minus}46}, Zr{sup {minus}88}, Nb{sup {minus}95}, Sc{sup {minus}46}, and Cr{sup {minus}51} was high, because of low mobility and translocation to shoot, their accumulation in the fruit fraction was negligible. The translocation of mobile nuclides in plants was demonstrated clearly by Rb{sup {minus}83}, Zn{sup {minus}65}, and Fe{sup {minus}59}. Data on the nuclide fraction mobilized from vegetative parts into edible parts was used to assess the percentage of accumulated radionuclides in plants that may reach humans through beans.

  16. Distribution, isomerization and enantiomer selectivity of hexabromocyclododecane (HBCD) diastereoisomers in different tissue and subcellular fractions of earthworms.

    Science.gov (United States)

    Li, Bing; Chen, Hao; Sun, Hongwen; Lan, Zhonghui

    2017-05-01

    In this study, earthworms Eisenia fetida (E. fetida) were exposed to a soil artificially contaminated with individual hexabromocyclododecane (HBCD) diastereoisomers (α-, β- and γ-HBCDs) to investigate the distribution, isomerization and enantiomer selectivity of HBCDs at tissue and subcellular levels. At the tissue level, the concentrations of HBCDs all followed the order of gut>bodyfluid>body wall, which suggested that earthworms accumulated HBCDs mainly via ingesting soil particles. At the subcellular level, the concentrations of HBCDs in an extracellular fraction consisting of granules, tissue fragment, cell membrane and intact cells (fraction A) were higher than those in an intracellular fractions consisting of the microsomal and cytosol (fraction B+C). This confirmed the passive diffusion during the distribution of HBCDs into the intracellular compartment. The distribution proportions of HBCDs varied among different tissue and subcellular fractions, and all changed over time within 14 days. The variable distributions of HBCDs in different fractions were a result of the comprehensive effects of dynamics and thermodynamics processes. The β- and γ-HBCDs were isomerized to α-HBCD in all tissue and subcellular fractions except for fraction C, and the isomerization ratios varied a lot, which seemed to be related to HBCDs residence time. The selective enrichment of (-) α-, (-) β and (-) γ-HBCDs was found in all fractions and this is consistent with that in the whole earthworm. Besides, the extents of enantio-selectivity did not change significantly among different tissue and subcellular fractions.

  17. Mercury Contamination: Fate and Risk Minimization Strategies

    Science.gov (United States)

    Charlet, L.

    Two river basins have been studied in French Guyana, which are subject to heavy mercury contamination, due to illegal gold mining. Within the framework of an interdisciplinary European project, the fate of mercury in water, air, soil, sediment has been studied, as well as its bio-accumulation in the food chain. This bioaccumulation results in the contamination of amerindian populations, through fish consumption. This study has been done in close contact with the economic and political actors. The results of the scientific interdisciplinary study has been translated in terms of risk minimization strategies, which are analyzed in the framework of the European Water Framework Directive.

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

    Science.gov (United States)

    Fan, Wenhong; Li, Qian; Yang, Xiuping; Zhang, Li

    2013-01-01

    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.

  19. Regulation of the MAPK family members: role of subcellular localization and architectural organization.

    Science.gov (United States)

    Fanger, G R

    1999-07-01

    The members of the mitogen-activated protein kinase (MAPK) family are regulated by a diverse array of extracellular cues ranging from cytokines, growth factors and neuropeptides, which activate cell surface receptors, to stresses such as cold, heat, osmolarity changes and irradiation. The MAPK pathways control genetic expression by modifying transcription factor activity and cue important cell fate decisions including survival, proliferation, and programmed cell death (apoptosis). One interesting feature of the MAPK pathways is that the components are evolutionarily conserved from yeast to human, and many of the pathways are similarly organized and regulated. Unlike previously imagined, architectural organization or the multimeric organization of signaling proteins into complexes which are localized to distinct subcellular regions is an important mechanism that influences the regulation of these pathways. In addition, extracellular stimuli can induce relocalization of specific signal transduction proteins. The formation of multimeric signaling complexes, as well as the dynamic movement of signaling proteins, contribute to determine signaling specificity and efficacy. This review describes what is currently known about the subcellular localization of MAPK pathway signaling proteins and the relocalization that occurs during events associated with activation of the MAPK family members.

  20. Subcellular localization of rickettsial invasion protein, InvA.

    Science.gov (United States)

    Gaywee, Jariyanart; Sacci, John B; Radulovic, Suzana; Beier, Magda S; Azad, Abdu F

    2003-01-01

    To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

  1. [Mechanism for subcellular localization of nuclear receptor CAR].

    Science.gov (United States)

    Kanno, Yuichiro; Inouye, Yoshio

    2011-03-01

    Animals including human beings have defense mechanisms against the toxicity of xenobiotics such as medicinal compounds and environmental pollutants. Receptor-type transcriptional factors, such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR), play important roles in the defense against xenobiotic toxicities. In the absence of stimuli, these receptors are distributed predominantly in the cytoplasmic compartment. Following xenobiotic stimuli, receptors translocate into the nucleus and transactivate its target genes. However, the exogenously expressed CAR translocates spontaneously into the nucleus in immortal cells. Previously, we identified subcellular localization signals in rat CAR: nuclear localization signal (NLS), nuclear export signal (NES) and cytoplasmic retention region (CRR). Lack of CRR function might be responsible for the spontaneous nuclear accumulation of CAR in immortal cells. Further, the nuclear import of CAR is regulated by the importin-Ran system, which is required for maintaining an intact microtubule network. Clarifying the mechanisms underlying the nuclear translocation of CAR would be useful for the establishment of novel assay systems for the screening of ligands and activators of CAR using immortal cells without sacrificing animals.

  2. Tuning the Catalytic Activity of Subcellular Nanoreactors.

    Science.gov (United States)

    Jakobson, Christopher M; Chen, Yiqun; Slininger, Marilyn F; Valdivia, Elias; Kim, Edward Y; Tullman-Ercek, Danielle

    2016-07-31

    Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity. Copyright © 2016. Published by Elsevier Ltd.

  3. 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 (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 components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root

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

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

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

  5. Influences of calcium silicate on chemical forms and subcellular distribution of cadmium in Amaranthus hypochondriacus L.

    Science.gov (United States)

    Lu, Huanping; Li, Zhian; Wu, Jingtao; Shen, Yong; Li, Yingwen; Zou, Bi; Tang, Yetao; Zhuang, Ping

    2017-01-01

    A pot experiment was conducted to investigate the effects of calcium silicate (CS) on the subcellular distribution and chemical forms of cadmium (Cd) in grain amaranths (Amaranthus hypochondriacus L. Cv. ‘K112’) grown in a Cd contaminated soil. Results showed that the dry weight and the photosynthetic pigments contents in grain amaranths increased significantly with the increasing doses of CS treatments, with the highest value found for the treatment of CS3 (1.65 g/kg). Compared with the control, application of CS4 (3.31 g/kg) significantly reduced Cd concentrations in the roots, stems and leaves of grain amaranths by 68%, 87% and 89%, respectively. At subcellular level, CS treatment resulted in redistribution of Cd, higher percentages of Cd in the chloroplast and soluble fractions in leaves of grain amaranths were found, while lower proportions of Cd were located at the cell wall of the leaves. The application of CS enhanced the proportions of pectate and protein integrated forms of Cd and decreased the percentages of water soluble Cd potentially associated with toxicity in grain amaranths. Changes of free Cd ions into inactive forms sequestered in subcellular compartments may indicate an important mechanism of CS for alleviating Cd toxicity and accumulation in plants.

  6. 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.

  7. Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Morimoto, Kazuhiro

    2013-01-01

    Azithromycin (AZM), a 15-membered ring macrolide antimicrobial agent, has an antibacterial spectrum that includes intracellular parasitic pathogens that survive or intracellularly multiply in alveolar macrophages (AMs). The subcellular distribution of AZM in AMs was evaluated in vitro in comparison with clarithromycin (CAM). AZM and CAM (50 µM) were applied to the NR8383 cells, used as an in vitro model of AMs, followed by incubation at 37°C or 4°C. The total amount of AZM in cells and subcellular distribution (cell fractionation) was determined after incubation. High level of AZM accumulation was observed in the NR8383 cells at 37°C, and the equilibrium intracellular to extracellular concentration ratio (I/E ratio) was approximately 680, which was remarkably higher than that of CAM (equilibrium I/E ratio=28). The intracellular accumulation of AZM and CAM was temperature dependent. In addition, AZM distributed to the granules fraction including organelles and soluble fraction including cytosol in the NR8383 cells, whereas CAM mainly distributed in soluble fraction. The amount of AZM in the granules fraction was markedly reduced in the presence of ammonium chloride for increase in intracellular pH. These results indicate that AZM is distributed in acidic compartment in AMs. This study suggests that high AZM accumulation in the NR8383 cells is due to the trapping and/or binding in acidic organelles, such as lysosomes.

  8. Heavy metal fates in laboratory bioretention systems.

    Science.gov (United States)

    Sun, Xueli; Davis, Allen P

    2007-01-01

    Key to managing heavy metals in bioretention is to understand their fates in bioretention facilities. In this study, pot prototypes filled with bioretention media were built to simulate the conditions of natural growth of plants. Synthetic runoff with different heavy metal loadings (copper, cadmium, lead, and zinc) was periodically applied. Metal accumulations in tissues of grasses -Panicum virgatum, Kentucky-31, and Bromus ciliatus, were investigated after 230d of growth and multiple runoff treatment events. After 183d of periodic runoff application, the concentrations of Zn, Cu, Pb and Cd with low and high loadings had the same trends in the plant tissues, Zn>Cu>Pb>Cd, following the trend of the input metal concentrations. The fates of input metals were 88-97% captured in soil media, 2.0-11.6% not captured by bioretention media, and 0.5-3.3% accumulated in plants. Compared to the metals retained by the soil, the percentages of input metals taken up by plants were relatively low due to the low plant biomass produced in this study. Greater biomass density would be required for the vegetation to have a valuable impact in prolonging the lifetime of a bioretention cell.

  9. Fate of Silver Nanoparticles in Lake Mesocosms

    Science.gov (United States)

    Furtado, Lindsay

    The fate of silver nanoparticles (AgNPs) in surface waters determines the ecological risk of this emerging contaminant. In this research, the fate of AgNPs in lake mesocosms was studied using both a continuous (i.e. drip) and one-time (i.e. plug) dosing regime. AgNPs were persistent in the tested lake environment as there was accumulation in the water column over time in drip mesocosms and slow dissipation from the water column (half life of 20 days) in plug mesocosms. In drip mesocosms, AgNPs were found to accumulate in the water column, periphtyon, and sediment according to loading rate; and, AgNP coating (PVP vs. CT) had no effect on agglomeration and dissolution based on filtration analysis. In plug mesocosms, cloud point extraction (CPE), single-particle-inductively coupled mass spectroscopy (spICP-MS), and asymmetrical flow field-flow fractionation (AF4-ICP-MS) confirmed the temporal dissolution of AgNPs into Ag+ over time; however, complexation is expected to reduce the toxicity of Ag + in natural waters.

  10. DBMLoc: a Database of proteins with multiple subcellular localizations

    Directory of Open Access Journals (Sweden)

    Zhou Yun

    2008-02-01

    Full Text Available Abstract Background Subcellular localization information is one of the key features to protein function research. Locating to a specific subcellular compartment is essential for a protein to function efficiently. Proteins which have multiple localizations will provide more clues. This kind of proteins may take a high proportion, even more than 35%. Description We have developed a database of proteins with multiple subcellular localizations, designated DBMLoc. The initial release contains 10470 multiple subcellular localization-annotated entries. Annotations are collected from primary protein databases, specific subcellular localization databases and literature texts. All the protein entries are cross-referenced to GO annotations and SwissProt. Protein-protein interactions are also annotated. They are classified into 12 large subcellular localization categories based on GO hierarchical architecture and original annotations. Download, search and sequence BLAST tools are also available on the website. Conclusion DBMLoc is a protein database which collects proteins with more than one subcellular localization annotation. It is freely accessed at http://www.bioinfo.tsinghua.edu.cn/DBMLoc/index.htm.

  11. Fate in intermittent claudication

    DEFF Research Database (Denmark)

    Jelnes, Rolf; Gaardsting, O; Hougaard Jensen, K

    1986-01-01

    The fate of 257 consecutive patients (100 women) aged 36-85 years (mean 65) first seen with intermittent claudication in 1977 was analysed after a mean of 6.5 (SD 0.5) years. When first seen none of the patients complained of rest pain or had ulcers or gangrenous lesions on the feet. At follow up....... The rate of clinical progression of the arteriosclerotic disease (that is, rest pain or gangrene) of the worst affected leg was 7.5% in the first year after referral. Thereafter the rate was 2.2% a year. An ankle systolic blood pressure below 70 mm Hg, a toe systolic blood pressure below 40 mm Hg......, or an ankle/arm pressure index below 50% were individually significantly associated with progression of the arteriosclerotic disease. These findings show the importance of peripheral blood pressure measurements in the management of patients with intermittent claudication due to arteriosclerotic disease....

  12. Domains involved in TAF15 subcellular localisation

    DEFF Research Database (Denmark)

    Marko, Marija; Vlassis, Arsenios; Guialis, Apostolia

    2012-01-01

    TAF15 (TBP associated factor 15) is a member of the highly conserved TET (also known as FET) protein family of RNA binding proteins (RBP), which comprises in addition FUS (fused in sarcoma, also known as TLS, translocated in liposarcoma) and EWS (Ewing sarcoma protein). The TET proteins are impli...... regulated in the HeLa and the neuronal HT22 cell lines and that TAF15 co-localised with a minor subset of RNA granules in the cytoplasm of HT22 cells, supporting a model whereupon TAF15 plays a role in RNA transport and/or local RNA translation....... to play important roles in the onset of specific tumours, certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this study we identified the domains of TAF15 responsible for its subcellular localisation in human (HeLa) cells and experimentally confirmed......, capable of being targeted to stress granules. We, moreover, showed that TAF15 cellular localisation depended on ongoing transcription and that independent domains of TAF15 engaged in nucleolar capping upon transcription inhibition. Finally, we demonstrated that TAF15 localisation was differentially...

  13. A formal ontology of subcellular neuroanatomy

    Directory of Open Access Journals (Sweden)

    Stephen D Larson

    2007-11-01

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

  14. A Formal Ontology of Subcellular Neuroanatomy

    Science.gov (United States)

    Larson, Stephen D.; Fong, Lisa L.; Gupta, Amarnath; Condit, Christopher; Bug, William J.; Martone, Maryann E.

    2007-01-01

    The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO), covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described. PMID:18974798

  15. 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.

  16. SPA Proteins Affect the Subcellular Localization of COP1 in the COP1/SPA Ubiquitin Ligase Complex during Photomorphogenesis.

    Science.gov (United States)

    Balcerowicz, Martin; Kerner, Konstantin; Schenkel, Christian; Hoecker, Ute

    2017-07-01

    The Arabidopsis (Arabidopsis thaliana) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant (spaQn). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1. © 2017 American Society of Plant Biologists. All Rights Reserved.

  17. Spatially patterned matrix elasticity directs stem cell fate

    Science.gov (United States)

    Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

    2016-08-01

    There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

  18. Predicting Protein Subcellular Location Using Digital Signal Processing

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  19. Predicting protein subcellular location using digital signal processing.

    Science.gov (United States)

    Pan, Yu-Xi; Li, Da-Wei; Duan, Yun; Zhang, Zhi-Zhou; Xu, Ming-Qing; Feng, Guo-Yin; He, Lin

    2005-02-01

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

  20. Genetic Circuit Architectures Underlying Cell Fate Choices for Immunity

    Science.gov (United States)

    Dinner, Aaron

    2009-03-01

    Antigen stimulated B cells follow an unusual developmental trajectory that transiently passes through a germinal center state, which promotes receptor affinity maturation and immunoglobulin class switching, before terminally differentiating into antibody secreting plasma cells. It was found that graded expression of the transcription factor IRF-4 regulates cell fate, but the relationship between antigen receptor signaling, the network of interactions with IRF-4, and cell fate was not known. This talk describes models that link ligand-receptor avidity with cell fate. The models have been validated experimentally by directly varying the levels and kinetics of IRF-4 accumulation. Furthermore, signaling through the antigen receptor is demonstrated to control the expression of IRF-4 and in turn the frequency of B cells that undergo class switching before differentiating into plasma cells. These findings provide an explanation for experiments that measure B cell numbers in transgenic mice. The architecture of our regulatory circuit provides a general mechanism for quantitative variations in a signal to be translated into a binary cell-fate choice involving transient expression of one of the two developmental fates. In collaboration with Aryeh Warmflash, Ying Li, Roger Sciammas, and Harinder Singh, The University of Chicago.

  1. SUBCELLULAR LOCALIZATION OF BETA CATENIN IN COLORECTAL NON NEOPLASTIC AND NEOPLASTIC LESIONS

    Directory of Open Access Journals (Sweden)

    Diah Rini Handjari

    2011-12-01

    Full Text Available Loss of adenomatous polyposis coli (APC function is typically an early event in sporadic colorectal cancer (CRC pathogenesis. The key tumor suppressor function of the APC protein lies in its ability to destabilize free cytoplasmic beta catenin. This lead to the accumulation of nuclear beta catenin, and together with the DNA binding protein Tcf-4, function as a transcriptional activator. Accumulation of stabilized free β-catenin is considered as an early event and perhaps initiating the process in intestinal tumorigenesis. Neoplastic transformation in the CRC associated chronic colitis is considered similar to the adenoma-carcinoma sequence in sporadic CRC. The distinguish feature from the CRC-related colitis is the difference in time and frequency changes. Loss of APC function, regarded as the beginning of a very common event in sporadic CRC, but the CRC associated chronic colitis generally occurs at the end of thedysplasia-carcinoma sequence. This research was conducted to determine the subcellular location of beta catenin expression in chronic colitis, colorectal adenomas and carcinomas that were evaluated by immunohistochemical staining. It can be concluded that beta-catenin is a component that plays a role in the development of the CRC and the subcellular location of beta-catenin can describe its oncogenic activity.

  2. 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.

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

    Science.gov (United States)

    Wang, Meng-Jiao; Wang, Wen-Xiong

    2011-01-25

    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+)], 0.01-84nM) 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.

  4. Support vector machine approach for protein subcellular localization prediction.

    Science.gov (United States)

    Hua, S; Sun, Z

    2001-08-01

    Subcellular localization is a key functional characteristic of proteins. A fully automatic and reliable prediction system for protein subcellular localization is needed, especially for the analysis of large-scale genome sequences. In this paper, Support Vector Machine has been introduced to predict the subcellular localization of proteins from their amino acid compositions. The total prediction accuracies reach 91.4% for three subcellular locations in prokaryotic organisms and 79.4% for four locations in eukaryotic organisms. Predictions by our approach are robust to errors in the protein N-terminal sequences. This new approach provides superior prediction performance compared with existing algorithms based on amino acid composition and can be a complementary method to other existing methods based on sorting signals. A web server implementing the prediction method is available at http://www.bioinfo.tsinghua.edu.cn/SubLoc/. Supplementary material is available at http://www.bioinfo.tsinghua.edu.cn/SubLoc/.

  5. 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.

  6. Subcellular localization of transglutaminase. Effect of collagen.

    Science.gov (United States)

    Juprelle-Soret, M; Wattiaux-De Coninck, S; Wattiaux, R

    1988-01-01

    1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together

  7. Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution.

    Science.gov (United States)

    De Marco, G; Lomartire, A; Calvo, A; Risso, A; De Luca, E; Mostert, M; Mandrioli, J; Caponnetto, C; Borghero, G; Manera, U; Canosa, A; Moglia, C; Restagno, G; Fini, N; Tarella, C; Giordana, M T; Rinaudo, M T; Chiò, A

    2017-02-01

    Cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 (TDP-43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP-43-coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP-43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS-linked mutant genes. TDP-43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin-containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2). TDP-43 cytoplasmic accumulation was found (P TDP-43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP-43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP-43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes. In ALS forms characterized by TDP-43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments. © 2016 British Neuropathological Society.

  8. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; Te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

  9. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions. PMID:28134274

  10. Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply.

    Science.gov (United States)

    Nie, Zhaojun; Zhao, Peng; Wang, Jia; Li, Jinfeng; Liu, Hongen

    2017-01-01

    Nitrogen (N) is critical for zinc (Zn) absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat (Triticum aestivum L.), an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn10) treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate (Vmax), root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N7.5) treatment were higher, but the Michaelis constant (Km) and minimum equilibrium concentrations (Cmin) in this treatment were lower than those in the low (N0.05) and high (N15) N treatments, when Zn was supplied at a high level (Zn10). Meanwhile, there were no pronounced differences in the above root traits between the N0.05Zn0 and N7.5Zn10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root development in

  11. Subcellular Proteomics of Soybean under Flooding Stress

    Institute of Scientific and Technical Information of China (English)

    Setsuko Komatsu

    2012-01-01

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

  12. Environmental Fate of Hydrazines

    Science.gov (United States)

    1989-12-01

    v/v. The absorbent cartridges removed trace gas contaminants , including remaining water and CO2 . Gas outlet-valves after each unit provided for air...largest kbkg was observed in Run A, presumably due to the interaction of hydrazine vapor with contamination on the chamber walls accumulated during its...64 UDMH Dry Air 2.8 19 13 1.1 a Ambirnt temperature, 50-75 ppm v/v initial concentrationb 24 m of added FEP c 14,700 ppm v/v water vapor Of these

  13. Effect of subcellular targeting on the expression and accumulation of Griffithsin in N. benthamiana

    CSIR Research Space (South Africa)

    Stark, T

    2010-09-01

    Full Text Available . Griffithsin (GRFT), a lectin isolated from the red algae Griffithsia, has shown extreme potency against the Human Immunodeficiency Virus (HIV) in vitro in very low quantities (Mori et al., 2005). The molecular target of GRFT is the high mannose residues...

  14. Predicting Protein Subcellular Localization: Past, Present, and Future

    Institute of Scientific and Technical Information of China (English)

    Pierre D(o)nnes; Annette H(o)glund

    2004-01-01

    Functional characterization of every single protein is a major challenge of the postgenomic era. The large-scale analysis of a cell's proteins, proteomics, seeks to provide these proteins with reliable annotations regarding their interaction partners and functions in the cellular machinery. An important step on this way is to determine the subcellular localization of each protein. Eukaryotic cells are divided into subcellular compartments, or organelles. Transport across the membrane into the organelles is a highly regulated and complex cellular process. Predicting the subcellular localization by computational means has been an area of vivid activity during recent years. The publicly available prediction methods differ mainly in four aspects: the underlying biological motivation, the computational method used, localization coverage, and reliability, which are of importance to the user.This review provides a short description of the main events in the protein sorting process and an overview of the most commonly used methods in this field.

  15. 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).

  16. Feature Fusion Based SVM Classifier for Protein Subcellular Localization Prediction.

    Science.gov (United States)

    Rahman, Julia; Mondal, Md Nazrul Islam; Islam, Md Khaled Ben; Hasan, Md Al Mehedi

    2016-12-18

    For the importance of protein subcellular localization in different branches of life science and drug discovery, researchers have focused their attentions on protein subcellular localization prediction. Effective representation of features from protein sequences plays a most vital role in protein subcellular localization prediction specially in case of machine learning techniques. Single feature representation-like pseudo amino acid composition (PseAAC), physiochemical property models (PPM), and amino acid index distribution (AAID) contains insufficient information from protein sequences. To deal with such problems, we have proposed two feature fusion representations, AAIDPAAC and PPMPAAC, to work with Support Vector Machine classifiers, which fused PseAAC with PPM and AAID accordingly. We have evaluated the performance for both single and fused feature representation of a Gram-negative bacterial dataset. We have got at least 3% more actual accuracy by AAIDPAAC and 2% more locative accuracy by PPMPAAC than single feature representation.

  17. 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

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

    Institute of Scientific and Technical Information of China (English)

    CHEN Chunying; ZHANG Peiqun; CHAI Zhifang

    2005-01-01

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

  19. Subcellular localization and rearrangement of endoplasmic reticulum by Brome mosaic virus capsid protein.

    Science.gov (United States)

    Bamunusinghe, Devinka; Seo, Jang-Kyun; Rao, A L N

    2011-03-01

    Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, as well as in other positive-strand RNA viruses pathogenic to humans (e.g., poliovirus) and animals (e.g., Flock House virus), is functionally coupled to replication. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. In this study, the application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a green fluorescent protein (GFP) fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV-infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP itself has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. The significance of CP-induced vesicles in relation to CP-organized viral functions that are linked to replication-coupled packaging is discussed.

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

    Directory of Open Access Journals (Sweden)

    Wang Ying-Nai

    2012-04-01

    Full Text Available Abstract 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.

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

    Directory of Open Access Journals (Sweden)

    Zhang Yi

    2010-09-01

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

  2. 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.

  3. 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.

  4. Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria

    Science.gov (United States)

    Raina, Jean-Baptiste; Clode, Peta L; Cheong, Soshan; Bougoure, Jeremy; Kilburn, Matt R; Reeder, Anthony; Forêt, Sylvain; Stat, Michael; Beltran, Victor; Thomas-Hall, Peter; Tapiolas, Dianne; Motti, Cherie M; Gong, Bill; Pernice, Mathieu; Marjo, Christopher E; Seymour, Justin R; Willis, Bette L; Bourne, David G

    2017-01-01

    Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate (DMSP). Despite their large-scale implications, these interactions remain unquantified at the cellular-scale. Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels, tracking the fate of a stable sulfur isotope (34S) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP, and finally its uptake and degradation by bacteria. Our results identify for the first time the storage locations of DMSP in microalgae, with high enrichments present in vacuoles, cytoplasm and chloroplasts. In addition, we quantify DMSP incorporation at the single-cell level, with DMSP-degrading bacteria containing seven times more 34S than the control strain. This study provides an unprecedented methodology to label, retain, and image small diffusible molecules, which can be transposable to other symbiotic systems. DOI: http://dx.doi.org/10.7554/eLife.23008.001 PMID:28371617

  5. Mathematical modeling of sub-cellular asymmetry of fat-dachsous heterodimer for generation of planar cell polarity.

    Directory of Open Access Journals (Sweden)

    Mohit Kumar Jolly

    Full Text Available Planar Cell Polarity (PCP is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft, Dachsous (Ds and Four-jointed (Fj. Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions

  6. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2016-01-01

    When predicting the subcellular localization of proteins from their amino acid sequences, there are basically three approaches: signal-based, global property-based, and homology-based. Each of these has its advantages and drawbacks, and it is important when comparing methods to know which approach...

  7. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    Science.gov (United States)

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  8. Measurement of endogenous subcellular concentration of steroids in tissue

    NARCIS (Netherlands)

    Poortman, J.; Landeghem, A.A.J. van; Helmond-Agema, A.; Thussen, J.H.H.

    1984-01-01

    A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different met

  9. 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.

  10. 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

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

    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

    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{sup 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{sup 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{sup 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{sub 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{sup 2+}] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.

  12. 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)

  13. 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

  14. Subcellular distribution and chemical forms of cadmium in Impatiens walleriana in relation to its phytoextraction potential.

    Science.gov (United States)

    Lai, Hung-Yu

    2015-11-01

    Impatiens (Impatiens walleriana) has been shown to be a potential cadmium (Cd) hyperaccumulator, but its mechanisms in accumulation and detoxification have not been reported. Rooted cuttings of Impatiens were planted in artificially Cd-contaminated soils for 50 days with total target concentrations of 0, 10, 20, 40, 80, and 120 mg/kg. The subcellular distribution and chemical forms of Cd in the different organs were analyzed after the pot experiment. Compared with the control group, various Cd treatments affected the growth exhibitions of Impatiens, but most of them were not statistically significant. The Cd accumulation of different organs increased with an increase in the soil Cd concentrations for most of the treatments, and it was in the decreasing order of root>stem>leaf. In the roots of Impatiens, Cd was mainly compartmentalized in the soluble fraction (Fs), which has a high migration capacity and will further translocate to the shoot. The Cd was mainly compartmentalized in the cell wall fraction (Fcw) in the shoots as a mechanism of tolerance. Most of the Cd in the various organs of Impatiens was mainly in the forms of pectate and protein-integrated (FNaCl), whereas a minor portion was a water soluble fraction (FW). The experimental results show that the Cd in the Fs, FW, and FNaCl in the roots of Impatiens had a high mobility and will further translocate to the shoot. They could be used to estimate the Cd accumulated in the shoots of Impatiens.

  15. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    Science.gov (United States)

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  16. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking

    Science.gov (United States)

    Lu, Yi-Ju; Schornack, Sebastian; Spallek, Thomas; Geldner, Niko; Chory, Joanne; Schellmann, Swen; Schumacher, Karin; Kamoun, Sophien; Robatzek, Silke

    2016-01-01

    Summary Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes. PMID:22233428

  17. From consumption to harvest: Environmental fate prediction of excreted ionizable trace organic chemicals

    DEFF Research Database (Denmark)

    Polesel, Fabio; Plósz, Benedek G.; Trapp, Stefan

    2015-01-01

    with freshwater or reclaimed wastewater. Recent research has shown the tendency for these substances to accumulate in food crops. In this study, we developed and applied a simulation tool to predict the fate of three ionizable trace chemicals (triclosan-TCS, furosemide-FUR, ciprofloxacin-CIP) from human...

  18. Modelling the Fate of Ionizable Trace Organic Chemicals from Consumption to Food Crops

    DEFF Research Database (Denmark)

    Polesel, Fabio; Plósz, Benedek G.; Trapp, Stefan

    In this study, we developed and applied a simulation tool to comprehensively predict the fate of three ionizable trace chemicals (triclosan—TCS, furosemide—FUR, ciprofloxacin—CIP) from human consumption/excretion up to the accumulation in wheat, following application of sewage sludge or irrigation...

  19. Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

    CERN Document Server

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

    2015-01-01

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

  20. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    Science.gov (United States)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

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

    Science.gov (United States)

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

    2016-02-01

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

  2. Crude subcellular fractionation of cultured mammalian cell lines

    OpenAIRE

    Holden Paul; Horton William A

    2009-01-01

    Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this...

  3. 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

  4. Convolutional LSTM Networks for Subcellular Localization of Proteins

    OpenAIRE

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

    2015-01-01

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

  5. Structural determinants of PINK1 topology and dual subcellular distribution

    OpenAIRE

    Kang Un; Lin William

    2010-01-01

    Abstract Background PINK1 is a mitochondria-targeted kinase that constitutively localizes to both the mitochondria and the cytosol. The mechanism of how PINK1 achieves cytosolic localization following mitochondrial processing remains unknown. Understanding PINK1 subcellular localization will give us insights into PINK1 functions and how mutations in PINK1 lead to Parkinson's disease. We asked how the mitochondrial localization signal, the transmembrane domain, and the kinase domain participat...

  6. The relative importance of water and diet for uptake and subcellular distribution of cadmium in the deposit-feeding polychaete, Capitella sp I

    DEFF Research Database (Denmark)

    Selck, Henriette; Forbes, Valery E.

    2004-01-01

    , starvation likewise influenced the distribution of protein between mitochondria and cytosol. Cutaneous uptake and accumulation of Cd from the water was related to surface area while dietary uptake was influenced by the amount of sediment passing through the gut. Irrespective of exposure route, Cd......The impact of dietary and water exposure on the accumulation and distribution of cadmium (Cd) in subcellular components of the polychaete Capitella sp. I was investigated. Worms were exposed to either dissolved Cd alone ('Water-Only' treatments; WO) or diet-bound Cd alone ('Algae-bound Only...

  7. Geometric modeling of subcellular structures, organelles, and multiprotein complexes

    Science.gov (United States)

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2013-01-01

    SUMMARY Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multi-protein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. PMID:23212797

  8. 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

  9. 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

  10. Zinc fate in animal husbandry systems.

    Science.gov (United States)

    Romeo, A; Vacchina, V; Legros, S; Doelsch, E

    2014-11-01

    Zinc (Zn) is considered in animal production systems as both an essential nutrient and a possible pollutant. While it is generally supplemented at low levels in animal diets, with less than 200 mg kg(-1) in complete feeds, it is under scrutiny due to potential accumulation in the environment. This explains why international regulations limit maximum supplementation levels in animal feeds in a stricter way. This article gives an overview of the current knowledge on the fate of zinc in animal production systems, from animal diets to animal wastes. Some analytical methods can be used for the quantification and qualification of Zn chemical forms: X-ray crystallography, electrospray tandem mass spectrometry, separation techniques, hyphenated techniques… Analysis of chelated forms issued from complex matrices, like hydrolysed proteins, remains difficult, and the speciation of Zn in diluted carriers (premix and feed) is a challenge. Our understanding of Zn absorption has made progress with recent research on ZnT/Zip families and metallothioneins. However, fine-tuned approaches towards the nutritional and metabolic interactions for Zn supplementation in farm conditions still require further studies. The speciation of zinc in pig manure and poultry litter has been a priority as monogastric animals are usually raised under intensive conditions and fed with high quantities of trace minerals, leading to high animal density and elevated quantities of zinc from animal wastes.

  11. Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function.

    Science.gov (United States)

    Fortugno, Paola; Wall, Nathan R; Giodini, Alessandra; O'Connor, Daniel S; Plescia, Janet; Padgett, Karen M; Tognin, Simona; Marchisio, Pier Carlo; Altieri, Dario C

    2002-02-01

    Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

  12. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Gonzalo P Solis

    Full Text Available Analyses of cultured cells and transgenic mice expressing prion protein (PrP deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1 the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2 the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and

  13. 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.

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

    Science.gov (United States)

    Chandra, Subhash

    2004-06-01

    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 13C and 15N 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 ( 13C15N) 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 39K, 23Na and 40Ca 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.

  15. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants.

    Science.gov (United States)

    Herrera, Rito; Álvarez, María C; Gelis, Samuel; Ramos, José

    2013-09-15

    Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

  16. The Fated Death of Tess

    Institute of Scientific and Technical Information of China (English)

    李莉

    2012-01-01

      The theme of this essay is about the fated deaths of Tess.The emphasis is‘inevitability’. Tess’s Death is mainly due to the social influence and man-made factors. Because of the low social status and moral conceptions, there is no living room left for Tess. Her parents push her get closer to her tragedy as well. Alec and Angel are the two persons who directly and indirectly cause Tess’s death. In addition, there are also some subjective causes for her doom. The symbolic red in the novel intentionally indicates the’inevitability’of her death.

  17. 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

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

    Science.gov (United States)

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

    2016-03-01

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

  19. Caenorhabditis elegans vulval cell fate patterning

    Science.gov (United States)

    Félix, Marie-Anne

    2012-08-01

    The spatial patterning of three cell fates in a row of competent cells is exemplified by vulva development in the nematode Caenorhabditis elegans. The intercellular signaling network that underlies fate specification is well understood, yet quantitative aspects remain to be elucidated. Quantitative models of the network allow us to test the effect of parameter variation on the cell fate pattern output. Among the parameter sets that allow us to reach the wild-type pattern, two general developmental patterning mechanisms of the three fates can be found: sequential inductions and morphogen-based induction, the former being more robust to parameter variation. Experimentally, the vulval cell fate pattern is robust to stochastic and environmental challenges, and minor variants can be detected. The exception is the fate of the anterior cell, P3.p, which is sensitive to stochastic variation and spontaneous mutation, and is also evolving the fastest. Other vulval precursor cell fates can be affected by mutation, yet little natural variation can be found, suggesting stabilizing selection. Despite this fate pattern conservation, different Caenorhabditis species respond differently to perturbations of the system. In the quantitative models, different parameter sets can reconstitute their response to perturbation, suggesting that network variation among Caenorhabditis species may be quantitative. Network rewiring likely occurred at longer evolutionary scales.

  20. Environmental fate and behaviour of nanomaterials

    DEFF Research Database (Denmark)

    Hartmann, Nanna Isabella Bloch; Skjolding, Lars Michael; Hansen, Steffen Foss

    In the current report, the existing knowledge on the fate of nanomaterials in the environment is reviewed and the major knowledge gaps are identified.......In the current report, the existing knowledge on the fate of nanomaterials in the environment is reviewed and the major knowledge gaps are identified....

  1. Convolutional LSTM Networks for Subcellular Localization of Proteins

    DEFF Research Database (Denmark)

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

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model...... on the other hand are designed to handle sequences. In this study we demonstrate that LSTM networks predict the subcellular location of proteins given only the protein sequence with high accuracy (0.902) outperforming current state of the art algorithms. We further improve the performance by introducing...... the LSTM networks....

  2. Convolutional LSTM Networks for Subcellular Localization of Proteins

    DEFF Research Database (Denmark)

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

    2015-01-01

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model...... on the other hand are designed to handle sequences. In this study we demonstrate that LSTM networks predict the subcellular location of proteins given only the protein sequence with high accuracy (0.902) outperforming current state of the art algorithms. We further improve the performance by introducing...

  3. 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.

  4. 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.

  5. Subcellular partitioning of cadmium in the freshwater bivalve, Pyganodon grandis, after separate short-term exposures to waterborne or diet-borne metal

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Sophie; Hare, Landis [INRS-Eau, Terre et Environnement, Universite du Quebec, 490 rue de la Couronne, Quebec, QC, G1K 9A9 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [INRS-Eau, Terre et Environnement, Universite du Quebec, 490 rue de la Couronne, Quebec, QC, G1K 9A9 (Canada)

    2010-11-15

    The dynamics of cadmium uptake and subcellular partitioning were studied in laboratory experiments conducted on Pyganodon grandis, a freshwater unionid bivalve that shows promise as a biomonitor for metal pollution. Bivalves were collected from an uncontaminated lake, allowed to acclimate to laboratory conditions ({>=}25 days), and then either exposed to a low, environmentally relevant, concentration of dissolved Cd (5 nM; 6, 12 and 24 h), or fed Cd-contaminated algae ({approx}70 nmol Cd g{sup -1} dry weight; 4 x 4 h). In this latter case, the bivalves were allowed to depurate for up to 8 days after the end of the feeding phase. As anticipated, the gills were the main target organ during the aqueous Cd exposure whereas the intestine was the initial site of Cd accumulation during the dietary exposure; during the subsequent depuration period, the dietary Cd accumulated in both the digestive gland and in the gills. For the gills, the distribution of Cd among the subcellular fractions (i.e., granules > heat-denatured proteins (HDP) {approx} heat-stable proteins (HSP) > mitochondria {approx} lysosomes + microsomes) was insensitive to the exposure route; both waterborne and diet-borne Cd ended up largely bound to the granule fraction. The subcellular distribution of Cd in the digestive gland differed markedly from that in the gills (HDP > HSP {approx} granules {approx} mitochondria > lysosomes + microsomes), but as in the case of the gills, this distribution was relatively insensitive to the exposure route. For both the gills and the digestive gland, the subcellular distributions of Cd differed from those observed in native bivalves that are chronically exposed to Cd in the field - in the short-term experimental exposures of P. grandis, metal detoxification was less effective than in chronically exposed native bivalves.

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

    CERN Document Server

    Thampi, Sabu M

    2007-01-01

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

  7. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

    Science.gov (United States)

    Magenau, Andrew J D; Saurabh, Saumya; Andreko, Susan K; Telmer, Cheryl A; Schmidt, Brigitte F; Waggoner, Alan S; Bruchez, Marcel P

    2015-10-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

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

    Science.gov (United States)

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

    2010-07-01

    Image analysis, machine learning and statistical modeling have become well established for the automatic recognition and comparison of the subcellular locations of proteins in microscope images. By using a comprehensive set of features describing static images, major subcellular patterns can be distinguished with near perfect accuracy. We now extend this work to time series images, which contain both spatial and temporal information. The goal is to use temporal features to improve recognition of protein patterns that are not fully distinguishable by their static features alone. We have adopted and designed five sets of features for capturing temporal behavior in 2D time series images, based on object tracking, temporal texture, normal flow, Fourier transforms and autoregression. Classification accuracy on an image collection for 12 fluorescently tagged proteins was increased when temporal features were used in addition to static features. Temporal texture, normal flow and Fourier transform features were most effective at increasing classification accuracy. We therefore extended these three feature sets to 3D time series images, but observed no significant improvement over results for 2D images. The methods for 2D and 3D temporal pattern analysis do not require segmentation of images into single cell regions, and are suitable for automated high-throughput microscopy applications. Images, source code and results will be available upon publication at http://murphylab.web.cmu.edu/software murphy@cmu.edu.

  10. Fate mapping of dendritic cells

    Directory of Open Access Journals (Sweden)

    Barbara Ursula Schraml

    2015-05-01

    Full Text Available Dendritic cells (DCs are a heterogeneous group of mononuclear phagocytes with versatile roles in immunity. They are classified predominantly based on phenotypic and functional properties, namely their stellate morphology, expression of the integrin CD11c and major histocompatibility class II molecules, as well as their superior capacity to migrate to secondary lymphoid organs and stimulate naïve T cells. However, these attributes are not exclusive to DCs and often change within inflammatory or infectious environments. This led to debates over cell identification and questioned even the mere existence of DCs as distinct leukocyte lineage. Here, we review experimental approaches taken to fate map DCs and discuss how these have shaped our understanding of DC ontogeny and lineage affiliation. Considering the ontogenetic properties of DCs will help to overcome the inherent shortcomings of purely phenotypic- and function-based approaches to cell definition and will yield a more robust way of DC classification.

  11. Research on the fates of drilling wastes

    Energy Technology Data Exchange (ETDEWEB)

    Munschenheim, D. K. [Department of Fisheries and Oceans, Dartmouth, NS (Canada). Bedford Inst. of Oceanography

    2000-07-01

    Dispersal characteristics and fates of fine particulate drilling wastes were studied in the Cohasset and Panuke oilfields lying 45 km west of Sable Island in 38 m of water. A second study was carried out at the Hibernia field in 1995 prior to the emplacement of the Hibernia platform. In the Cohasset and Panuke study the wells were relatively shallow and drilling operations were done with a minimum of barite. The initial trial comparing samples from within the discharge plume with those taken from clear water revealed that drilling waste fines accumulate in the benthic boundary layer. Subsequent studies at Copan delineated the extent of drilling wastes coverage on the seabed, showing flocculation of the waste particles, with subsequent increase in their settling rate. There was also evidence of an increase in water column turbidity with increasing proximity to the platform. In the case of Hibernia drilling wastes were detected only in the upper water column. Subsequent information revealed that this was due to the fact that at the time of sampling silica-based drilling mud, formulated with fresh water, was used. In 1998 Hibernia switched to synthetic muds. Since that time, no drilling mud waste was detected in the upper water column and only slight amounts in the benthic boundary layer. The explanation lies in the differences in discharge practices, such as for example pre-dilution with sea water. Overall, the results confirm the suitability of particle size analysis for the detection of particulate drilling wastes on energetic offshore banks. The technique also confirms the role of flocculation in retarding the dispersion of fine particles through increasing the settling rate. 4 refs., 6 figs.

  12. 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

  13. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-03-09

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

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

    Science.gov (United States)

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

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  17. Organ, cellular, and subcellular localization of brain-specific anion transporter BSAT1.

    Science.gov (United States)

    Baklaushev, V P; Kardashova, K Sh; Gurina, O I; Yusubaliyeva, G M; Zorkina, Ya A; Chekhonin, V P

    2013-08-01

    Organ, cellular, and subcellular localization of brain-specific anion transporter BSAT1 was studied in rats using antibodies to the extracellular fragment (451-557 a.a). The antibodies were shown to recognize the antigen predominantly localized in the nervous tissue, tumors of glial origin, and primordial ovarian follicles. The absence of BSAT1 immunofluorescence signal in kidney and liver sections and accumulation of (125)I labeled antibodies to BSAT1 in these organs indicate that these antibodies do not cross-react with the most common isoforms of OATP expressed in these organs. Analysis of the cellular localization suggests that in the brain, BSAT1 is localized predominantly in astrocytes, but not in endothelial cells, as was previously reported. Laser scanning confocal microscopy with a set of relevant trackers revealed membrane localization of BSAT1. Taking into account the data on the of localization, we can conclude that antibodies to BSAT1 451-557 can be used for basic research of the transport of thyroxin and prostaglandins across the blood brain barrier and for testing the systems for targeted transport of diagnostic preparations and drugs across the blood brain barrier, e.g. to astroglial tumors.

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

    Science.gov (United States)

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

    2015-05-20

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

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

    Institute of Scientific and Technical Information of China (English)

    朱寿彭; 汪源长

    1994-01-01

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

  20. An image analysis method to quantify CFTR subcellular localization.

    Science.gov (United States)

    Pizzo, Lucilla; Fariello, María Inés; Lepanto, Paola; Aguilar, Pablo S; Kierbel, Arlinet

    2014-08-01

    Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify

  1. Microscopy with spatial filtering for monitoring subcellular morphology

    Science.gov (United States)

    Zheng, Jing-Yi

    Dynamic alteration in organelle morphology is an important indicator of cellular function and many efforts have been made to monitor the subcellular morphology. Optical scatter imaging (OSI), which combines light scattering spectroscopy with microscopic imaging, was developed to non-invasively track real-time changes in particle morphology in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter (OSIR, optical scatter imaging ratio) at each pixel in the full field of view. For spherical particles, the OSIR was shown to decrease monotonically with diameter. In living cells, we reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl- xL transmembrane domain, but could not be observed by fluorescence or DIC images1. However, the initial design was based on Mie theory of scattering by spheres, and hence only adequate for measuring spherical particles. This limits the applicability of OSI to cellular functional studies involving organelles, which are naturally non-spherical. In this project, we aim to enhance the current capability of the existing optical scatter microscope to assess size and shape information for both spherical and non-spherical particles, and eventually apply this technique for monitoring and quantifying subcellular morphology within living cells. To reach this goal, we developed an improved system, in which the variable diameter iris is replaced with a digital micromirror device and adopted the concept of Gabor filtering to extend our assessment of morphology to the characterization of particle shape and orientation. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This

  2. 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.

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

    Science.gov (United States)

    Wang, Meng-Jiao; Wang, Wen-Xiong

    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(2+)] ([Cd(2+)]-based-IC(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.

  4. Homeostatic Plasticity of Subcellular Neuronal Structures: From Inputs to Outputs.

    Science.gov (United States)

    Wefelmeyer, Winnie; Puhl, Christopher J; Burrone, Juan

    2016-10-01

    Neurons in the brain are highly plastic, allowing an organism to learn and adapt to its environment. However, this ongoing plasticity is also inherently unstable, potentially leading to aberrant levels of circuit activity. Homeostatic forms of plasticity are thought to provide a means of controlling neuronal activity by avoiding extremes and allowing network stability. Recent work has shown that many of these homeostatic modifications change the structure of subcellular neuronal compartments, ranging from changes to synaptic inputs at both excitatory and inhibitory compartments to modulation of neuronal output through changes at the axon initial segment (AIS) and presynaptic terminals. Here we review these different forms of structural plasticity in neurons and the effects they may have on network function. Copyright © 2016. Published by Elsevier Ltd.

  5. The sub-cellular localization of Sulfolobus DNA replication.

    Science.gov (United States)

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  6. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

    Science.gov (United States)

    Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S.

    2001-01-01

    Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

  7. Fate of metal-associated POM in a soil under arable land use contaminated by metallurgical fallout in northern France

    NARCIS (Netherlands)

    Labanowski, J.; Sebastia, J.; Foy, E.; Jongmans, T.; Lamy, I.; Oort, van F.

    2007-01-01

    Organic matter is a major metal-retaining constituent in soils. Among the diversity of organic components in soils, particulate organic matter (POM) accumulates large amounts of metals, but the fate of such metal-associated POM is unknown. We studied different POM size fractions and their

  8. Subcellular distribution of glutathione and cysteine in cyanobacteria

    Science.gov (United States)

    Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    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. PMID:20349253

  9. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Subcellular partitioning kinetics, metallothionein response and oxidative damage in the marine mussel Mytilus galloprovincialis exposed to cadmium-based quantum dots.

    Science.gov (United States)

    Rocha, Thiago Lopes; Gomes, Tânia; Durigon, Emerson Giuliani; Bebianno, Maria João

    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(-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.

  12. The fate of pancake vortices

    Science.gov (United States)

    Sutyrin, G. G.; Radko, T.

    2017-03-01

    Nonlinear evolution of pancake-like vortices in a uniformly rotating and stratified fluid is studied using a 3D Boussinesq numerical model at large Rossby numbers. After the initial stage of viscous decay, the simulations reveal exponential growth of toroidal circulation cells (aka Taylor vortices) at the peripheral annulus with a negative Rayleigh discriminant. At the nonlinear stage, these thin cells redistribute the angular momentum and density differently at the levels of radial outflow and inflow. Resulting layering, with a vertical stacking of sharp variations in velocity and density, enhances small-scale mixing and energy decay. Characteristic detectable stretching patterns are produced in the density field. The circulation patterns, induced by centrifugal instability, tend to homogenize the angular momentum in the vicinity of the unstable region. We demonstrate that the peak intensity of the cells and the vortex energy decay are dramatically reduced by the earth's rotation due to conservation of total absolute angular momentum. The results have important implications for better understanding the fate of pancake vortices and physical mechanisms of energy transfer in stratified fluids.

  13. Glyphosate fate in soils when arriving in plant residues.

    Science.gov (United States)

    Mamy, Laure; Barriuso, Enrique; Gabrielle, Benoît

    2016-07-01

    A significant fraction of pesticides sprayed on crops may be returned to soils via plant residues, but its fate has been little documented. The objective of this work was to study the fate of glyphosate associated to plants residues. Oilseed rape was used as model plant using two lines: a glyphosate-tolerant (GT) line and a non-GT one, considered as a crucifer weed. The effects of different fragmentation degrees and placements in soil of plant residues were tested. A control was set up by spraying glyphosate directly on the soil. The mineralization of glyphosate in soil was slower when incorporated into plant residues, and the amounts of extractable and non-extractable glyphosate residues increased. Glyphosate availability for mineralization increased when the size of plant residues decreased, and as the distribution of plant residues in soil was more homogeneous. After 80 days of soil incubation, extractable (14)C-residues mostly involved one metabolite of glyphosate (AMPA) but up to 2.6% of initial (14)C was still extracted from undecayed leaves as glyphosate. Thus, the trapping of herbicides in plant materials provided a protection against degradation, and crops residues returns may increase the persistence of glyphosate in soils. This pattern appeared more pronounced for GT crops, which accumulated more non-degraded glyphosate in their tissues.

  14. Molecular trajectories leading to the alternative fates of duplicate genes.

    Directory of Open Access Journals (Sweden)

    Michael Marotta

    Full Text Available Gene duplication generates extra gene copies in which mutations can accumulate without risking the function of pre-existing genes. Such mutations modify duplicates and contribute to evolutionary novelties. However, the vast majority of duplicates appear to be short-lived and experience duplicate silencing within a few million years. Little is known about the molecular mechanisms leading to these alternative fates. Here we delineate differing molecular trajectories of a relatively recent duplication event between humans and chimpanzees by investigating molecular properties of a single duplicate: DNA sequences, gene expression and promoter activities. The inverted duplication of the Glutathione S-transferase Theta 2 (GSTT2 gene had occurred at least 7 million years ago in the common ancestor of African great apes and is preserved in chimpanzees (Pan troglodytes, whereas a deletion polymorphism is prevalent in humans. The alternative fates are associated with expression divergence between these species, and reduced expression in humans is regulated by silencing mutations that have been propagated between duplicates by gene conversion. In contrast, selective constraint preserved duplicate divergence in chimpanzees. The difference in evolutionary processes left a unique DNA footprint in which dying duplicates are significantly more similar to each other (99.4% than preserved ones. Such molecular trajectories could provide insights for the mechanisms underlying duplicate life and death in extant genomes.

  15. Early perturbation in mitochondria redox homeostasis in response to environmental stress predicts cell fate in diatoms.

    Science.gov (United States)

    van Creveld, Shiri Graff; Rosenwasser, Shilo; Schatz, Daniella; Koren, Ilan; Vardi, Assaf

    2015-02-01

    Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of global photosynthesis. Nevertheless, little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a redox-sensitive green fluorescent protein sensor targeted to various subcellular organelles in the marine diatom Phaeodactylum tricornutum, to map the spatial and temporal oxidation patterns in response to environmental stresses. Specific organelle oxidation patterns were found in response to various stress conditions such as oxidative stress, nutrient limitation and exposure to diatom-derived infochemicals. We found a strong correlation between the mitochondrial glutathione (GSH) redox potential (EGSH) and subsequent induction of cell death in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), and a volatile halocarbon (BrCN) that mediate trophic-level interactions in marine diatoms. Induction of cell death in response to DD was mediated by oxidation of mitochondrial EGSH and was reversible by application of GSH only within a narrow time frame. We found that cell fate can be accurately predicted by a distinct life-death threshold of mitochondrial EGSH (-335 mV). We propose that compartmentalized redox-based signaling can integrate the input of diverse environmental cues and will determine cell fate decisions as part of algal acclimation to stress conditions.

  16. Production, use, and fate of all plastics ever made

    Science.gov (United States)

    Geyer, Roland; Jambeck, Jenna R.; Law, Kara Lavender

    2017-01-01

    Plastics have outgrown most man-made materials and have long been under environmental scrutiny. However, robust global information, particularly about their end-of-life fate, is lacking. By identifying and synthesizing dispersed data on production, use, and end-of-life management of polymer resins, synthetic fibers, and additives, we present the first global analysis of all mass-produced plastics ever manufactured. We estimate that 8300 million metric tons (Mt) as of virgin plastics have been produced to date. As of 2015, approximately 6300 Mt of plastic waste had been generated, around 9% of which had been recycled, 12% was incinerated, and 79% was accumulated in landfills or the natural environment. If current production and waste management trends continue, roughly 12,000 Mt of plastic waste will be in landfills or in the natural environment by 2050. PMID:28776036

  17. 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

  18. lin-28 controls the succession of cell fate choices via two distinct activities.

    Directory of Open Access Journals (Sweden)

    Bhaskar Vadla

    Full Text Available lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

  19. Addressing the complexity of water chemistry in environmental fate modeling for engineered nanoparticles.

    Science.gov (United States)

    Sani-Kast, Nicole; Scheringer, Martin; Slomberg, Danielle; Labille, Jérôme; Praetorius, Antonia; Ollivier, Patrick; Hungerbühler, Konrad

    2015-12-01

    Engineered nanoparticle (ENP) fate models developed to date - aimed at predicting ENP concentration in the aqueous environment - have limited applicability because they employ constant environmental conditions along the modeled system or a highly specific environmental representation; both approaches do not show the effects of spatial and/or temporal variability. To address this conceptual gap, we developed a novel modeling strategy that: 1) incorporates spatial variability in environmental conditions in an existing ENP fate model; and 2) analyzes the effect of a wide range of randomly sampled environmental conditions (representing variations in water chemistry). This approach was employed to investigate the transport of nano-TiO2 in the Lower Rhône River (France) under numerous sets of environmental conditions. The predicted spatial concentration profiles of nano-TiO2 were then grouped according to their similarity by using cluster analysis. The analysis resulted in a small number of clusters representing groups of spatial concentration profiles. All clusters show nano-TiO2 accumulation in the sediment layer, supporting results from previous studies. Analysis of the characteristic features of each cluster demonstrated a strong association between the water conditions in regions close to the ENP emission source and the cluster membership of the corresponding spatial concentration profiles. In particular, water compositions favoring heteroaggregation between the ENPs and suspended particulate matter resulted in clusters of low variability. These conditions are, therefore, reliable predictors of the eventual fate of the modeled ENPs. The conclusions from this study are also valid for ENP fate in other large river systems. Our results, therefore, shift the focus of future modeling and experimental research of ENP environmental fate to the water characteristic in regions near the expected ENP emission sources. Under conditions favoring heteroaggregation in these

  20. Emerging patterns for engineered nanomaterials in the environment: a review of fate and toxicity studies

    Science.gov (United States)

    Garner, Kendra L.; Keller, Arturo A.

    2014-08-01

    A comprehensive assessment of the environmental risks posed by engineered nanomaterials (ENMs) entering the environment is necessary, due in part to the recent predictions of ENM release quantities and because ENMs have been identified in waste leachate. The technical complexity of measuring ENM fate and transport processes in all environments necessitates identifying trends in ENM processes. Emerging information on the environmental fate and toxicity of many ENMs was collected to provide a better understanding of their environmental implications. Little research has been conducted on the fate of ENMs in the atmosphere; however, most studies indicate that ENMs will in general have limited transport in the atmosphere due to rapid settling. Studies of ENM fate in realistic aquatic media indicates that in general, ENMs are more stable in freshwater and stormwater than in seawater or groundwater, suggesting that transport may be higher in freshwater than in seawater. ENMs in saline waters generally sediment out over the course of hours to days, leading to likely accumulation in sediments. Dissolution is significant for specific ENMs (e.g., Ag, ZnO, copper ENMs, nano zero-valent iron), which can result in their transformation from nanoparticles to ions, but the metal ions pose their own toxicity concerns. In soil, the fate of ENMs is strongly dependent on the size of the ENM aggregates, groundwater chemistry, as well as the pore size and soil particle size. Most groundwater studies have focused on unfavorable deposition conditions, but that is unlikely to be the case in many natural groundwaters with significant ionic strength due to hardness or salinity. While much still needs to be better understood, emerging patterns with regards to ENM fate, transport, and exposure combined with emerging information on toxicity indicate that risk is low for most ENMs, though current exposure estimates compared with current data on toxicity indicates that at current production and

  1. Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo.

    Science.gov (United States)

    Medina, David X; Householder, Kyle T; Ceton, Ricki; Kovalik, Tina; Heffernan, John M; Shankar, Rohini V; Bowser, Robert P; Wechsler-Reya, Robert J; Sirianni, Rachael W

    2017-03-02

    Understanding of the mechanisms by which systemically administered nanoparticles achieve delivery across biological barriers remains incomplete, due in part to the challenge of tracking nanoparticle fate in the body. Here, we develop a new approach for "barcoding" nanoparticles (NPs) composed of poly(lactic-co-glycolic acid) (PLGA) with bright, spectrally defined quantum dots (QDs) to enable direct, fluorescent detection of NP fate with subcellular resolution. We show that QD labeling does not affect major biophysical properties of NPs or their interaction with cells and tissues. Live cell imaging enabled simultaneous visualization of the interaction of control and targeted nanoparticles with bEnd.3 cells in a flow chamber, providing direct evidence that surface modification of nanoparticles with the cell-penetrating peptide TAT increases their biophysical association with cell surfaces over very short time periods under convective current. We next developed this technique for quantitative biodistribution analysis in vivo. These studies demonstrate NP surface modification with the cell penetrating peptide TAT facilitates brain-specific delivery that is restricted to brain vasculature. Although nanoparticle entry into the healthy brain parenchyma is minimal, with no evidence for movement of NPs across the blood-brain barrier (BBB), we observed nanoparticles are able to enter to the central nervous system (CNS) through regions of altered BBB permeability - for example, into circumventricular organs in the brain or leaky vasculature of late-stage intracranial tumors. In sum, these data demonstrate a new, multispectral approach for barcoding PLGA, which enables simultaneous, quantitative analysis of the fate of multiple nanoparticle formulations in vivo.

  2. Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids.

    Science.gov (United States)

    Zechmann, B; Mauch, F; Sticher, L; Müller, M

    2008-01-01

    The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly

  3. Telomerase RNA accumulates in Cajal bodies in human cancer cells.

    Science.gov (United States)

    Zhu, Yusheng; Tomlinson, Rebecca L; Lukowiak, Andrew A; Terns, Rebecca M; Terns, Michael P

    2004-01-01

    Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

  4. Subcellular and in-vivo Nano-Endoscopy

    Science.gov (United States)

    Cheemalapati, Surya Venkatasekhar; Winskas, John; Wang, Hao; Konnaiyan, Karthik; Zhdanov, Arseny; Roth, Alison; Adapa, Swamy Rakesh; Deonarine, Andrew; Noble, Mark; Das, Tuhin; Gatenby, Robert; Westerheide, Sandy D.; Jiang, Rays H. Y.; Pyayt, Anna

    2016-01-01

    Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level. PMID:27694854

  5. Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and non-methylating cells.

    Science.gov (United States)

    Dopp, E; von Recklinghausen, U; Diaz-Bone, R; Hirner, A V; Rettenmeier, A W

    2010-07-01

    Arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, liver and lung. Inorganic arsenic, existing in highly toxic trivalent and significantly less toxic pentavalent forms, is methylated to mono- and di-methylated species mainly in the liver. Due to the low toxicity of pentavalent methylated species, methylation has been regarded as a detoxification process for many years; however, recent findings of a high toxicity of trivalent methylated species have indicated the contrary. In order to elucidate the role of speciation and methylation for the toxicity and carcinogenicity of arsenic, systematic studies were conducted comparing cellular uptake, subcellular distribution as well as toxic and genotoxic effects of organic and inorganic pentavalent and trivalent arsenic species in both non-methylating (urothelial cells and fibroblasts) and methylating cells (hepatocytes). The membrane permeability was found to be dependent upon both the arsenic species and the cell type. Uptake rates of trivalent methylated species were highest and exceeded those of their pentavalent counterparts by several orders of magnitude. Non-methylating cells (urothelial cells and fibroblasts) seem to accumulate higher amounts of arsenic within the cell than the methylating hepatocytes. Cellular uptake and extrusion seem to be faster in hepatocytes than in urothelial cells. The correlation of uptake with toxicity indicates a significant role of membrane permeability towards toxicity. Furthermore, cytotoxic effects are more distinct in hepatocytes. Differential centrifugation studies revealed that elevated concentrations of arsenic are present in the ribosomal fraction of urothelial cells and in nucleic and mitochondrial fractions of hepatic cells. Further studies are needed to define the implications of the observed enrichment of arsenic in specific cellular organelles for its carcinogenic activity. This review summarizes our recent research on cellular uptake

  6. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    Science.gov (United States)

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  7. Optimization of ruminococcus albus endoglucanase cel5-cbm6 production in plants by incorporating an elp tag and targeting to different subcellular compartments

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, E.O.; Menassa, R. [Western Ontario Univ., London, ON (Canada). Dept. of Biology; Agriculture and Agri-Food Canada, London, ON (Canada); Kolotilin, I. [Agriculture and Agri-Food Canada, London, ON (Canada)

    2009-07-01

    The production of biomass-based biofuel such as ethanol depends on the deconstruction of a cellulosic matrix and requires a variety of enzymes that hydrolyze glycosidic bonds to release fermentable sugars. Endoglucanases are one of most important groups of natural cellulosic hydrolytic enzymes that act on cellulose. In order to decrease ethanol production costs, the cost of producing cellulases must also be reduced. Genetically engineered transgenic plants are among the most economical systems for large scale production of recombinant proteins because of the large amount of enzymes that can be produced with minimal input. Cellulases present different levels of expression in different subcellular compartments. Cel5-CBM6 is a fused protein containing an endocellulase from Ruminococus albus (Cel5) and a cellulose binding domain (CBD) of Clostridium stercorarium. It accumulates in both the chloroplast and cytoplasm, but severe growth defects occur when expressed in the cytoplasm. Therefore, other subcellular compartments such as endoplasmic reticulum (ER) and vacuole must be evaluated and compared to determine the best co partment for production and activity of cellulases. Since elastin-like polypeptide (ELP) has also been shown to increase recombinant protein accumulation in plants, this study evaluated the effects of incorporating an ELP tag and a retrieval signal peptide on the expression levels of Cel5-CBM6.

  8. A multiresolution approach to automated classification of protein subcellular location images

    National Research Council Canada - National Science Library

    Chebira, Amina; Barbotin, Yann; Jackson, Charles; Merryman, Thomas; Srinivasa, Gowri; Murphy, Robert F; Kovacević, Jelena

    2007-01-01

    .... Over the past ten years, the feasibility of using machine learning methods to recognize all major subcellular location patterns has been convincingly demonstrated, using diverse feature sets and classifiers...

  9. Correction: 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

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  10. Fate of Pyrethroids in Farmland Ponds

    DEFF Research Database (Denmark)

    Mogensen, B. B.; Sørensen, P. B.; Stuer-Lauridsen, F.;

    Pyrethroids constitute a group of widely used insecticides, which are toxic to aquatic organisms. This report presents the results from a 2-year study of the fate of pyrethroids in ponds, i.e. their distribution in the water column, the sediment and the surface microlayer respectively. The measur......Pyrethroids constitute a group of widely used insecticides, which are toxic to aquatic organisms. This report presents the results from a 2-year study of the fate of pyrethroids in ponds, i.e. their distribution in the water column, the sediment and the surface microlayer respectively...

  11. Fate of HERS during Tooth Root Development

    OpenAIRE

    HUANG, XIAOFENG; BRINGAS, PABLO; Slavkin, Harold C.; Chai, Yang

    2009-01-01

    Tooth root development begins after the completion of crown formation in mammals. Previous studies have shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of the root throughout root formation and do not disappear. Most of the HERS c...

  12. On the Formation of Tess’ Tragic Fate

    Institute of Scientific and Technical Information of China (English)

    张颖

    2014-01-01

    "Tess of the d’Urbervilles"is one of the most important books of Thomas Hardy.The heroine Tess is the most outstanding female image created by Hardy and also an idealized artistic model.Her fate moves the readers deeply and represents the beauty of the book.By analysing the special social conditions,we can see the typicalness and the inevitability of Tess’tragic fate.Tess naturally becomes the typical victim of the cruel social reality.Hardy’s realistic thought about the society and his progressive ideas to which oughts to be paid attention by us embodies in the novel.

  13. Sub-cellular partitioning of Zn, Cu, Cd and Pb in the digestive gland of native Octopus vulgaris exposed to different metal concentrations (Portugal)

    Energy Technology Data Exchange (ETDEWEB)

    Raimundo, J. [National Institute for Agronomy and Fisheries Research - IPIMAR, Av. Brasilia, 1449-006 Lisbon (Portugal)], E-mail: jraimundo@ipimar.pt; Vale, C. [National Institute for Agronomy and Fisheries Research - IPIMAR, Av. Brasilia, 1449-006 Lisbon (Portugal); Duarte, R.; Moura, I. [REQUIMTE - CQFB, Department of Chemistry, Faculty of Sciences and Technology, New University of Lisbon, Qta Torre, 2829-516 Monte da Caparica (Portugal)

    2008-02-15

    Concentrations of Zn, Cu, Cd and Pb and their sub-cellular distributions were determined in composite samples of digestive glands of the common octopus, Octopus vulgaris caught from two areas of the Portuguese coast characterised by contrasting metal contamination. Minor contents of Zn (1%), Cu (2%), Cd (6%) and Pb (7%) were found in the insoluble fraction, consisting of nuclei, mitochondria, lysosomes and microsome operationally separated from the whole digestive gland through a sequential centrifugation. A tendency for linear relationships between metal concentrations in nuclei, mitochondria, lysosomes and whole digestive gland was observed. These relationships suggest that despite low metal content organelles responded to the increasing accumulated metals, which means that detoxifying mechanism in cytosol was incomplete. Poorer correlations between microsome and whole digestive gland did not point to metal toxicity in the analysed compartments. However, the high accumulated Cd indicated that O. vulgaris is an important vehicle of this element to its predators in the coastal environment.

  14. Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes.

    Science.gov (United States)

    1979-08-01

    subcellular marker, we will compare the subcellular distribution of activity using two other substrates, inosine diphosphate and thiamine pyrophosphate, also...diphosphatase and its identity with thiamine pyrophosphatase. J. Biol. Chem. 243 2934-2942. -’/ t.~ i B6 .~ 4. jo 4 A IN ie %f I.., Ad~ ,de~M 1. ~A~1’ -. 44,t * ’j " *’ 7A-. lb. Ai

  15. 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.

  16. Glycyrrhetic acid, but not glycyrrhizic acid, strengthened entecavir activity by promoting its subcellular distribution in the liver via efflux inhibition.

    Science.gov (United States)

    Chen, Qianying; Chen, Hongzhu; Wang, Wenjie; Liu, Jiali; Liu, Wenyue; Ni, Ping; Sang, Guowei; Wang, Guangji; Zhou, Fang; Zhang, Jingwei

    2017-08-30

    Entecavir (ETV) is a superior nucleoside analogue used to treat hepatitis B virus (HBV) infection. Although its advantages over other agents include low viral resistance and the elicitation of a sharp decrease in HBV DNA, adverse effects such as hepatic steatosis, hepatic damage and lactic acidosis have also been reported. Glycyrrhizin has long been used as hepato-protective medicine. The clinical combination of ETV plus glycyrrhizin in China displays better therapeutic effects and lower rates of liver damage. However, there is little evidence explaining the probable synergistic mechanism that exists between these two drugs from a pharmacokinetics view. Here, alterations in the plasma pharmacokinetics, tissue distribution, subcellular distribution, and in vitro and in vivo antiviral activity of ETV after combination with glycyrrhizic acid (GL) were analysed to determine the synergistic mechanisms of these two drugs. Specific efflux transporter membrane vesicles were also used to elucidate their interactions. The primary active GL metabolite, glycyrrhetic acid (GA), did not affect the plasma pharmacokinetics of ETV but promoted its accumulation in hepatocytes, increasing its distribution in the cytoplasm and nucleus and augmenting the antiviral efficiency of ETV. These synergistic actions were primarily due to the inhibitory effect of GA on MRP4 and BCRP, which transport ETV out of hepatocytes. In conclusion, GA interacted with ETV at cellular and subcellular levels in the liver through MRP4 and BCRP inhibition, which enhanced the antiviral activity of ETV. Our results partially explain the synergistic mechanism of ETV and GL from a pharmacokinetics view, providing more data to support the use of these compounds together in clinical HBV treatment. Copyright © 2017. Published by Elsevier B.V.

  17. 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

  18. 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.

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

    Science.gov (United States)

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

    2014-09-01

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

  20. Non-target-site glyphosate resistance in Conyza bonariensis is based on modified subcellular distribution of the herbicide.

    Science.gov (United States)

    Kleinman, Ziv; Rubin, Baruch

    2017-01-01

    Conyza spp. were the first broadleaf weeds reported to have evolved glyphosate resistance. Several mechanisms have been proposed for glyphosate resistance. In an effort to elucidate the mechanism of this resistance in Conyza bonariensis, possible target-site and non-target-site mechanisms were investigated in glyphosate-resistant (GR) C. bonariensis biotypes. Using differential glyphosate applications and analyses of shikimate accumulation, we followed the herbicide effect in different plant organs and monitored the herbicide's apparent mobility. We found high shikimate levels in the roots and young leaves of glyphosate-sensitive (GS) plants, regardless of the site of application, whereas in GR plants, shikimate accumulated mainly in treated young leaves. (14) C-glyphosate studies, however, revealed the expected source-to-sink translocation pattern in both GS and GR plants. Sequencing of the appropriate EPSPS DNA fragments of GR and GS plants revealed no alteration at the Pro106 position. These data support the hypothesis that the glyphosate resistance of our C. bonariensis GR biotypes is associated with altered subcellular distribution of glyphosate, which keeps the herbicide sequestered away from the EPSPS target site in the chloroplast. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

    Science.gov (United States)

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

    2013-11-01

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

  2. [Effects of exogenous nitric oxide on the subcellular distribution and chemical forms of copper in tomato seedlings under copper stress].

    Science.gov (United States)

    Jiang, Chun-Hui; Wang, Xiu-Feng; Yin, Bo; Li, Xiao-Yun; Cui, Xiu-Min

    2012-11-01

    A nutrient solution culture experiment was conducted to study the effects of exogenous NO donor (sodium nitroprusside) on the subcellular distribution and chemical form of copper (Cu) in tomato seedlings under the stress of 50 micromol x L(-1) of Cu2+ (CuCl2). Under this stress, the biomass and plant height of tomato seedlings decreased by 33.7% and 23.1%, respectively. Exogenous NO alleviated this inhibition effect significantly, but the Cu concentration and accumulation in the seedling organs still had a significant increase. Under the Cu stress, the Cu concentration and accumulation in the seedling organs were in the order of root > leaf > stem > petiole. Exogenous NO limited the absorbed Cu transferred from root to shoot, but could not remove this translocation. Exogenous NO increased the Cu concentration in vacuole and cell wall significantly, and decreased the Cu concentration in organelle, which lessened the damage of Cu on the regular metabolic balance in cytoplasm and increased the tolerance of organelle against Cu. Exogenous NO increased the acetic acid-extractable Cu (F(HAc)) in root, sodium chloride-extractable Cu (F(NaCl)) in stem, F(HAc) in petiole, and ethanol-extractable Cu (F(E)) and F(NaCl) in leaf, while decreased the concentration and distribution of water-extractable Cu (F(W)) in different organs, which efficiently reduced the bio-toxicity of excessive copper.

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

    Directory of Open Access Journals (Sweden)

    Yousef Jamali

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

  4. 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.

  5. Oleosin of subcellular lipid droplets evolved in green algae.

    Science.gov (United States)

    Huang, Nan-Lan; Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-04-01

    In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants.

  6. SIMPLEBOX: a generic multimedia fate evaluation model

    NARCIS (Netherlands)

    Meent D van de

    1993-01-01

    This document describes the technical details of the multimedia fate model SimpleBox, version 1.0 (930801). SimpleBox is a multimedia box model of what is commonly referred to as a "Mackay-type" model ; it assumes spatially homogeneous environmental compartments (air, water, suspended matter, aquati

  7. SIMPLEBOX: a generic multimedia fate evaluation model

    NARCIS (Netherlands)

    van de Meent D

    1993-01-01

    This document describes the technical details of the multimedia fate model SimpleBox, version 1.0 (930801). SimpleBox is a multimedia box model of what is commonly referred to as a "Mackay-type" model ; it assumes spatially homogeneous environmental compartments (air, water, suspended m

  8. Muscle Stem Cell Fate Is Controlled by the Cell-Polarity Protein Scrib

    Directory of Open Access Journals (Sweden)

    Yusuke Ono

    2015-02-01

    Full Text Available Satellite cells are resident skeletal muscle stem cells that supply myonuclei for homeostasis, hypertrophy, and repair in adult muscle. Scrib is one of the major cell-polarity proteins, acting as a potent tumor suppressor in epithelial cells. Here, we show that Scrib also controls satellite-cell-fate decisions in adult mice. Scrib is undetectable in quiescent cells but becomes expressed during activation. Scrib is asymmetrically distributed in dividing daughter cells, with robust accumulation in cells committed to myogenic differentiation. Low Scrib expression is associated with the proliferative state and preventing self-renewal, whereas high Scrib levels reduce satellite cell proliferation. Satellite-cell-specific knockout of Scrib in mice causes a drastic and insurmountable defect in muscle regeneration. Thus, Scrib is a regulator of tissue stem cells, controlling population expansion and self-renewal with Scrib expression dynamics directing satellite cell fate.

  9. Evolution of a system sensitive to stochastic noise: P3.p cell fate in Caenorhabditis.

    Science.gov (United States)

    Pénigault, Jean-Baptiste; Félix, Marie-Anne

    2011-09-15

    The C. elegans cell lineage is overall invariant. One rare instance of variability concerns P3.p, the most anterior vulva precursor cell, which may either fuse with the epidermis without dividing, or remain competent to form vulval tissue and divide. Here we examine the evolutionary properties of this stochastic variation in P3.p fate. In the Caenorhabditis genus, high P3.p competence is ancestral and reduction in P3.p competence and division frequency occurred in C. sp. 14 and in a clade of nine species. Within this clade, the frequency of P3.p division further varies within and among species, being intermediate in C. elegans and low in C. briggsae. P3.p fate frequency is sensitive to random mutation accumulation, suggesting that this trait may evolve rapidly because of its sensitivity to mutational impact. P3.p fate depends on LIN-39/Hox5 expression and we find that the peak of LIN-39/Hox5 protein level is displaced posteriorly in C. briggsae compared to C. elegans. However, P3.p fate specification is most sensitive to the dose of EGL-20 and CWN-1, two Wnts that are secreted in a long-range gradient from the posterior end of C. elegans larvae (accompanying article). A half-dose of either of these Wnts is sufficient to affect division frequency in C. elegans N2 to levels similar to those in C. briggsae. Symmetrically, we show that an increase in Wnt dose rescues anterior competence in C. briggsae. We propose that evolutionary variation in the concentration or interpretation of the long-range Wnt gradient may be involved in the rapid evolution of P3.p fate in Caenorhabditis.

  10. The accumulation and localization of chalcone synthase in grapevine (Vitis vinifera L.).

    Science.gov (United States)

    Wang, Huiling; Wang, Wei; Zhan, JiCheng; Yan, Ailing; Sun, Lei; Zhang, Guojun; Wang, Xiaoyue; Ren, Jiancheng; Huang, Weidong; Xu, Haiying

    2016-09-01

    Chalcone synthase (CHS, E.C.2.3.1.74) is the first committed enzyme in the flavonoid pathway. Previous studies have primarily focused on the cloning, expression and regulation of the gene at the transcriptional level. Little is yet known about the enzyme accumulation, regulation at protein level, as well as its localization in grapevine. In present study, the accumulation, tissue and subcellular localization of CHS in different grapevine tissues (Vitis vinifera L. Cabernet Sauvignon) were investigated via the techniques of Western blotting, immunohistochemical localization, immunoelectron microscopy and confocal microscopy. The results showed that CHS were mainly accumulated in the grape berry skin, leaves, stem tips and stem phloem, correlated with flavonoids accumulation. The accumulation of CHS is developmental dependent in grape berry skin and flesh. Immunohistochemical analysis revealed that CHS were primarily localized in the exocarp and vascular bundles of the fruits during berry development; in palisade, spongy tissues and vascular bundles of the leaves; in the primary phloem and pith ray in the stems; in the growth point, leaf primordium, and young leaves of leaf buds; and in the endoderm and primary phloem of grapevine roots. Furthermore, at the subcellular level, the cell wall, cytoplasm and nucleus localized patterns of CHS were observed in the grapevine vegetative tissue cells. Results above indicated that distribution of CHS in grapevine was organ-specific and tissue-specific. This work will provide new insight for the biosynthesis and regulation of diverse flavonoid compounds in grapevine. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate

    Directory of Open Access Journals (Sweden)

    Timothy J. Petros

    2015-11-01

    Full Text Available Fate determination in the mammalian telencephalon, with its diversity of neuronal subtypes and relevance to neuropsychiatric disease, remains a critical area of study in neuroscience. Most studies investigating this topic focus on the diversity of neural progenitors within spatial and temporal domains along the lateral ventricles. Often overlooked is whether the location of neurogenesis within a fate-restricted domain is associated with, or instructive for, distinct neuronal fates. Here, we use in vivo fate mapping and the manipulation of neurogenic location to demonstrate that apical versus basal neurogenesis influences the fate determination of major subgroups of cortical interneurons derived from the subcortical telencephalon. Somatostatin-expressing interneurons arise mainly from apical divisions along the ventricular surface, whereas parvalbumin-expressing interneurons originate predominantly from basal divisions in the subventricular zone. As manipulations that shift neurogenic location alter interneuron subclass fate, these results add an additional dimension to the spatial-temporal determinants of neuronal fate determination.

  12. Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

    Directory of Open Access Journals (Sweden)

    Ildiko M L Somorjai

    Full Text Available BACKGROUND: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. METHODOLOGY/PRINCIPAL FINDINGS: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10 displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155 caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155 produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10. These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and

  13. Subcellular distribution of central carbohydrate metabolism pathways in the red alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Moriyama, Takashi; Sakurai, Kenta; Sekine, Kohsuke; Sato, Naoki

    2014-09-01

    Comprehensive subcellular localization analysis revealed that the subcellular distribution of carbohydrate metabolic pathways in the red alga Cyanidioschyzon is essentially identical with that in Arabidopsis , except the lack of transaldolase. In plants, the glycolysis and oxidative pentose phosphate pathways (oxPPP) are located in both cytosol and plastids. However, in algae, particularly red algae, the subcellular localization of enzymes involved in carbon metabolism is unclear. Here, we identified and examined the localization of enzymes related to glycolysis, oxPPP, and tricarboxylic acid (TCA) and Calvin-Benson cycles in the red alga Cyanidioschyzon merolae. A gene encoding transaldolase of the oxPPP was not found in the C. merolae genome, and no transaldolase activity was detected in cellular extracts. The subcellular localization of 65 carbon metabolic enzymes tagged with green fluorescent protein or hemagglutinin was examined in C. merolae cells. As expected, TCA and Calvin-Benson cycle enzymes were localized to mitochondria and plastids, respectively. The analyses also revealed that the cytosol contains the entire glycolytic pathway and partial oxPPP, whereas the plastid contains a partial glycolytic pathway and complete oxPPP, with the exception of transaldolase. Together, these results suggest that the subcellular distribution of carbohydrate metabolic pathways in C. merolae is essentially identical with that reported in the photosynthetic tissue of Arabidopsis thaliana; however, it appears that substrates typically utilized by transaldolase are consumed by glycolytic enzymes in the plastidic oxPPP of C. merolae.

  14. Distinct MicroRNA Subcellular Size and Expression Patterns in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Beibei Chen

    2012-01-01

    Full Text Available Introduction. Small noncoding RNAs have important regulatory functions in different cell pathways. It is believed that most of them mainly play role in gene post-transcriptional regulation in the cytoplasm. Recent evidence suggests miRNA and siRNA activity in the nucleus. Here, we show distinct genome-wide sub-cellular localization distribution profiles of small noncoding RNAs in human breast cancer cells. Methods. We separated breast cancer cell nuclei from cytoplasm, and identified small RNA sequences using a high-throughput sequencing platform. To determine the relationship between miRNA sub-cellular distribution and cancer progression, we used microarray analysis to examine the miRNA expression levels in nucleus and cytoplasm of three human cell lines, one normal breast cell line and two breast cancer cell lines. Logistic regression and SVM were used for further analysis. Results. The sub-cellular distribution of small noncoding RNAs shows that numerous miRNAs and their isoforms (isomiR not only locate to the cytoplasm but also appeare in the nucleus. Subsequent microarray analyses indicated that the miRNA nuclear-cytoplasmic-ratio is a significant characteristic of different cancer cell lines. Conclusions. Our results indicate that the sub-cellular distribution is important for miRNA function, and that the characterization of the small RNAs sub-cellular localizome may contribute to cancer research and diagnosis.

  15. Paleoproterozoic pyrobitumen: Re-Os goechemistry reveals the fate of giant carbon accumulations in Russian Karelia

    Science.gov (United States)

    Hannah, J. L.; Stein, H. J.; Yang, G.; Zimmerman, A.

    2010-12-01

    Carbon-rich pyrobitumen is exposed in abundance in the Onega Basin of Karelia, northwestern Russia - the fossil remains of a giant oil field [1]. The pyrobitumen, locally termed “shungite”, occurs as veins, lenses, and droplets within the Zaonezhskaya and Kondopozhskaya Formations, part of a Paleoproterozoic platform sequence developed on a rifted continental margin. In its purest form, this material approaches 100% C. Less pure pyrobitumen occurs in Corg-rich siltstones, which are intercalated with basaltic flows and sills, including peperites. Locally, the pyrobitumen forms a massive organo-siliceous diapir, cross-cutting the sedimentary section. A Re-Os isochron for six samples of Corg-rich siltstone taken from a mine adit in Shunga village yields a precise age of 2.05 Ga [2], fitting existing constraints for the depositional age. The initial 187Os/188Os ratio given by the isochron for the Shunga adit samples is within uncertainty of chondritic (0.113 at 2.05 Ga), as expected in a restricted rift basin dominated by hydrothermal Os. Re-Os data for samples from ICDP FAR-DEEP drillhole 13A near Shunga village, tell the rest of the story. Eleven ~500 mg samples, drilled from two 3-cm intervals 0.5 m apart in a single core, yield an isochron age of ~1.73 Ga with initial 187Os/188Os = 5. This age is consistent with the latest phase of the Svecofennian orogeny [3], known to have impacted the Onega Basin. Assuming isotopic homogenization (maturation) of the pyrobitumen took place during a relatively brief thermal maximum, the largest control on the present-day 187Os/188Os is the 187Re/188Os. The 187Re/188Os for the 17 siltstone samples analyzed to date range from 280 to 1250. An average 187Re/188Os of 900 will raise the 187Os/188Os from 0.113 to about 5 during the 320 m.y. between deposition (2.05 Ga) and maturation (1.73 Ga) - precisely the value determined by our isochron. Similarly, the lower 187Re/188Os of 280 for three samples of lustrous pyrobitumen from the Shunga adit yields the measured present-day 187Os/188Os of 10.2 for a maturation age of 1.73 Ga. Other sections from the FAR-DEEP drill core have yielded scattered Re-Os data, most likely reflecting mixtures of primary and migrated organic material. Targeted drilling can help avoid analysis of mixtures, but is not always fool-proof in complex systems. Although precision may be conpromised by dating samples that contain migrated hydrocarbons, objective interpretations may nonetheless be obtained by forward-modeling in-growth of 187Os based on timing of known regional events, and comparing the model output with measured isotopic data. Generation of pyrobitumen from organic material during the waning stage of the Svecofennian orogney fits both regional geology and Re-Os isotopic data. [1] Melezhik et al. (2009) Terra Nova 21: 119-126. [2] Hannah et al. (2008) 33rd IGC, Oslo, #1352701. [3] Stein (2006) Lithos 87: 300-327. Supported by ICDP FAR-DEEP Project (Melezhik et al.) and NSF award EAR-0820848 to J. Hannah and H. Stein.

  16. Intermittent rainfall in dynamic multimedia fate modeling.

    Science.gov (United States)

    Hertwich, E G

    2001-03-01

    It has been shown that steady-state multimedia models (level III fugacity models) lead to a substantial underestimate of air concentrations for chemicals with a low Henry's law constant (H multimedia models are used to estimate the spatial range or inhalation exposure. A dynamic model of pollutant fate is developed for conditions of intermittent rainfall to calculate the time profile of pollutant concentrations in different environmental compartments. The model utilizes a new, mathematically efficient approach to dynamic multimedia fate modeling that is based on the convolution of solutions to the initial conditions problem. For the first time, this approach is applied to intermittent conditions. The investigation indicates that the time-averaged pollutant concentrations under intermittent rainfall can be approximated by the appropriately weighted average of steady-state concentrations under conditions with and without rainfall.

  17. Accumulation by Conservation

    NARCIS (Netherlands)

    Büscher, Bram; Fletcher, Robert

    2014-01-01

    Following the financial crisis and its aftermath, it is clear that the inherent contradictions of capitalist accumulation have become even more intense and plunged the global economy into unprecedented turmoil and urgency. Governments, business leaders and other elite agents are frantically searchin

  18. Biological fate of low-calorie sweeteners.

    Science.gov (United States)

    Magnuson, Bernadene A; Carakostas, Michael C; Moore, Nadia H; Poulos, Sylvia P; Renwick, Andrew G

    2016-11-01

    With continued efforts to find solutions to rising rates of obesity and diabetes, there is increased interest in the potential health benefits of the use of low- and no-calorie sweeteners (LNCSs). Concerns about safety often deter the use of LNCSs as a tool in helping control caloric intake, even though the safety of LNCS use has been affirmed by regulatory agencies worldwide. In many cases, an understanding of the biological fate of the different LNSCs can help health professionals to address safety concerns. The objectives of this review are to compare the similarities and differences in the chemistry, regulatory status, and biological fate (including absorption, distribution, metabolism, and excretion) of the commonly used LNCSs: acesulfame potassium, aspartame, saccharin, stevia leaf extract (steviol glycoside), and sucralose. Understanding the biological fate of the different LNCSs is helpful in evaluating whether reports of biological effects in animal studies or in humans are indicative of possible safety concerns. Illustrations of the usefulness of this information to address questions about LNCSs include discussion of systemic exposure to LNCSs, the use of sweetener combinations, and the potential for effects of LNCSs on the gut microflora. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. SIMS ion microscopy as a novel, practical tool for subcellular chemical imaging in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, S

    2003-01-15

    The development of cryogenic sample preparations, subcellular image quantification schemes, and correlative confocal laser scanning microscopy and ion microscopy have made dynamic SIMS a versatile tool in biology and medicine. For example, ion microscopy can provide much needed, novel information on calcium influx and intracellular calcium stores at organelle resolution in normal and transformed cells in order to better understand the altered calcium signaling in malignant cells. 3-D SIMS imaging of cells revealed dynamic gradients of calcium in cells undergoing mitosis and cytokinesis. Studies of subcellular localization of anticancer drugs is another area of research where ion microscopy can provide novel observations in many types of cancers. Ion microscopy is already an essential tool in boron neutron capture therapy (BNCT) of brain cancer as it can be used to quantitatively image the subcellular location of boron in cells and tissues. This information is critically needed for testing the efficacy of boronated agents and for calculations of radiation dosimetry.

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

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2016-12-01

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

  1. Geary autocorrelation and DCCA coefficient: Application to predict apoptosis protein subcellular localization via PSSM

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2017-02-01

    Apoptosis is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death. Predicting subcellular location of apoptosis proteins is very helpful for understanding its mechanism of programmed cell death. Prediction of apoptosis protein subcellular location is still a challenging and complicated task, and existing methods mainly based on protein primary sequences. In this paper, we propose a new position-specific scoring matrix (PSSM)-based model by using Geary autocorrelation function and detrended cross-correlation coefficient (DCCA coefficient). Then a 270-dimensional (270D) feature vector is constructed on three widely used datasets: ZD98, ZW225 and CL317, and support vector machine is adopted as classifier. The overall prediction accuracies are significantly improved by rigorous jackknife test. The results show that our model offers a reliable and effective PSSM-based tool for prediction of apoptosis protein subcellular localization.

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Automated identification of subcellular organelles by coherent anti-stokes Raman scattering.

    Science.gov (United States)

    El-Mashtoly, Samir F; Niedieker, Daniel; Petersen, Dennis; Krauss, Sascha D; Freier, Erik; Maghnouj, Abdelouahid; Mosig, Axel; Hahn, Stephan; Kötting, Carsten; Gerwert, Klaus

    2014-05-06

    Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the "random forest" ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.

  5. Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

    Science.gov (United States)

    Perrière, Guy; Thioulouse, Jean

    2003-02-01

    Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

  6. Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28

    DEFF Research Database (Denmark)

    Hernebring, Malin; Fredriksson, Asa; Liljevald, Maria

    2013-01-01

    In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator...... PA28aß (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNFa. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28a using miRNA counteracted the removal of damaged proteins demonstrating...

  7. Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

    Science.gov (United States)

    Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

    2015-05-01

    Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1

  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 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

  10. Protein subcellular localization prediction using multiple kernel learning based support vector machine.

    Science.gov (United States)

    Hasan, Md Al Mehedi; Ahmad, Shamim; Molla, Md Khademul Islam

    2017-03-28

    Predicting the subcellular locations of proteins can provide useful hints that reveal their functions, increase our understanding of the mechanisms of some diseases, and finally aid in the development of novel drugs. As the number of newly discovered proteins has been growing exponentially, which in turns, makes the subcellular localization prediction by purely laboratory tests prohibitively laborious and expensive. In this context, to tackle the challenges, computational methods are being developed as an alternative choice to aid biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging issue, particularly, when query proteins have multi-label characteristics, i.e., if they exist simultaneously in more than one subcellular location or if they move between two or more different subcellular locations. To date, to address 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 to the protein subcellular localization prediction problem. However, the practicability of an SVM is affected by the challenges of selecting an appropriate kernel and selecting the parameters of the selected kernel. To address this difficulty, in this study, we aimed to develop an efficient multi-label protein subcellular localization prediction system, named as MKLoc, by introducing multiple kernel learning (MKL) based SVM. We evaluated MKLoc using a combined dataset containing 5447 single-localized proteins (originally published as part of the Höglund dataset) and 3056 multi-localized proteins (originally published as part of the DBMLoc set). Note that this dataset was used by Briesemeister et al. in their extensive comparison of multi-localization prediction systems. Finally, our experimental results indicate that

  11. [Prediction of protein subcellular locations by ensemble of improved K-nearest neighbor].

    Science.gov (United States)

    Xue, Wei; Wang, Xiongfei; Zhao, Nan; Yang, Rongli; Hong, Xiaoyu

    2017-04-25

    Adaboost algorithm with improved K-nearest neighbor classifiers is proposed to predict protein subcellular locations. Improved K-nearest neighbor classifier uses three sequence feature vectors including amino acid composition, dipeptide and pseudo amino acid composition of protein sequence. K-nearest neighbor uses Blast in classification stage. The overall success rates by the jackknife test on two data sets of CH317 and Gram1253 are 92.4% and 93.1%. Adaboost algorithm with the novel K-nearest neighbor improved by Blast is an effective method for predicting subcellular locations of proteins.

  12. Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae

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    Dick Jeffrey M

    2009-07-01

    Full Text Available Abstract Background Protein subcellular localization and differences in oxidation state between subcellular compartments are two well-studied features of the the cellular organization of S. cerevisiae (yeast. Theories about the origin of subcellular organization are assisted by computational models that can integrate data from observations of compositional and chemical properties of the system. Presentation and implications of the hypothesis I adopt the hypothesis that the state of yeast subcellular organization is in a local energy minimum. This hypothesis implies that equilibrium thermodynamic models can yield predictions about the interdependence between populations of proteins and their subcellular chemical environments. Testing the hypothesis Three types of tests are proposed. First, there should be correlations between modeled and observed oxidation states for different compartments. Second, there should be a correspondence between the energy requirements of protein formation and the order the appearance of organelles during cellular development. Third, there should be correlations between the predicted and observed relative abundances of interacting proteins within compartments. Results The relative metastability fields of subcellular homologs of glutaredoxin and thioredoxin indicate a trend from less to more oxidizing as mitochondrion – cytoplasm – nucleus. Representing the overall amino acid compositions of proteins in 23 different compartments each with a single reference model protein suggests that the formation reactions for proteins in the vacuole (in relatively oxidizing conditions, ER and early Golgi (in relatively reducing conditions are relatively highly favored, while that for the microtubule is the most costly. The relative abundances of model proteins for each compartment inferred from experimental data were found in some cases to correlate with the predicted abundances, and both positive and negative correlations were

  13. Predicting gram-positive bacterial protein subcellular localization based on localization motifs.

    Science.gov (United States)

    Hu, Yinxia; Li, Tonghua; Sun, Jiangming; Tang, Shengnan; Xiong, Wenwei; Li, Dapeng; Chen, Guanyan; Cong, Peisheng

    2012-09-07

    The subcellular localization of proteins is closely related to their functions. In this work, we propose a novel approach based on localization motifs to improve the accuracy of predicting subcellular localization of Gram-positive bacterial proteins. Our approach performed well on a five-fold cross validation with an overall success rate of 89.5%. Besides, the overall success rate of an independent testing dataset was 97.7%. Moreover, our approach was tested using a new experimentally-determined set of Gram-positive bacteria proteins and achieved an overall success rate of 96.3%.

  14. Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

    Science.gov (United States)

    Datta, R K; Antopol, W; Ghosh, J J

    1977-01-01

    Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

  15. 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.

  16. Iron accumulates in Huntington's disease neurons: protection by deferoxamine.

    Directory of Open Access Journals (Sweden)

    Jianfang Chen

    Full Text Available Huntington's disease (HD is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull's staining for ferrous iron (II combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2 and accordingly decreased expression of iron uptake transferrin receptor (TfR and increased levels of neuronal iron export protein ferroportin (FPN. Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

  17. Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

    Science.gov (United States)

    Overdijk, Elysa J R; DE Keijzer, Jeroen; DE Groot, Deborah; Schoina, Charikleia; Bouwmeester, Klaas; Ketelaar, Tijs; Govers, Francine

    2016-08-01

    Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.

  18. Exogenous Nitric Oxide Involved in Subcellular Distribution and Chemical Forms of Cu2+Under Copper Stress in Tomato Seedlings

    Institute of Scientific and Technical Information of China (English)

    DONG Yu-xiu; WANG Xiu-feng; CUI Xiu-min

    2013-01-01

    Nitric oxide (NO), a bioactive signaling molecule, serves as an antioxidant and anti-stress agent under abiotic stress. A hydroponics experiment was conducted to investigate the effects of sodium nitroprusside (SNP), a NO donor, on tomato seedlings exposed to 50 µmol L-1 CuCl2. The results show that copper is primarily stored in the soluble cell sap fraction in the roots, especially after treatment with Cu+SNP treatment, which accounted for 66.2%of the total copper content. The copper concentration gradually decreased from the roots to the leaves. In the leaves, exogenous NO induces the storage of excess copper in the cell walls. Copper stress decreases the proportion of copper integrated with pectates and proteins, but exogenous NO remarkably reverses this trend. The alleviating effect of NO is blocked by hemoglobin. Thus, exogenous NO is likely involved in the regulation of the subcellular copper concentrations and its chemical forms under copper stress. Although exogenous NO inhibited the absorption and transport of excess copper to some extent, the copper accumulation in tomato seedlings signiifcantly increased under copper stress. The use of exogenous NO to enhance copper tolerance in some plants is a promising method for copper remediation.

  19. Subcellular distribution and uptake mechanism of di-n-butyl phthalate in roots of pumpkin (Cucurbita moschata) seedlings.

    Science.gov (United States)

    Lin, Qingqi; Yang, Xiuhong; Huang, Xiongfei; Wang, Shizhong; Chao, Yuanqing; Qiu, Rongliang

    2016-01-01

    Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins.

  20. Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development.

    Directory of Open Access Journals (Sweden)

    Mohd A Mohd Ridzuan

    Full Text Available An actomyosin motor complex assembled below the parasite's plasma membrane drives erythrocyte invasion by Plasmodium falciparum merozoites. The complex is comprised of several proteins including myosin (MyoA, myosin tail domain interacting protein (MTIP and glideosome associated proteins (GAP 45 and 50, and is anchored on the inner membrane complex (IMC, which underlies the plasmalemma. A ternary complex of MyoA, MTIP and GAP45 is formed that then associates with GAP50. We show that full length GAP45 labelled internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony, where it accumulates during intracellular development until merozoite release. We show that GAP45 is phosphorylated by calcium dependent protein kinase 1 (CDPK1, and identify the modified serine residues. Replacing these serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor protein complex or its subcellular location in the parasite. The early assembly of the motor complex suggests that it has functions in addition to its role in erythrocyte invasion.

  1. Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways.

    Directory of Open Access Journals (Sweden)

    Irena Moserova

    Full Text Available We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS. Para derivatives pTPP(EG4 and pTPPF(EG4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG4 localized in the endoplasmic reticulum (ER induced dramatic changes in Ca(2+ homeostasis manifested by Ca(2+ rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG4-mediated apoptosis, confirming the causative role of the PERK pathway.

  2. Fate of pharmaceuticals and pesticides in fly larvae composting

    Energy Technology Data Exchange (ETDEWEB)

    Lalander, C., E-mail: cecilia.lalander@slu.se [Department of Energy and Technology, Swedish University of Agricultural Sciences (Sweden); Senecal, J.; Gros Calvo, M. [Department of Energy and Technology, Swedish University of Agricultural Sciences (Sweden); Ahrens, L.; Josefsson, S.; Wiberg, K. [Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences (Sweden); Vinnerås, B. [Department of Energy and Technology, Swedish University of Agricultural Sciences (Sweden)

    2016-09-15

    A novel and efficient organic waste management strategy currently gaining great attention is fly larvae composting. High resource recovery efficiency can be achieved in this closed-looped system, but pharmaceuticals and pesticides in waste could potentially accumulate in every loop of the treatment system and spread to the environment. This study evaluated the fate of three pharmaceuticals (carbamazepine, roxithromycin, trimethoprim) and two pesticides (azoxystrobin, propiconazole) in a fly larvae composting system and in a control treatment with no larvae. It was found that the half-life of all five substances was shorter in the fly larvae compost (< 10% of control) and no bioaccumulation was detected in the larvae. Fly larvae composting could thus impede the spread of pharmaceuticals and pesticides into the environment. - Highlights: • Degradation of pharmaceuticals and pesticides in fly larvae composting (FLC). • Half-life considerably shorter in FLC than in control with no larvae. • Half-life of carbamazepine was less than two days in FLC. • No bioaccumulation in larvae detected. • FLC could impede the spreading of pharmaceuticals and pesticide in the environment.

  3. Fate of higher brominated PBDEs in lactating cows.

    Science.gov (United States)

    Kierkegaard, Amelie; Asplund, Lillemor; de Wit, Cynthia A; McLachlan, Michael S; Thomas, Gareth O; Sweetman, Andrew J; Jones, Kevin C

    2007-01-15

    Dietary intake studies of lower brominated diphenyl ethers (BDEs) have shown that fish and animal products are important vectors of human exposure, but almost no data exist for higher brominated BDEs. Therefore, the fate of hepta- to decaBDEs was studied in lactating cows exposed to a naturally contaminated diet by analyzing feed, feces, and milk samples from a previous mass balance study of PCB. Tissue distribution was studied in one cow slaughtered after the experiment. BDE-209 was the dominant congener in feed, organs, adipose tissues, and feces, but not in milk. In contrast to PCBs and lower brominated BDEs, concentrations of hepta- to decaBDEs in adipose tissue were 9-80 times higher than in milk fat and the difference increased with degree of bromination/log K(OW). The congener profiles in adipose tissue and feed differed; BDE-207, BDE-196, BDE-197, and BDE-182 accumulated to a surprisingly greater extent in the fat compared to their isomers, suggesting metabolic debromination of BDE-209 to these BDEs. The results indicate that meat rather than dairy product consumption may be an important human exposure route to higher brominated BDEs.

  4. Concentration Sensing by the Moving Nucleus in Cell Fate Determination: A Computational Analysis.

    Directory of Open Access Journals (Sweden)

    Varun Aggarwal

    Full Text Available During development of the vertebrate neuroepithelium, the nucleus in neural progenitor cells (NPCs moves from the apex toward the base and returns to the apex (called interkinetic nuclear migration at which point the cell divides. The fate of the resulting daughter cells is thought to depend on the sampling by the moving nucleus of a spatial concentration profile of the cytoplasmic Notch intracellular domain (NICD. However, the nucleus executes complex stochastic motions including random waiting and back and forth motions, which can expose the nucleus to randomly varying levels of cytoplasmic NICD. How nuclear position can determine daughter cell fate despite the stochastic nature of nuclear migration is not clear. Here we derived a mathematical model for reaction, diffusion, and nuclear accumulation of NICD in NPCs during interkinetic nuclear migration (INM. Using experimentally measured trajectory-dependent probabilities of nuclear turning, nuclear waiting times and average nuclear speeds in NPCs in the developing zebrafish retina, we performed stochastic simulations to compute the nuclear trajectory-dependent probabilities of NPC differentiation. Comparison with experimentally measured nuclear NICD concentrations and trajectory-dependent probabilities of differentiation allowed estimation of the NICD cytoplasmic gradient. Spatially polarized production of NICD, rapid NICD cytoplasmic consumption and the time-averaging effect of nuclear import/export kinetics are sufficient to explain the experimentally observed differentiation probabilities. Our computational studies lend quantitative support to the feasibility of the nuclear concentration-sensing mechanism for NPC fate determination in zebrafish retina.

  5. Microcosm study on fate of polybrominated diphenyl ethers (PBDEs) in contaminated mangrove sediment.

    Science.gov (United States)

    Zhu, Haowen; Wang, Ying; Tam, Nora F Y

    2014-01-30

    Polybrominated diphenyl ethers (PBDEs) are toxic and ubiquitous environmental contaminants, but their fate in aquatic environments is not clear. A mangrove microcosm study was employed to investigate the fate of two abundant congeners, BDE-47 and BDE-209, in contaminated sediment. After seven months, more than 90% of the spiked BDE-47 in the mangrove sediment was removed with the formation of lower brominated PBDEs, including BDE-28, -17, -15, -8, -7/4, suggesting that microbial debromination was the main contributor. Debromination of BDE-209 was also observed in the sediment but its dissipation rate was significantly lower than BDE-47. All these congeners were taken up, translocated and accumulated into the tissues of two typical mangrove plants, Kandelia obovata and Avicennia marina. PBDEs, even at very high contamination levels, in the sediment (5000ngg(-1)) and the debrominated congeners did not pose any adverse effect on the dry weight, augmentation and root/shoot ratio of either mangrove species. This is the first study to reveal that anaerobic microbial debromination and uptake by mangrove plants are the key processes controlling the fate of PBDEs in mangrove sediment.

  6. Biokinetics of zinc oxide nanoparticles: toxicokinetics, biological fates, and protein interaction

    Directory of Open Access Journals (Sweden)

    Choi SJ

    2014-12-01

    Full Text Available Soo-Jin Choi,1 Jin-Ho Choy2 1Department of Food Science and Technology, Seoul Women's University, 2Center for Intelligent Nano Bio Materials (CINBM, Department of Bioinspired Science and Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, South Korea Abstract: Biokinetic studies of zinc oxide (ZnO nanoparticles involve systematic and quantitative analyses of absorption, distribution, metabolism, and excretion in plasma and tissues of whole animals after exposure. A full understanding of the biokinetics provides basic information about nanoparticle entry into systemic circulation, target organs of accumulation and toxicity, and elimination time, which is important for predicting the long-term toxic potential of nanoparticles. Biokinetic behaviors can be dependent on physicochemical properties, dissolution property in biological fluids, and nanoparticle–protein interaction. Moreover, the determination of biological fates of ZnO nanoparticles in the systemic circulation and tissues is critical in interpreting biokinetic behaviors and predicting toxicity potential as well as mechanism. This review focuses on physicochemical factors affecting the biokinetics of ZnO nanoparticles, in concert with understanding bioavailable fates and their interaction with proteins. Keywords: ZnO nanoparticles, biokinetics, distribution, excretion, fate, interaction

  7. Antiproton Accumulator (AA)

    CERN Multimedia

    Photographic Service

    1980-01-01

    The AA in its final stage of construction, before it disappeared from view under concrete shielding. Antiprotons were first injected, stochastically cooled and accumulated in July 1980. From 1981 on, the AA provided antiprotons for collisions with protons, first in the ISR, then in the SPS Collider. From 1983 on, it also sent antiprotons, via the PS, to the Low-Energy Antiproton Ring (LEAR). The AA was dismantled in 1997 and shipped to Japan.

  8. Microbial Uptake, Toxicity, and Fate of Biofabricated ZnS:Mn Nanocrystals.

    Directory of Open Access Journals (Sweden)

    Brian J F Swift

    Full Text Available Despite their importance in nano-environmental health and safety, interactions between engineered nanomaterials and microbial life remain poorly characterized. Here, we used the model organism E. coli to study the penetration requirements, subcellular localization, induction of stress responses, and long-term fate of luminescent Mn-doped ZnS nanocrystals fabricated under "green" processing conditions with a minimized ZnS-binding protein. We find that such protein-coated quantum dots (QDs are unable to penetrate the envelope of unmodified E. coli but readily translocate to the cytoplasm of cells that have been made competent by chemical treatment. The process is dose-dependent and reminiscent of bacterial transformation. Cells that have internalized up to 0.5 μg/mL of nanocrystals do not experience a significant activation of the unfolded protein or SOS responses but undergo oxidative stress when exposed to high QD doses (2.5 μg/mL. Finally, although they are stable in quiescent cells over temperatures ranging from 4 to 42°C, internalized QDs are rapidly diluted by cell division in a process that does not involve TolC-dependent efflux. Taken together, our results suggest that biomimetic QDs based on low toxicity inorganic cores capped by a protein shell are unlikely to cause significant damage to the microbial ecosystem.

  9. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

    Science.gov (United States)

    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  10. An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

    Directory of Open Access Journals (Sweden)

    David A. Turner

    2014-06-01

    Full Text Available Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage.

  11. A functional dissection of PTEN N-terminus : Implications in PTEN subcellular targeting and tumor suppressor activity

    NARCIS (Netherlands)

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J.; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization

  12. 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, allanto

  13. Toxicity and the fractional distribution of trace metals accumulated from contaminated sediments by the clam Scrobicularia plana exposed in the laboratory and the field

    Energy Technology Data Exchange (ETDEWEB)

    Kalman, J., E-mail: judit.kalman@uca.es [Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD (United Kingdom); Bonnail-Miguel, E. [Department of Physical-Chemistry, University of Cadiz, Poligono Industrial Rio San Pedro s/n, 11,510 Puerto Real, Cadiz (Spain); Smith, B.D. [Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD (United Kingdom); Bury, N.R. [Division of Diabetes and Nutritional Science, King' s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH (United Kingdom); Rainbow, P.S. [Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD (United Kingdom)

    2015-02-15

    The relationship between the subcellular distribution of accumulated toxic metals into five operational fractions (subsequently combined into presumed detoxified and non-detoxified components) and toxicity in the clam Scrobicularia plana was investigated under different laboratory exposures. Clams were exposed to metal contaminated media (water and diet) and analysed for the partitioning of accumulated As, Cu and Zn into subcellular fractions. In general, metallothionein-like proteins, metal-rich granules and cellular debris in different proportions acted as main storage sites of accumulated metals in the clam soft tissues for these three metals. No significant differences were noted in the accumulation rates of As, Cu and Zn of groups of individuals with or without apparent signs of toxicity after up to 30 days of exposure to naturally contaminated sediment mixtures. There was, however, an increased proportional accumulation of Cu in the non-detoxified fraction with increased Cu accumulation rate in the clams, suggesting that the Cu uptake rate from contaminated sediments exceeded the combined rates of elimination and detoxification of Cu, with the subsequent likelihood for toxic effects in the clams. - Highlights: • Scrobicularia plana accumulated As, Cu and Zn from naturally toxic sediments. • Toxic metals were accumulated in detoxified and non-detoxified components. • Cu accumulation in the non-detoxified pool increased with increased Cu uptake rate. • Cu uptake rate exceeded combined loss and detoxification rates to cause toxicity.

  14. A Motif in the F Homomorph of Rabbit Haemorrhagic Disease Virus Polymerase Is Important for the Subcellular Localisation of the Protein and Its Ability to Induce Redistribution of Golgi Membranes

    Directory of Open Access Journals (Sweden)

    Nadya Urakova

    2017-08-01

    Full Text Available Rabbit haemorrhagic disease virus (RHDV is a calicivirus that infects and frequently kills rabbits. Previously, we showed that the RHDV RNA-dependent RNA polymerase (RdRp is associated with distinct, but yet uncharacterised subcellular structures and is capable of inducing a redistribution of Golgi membranes. In this study, we identified a partially hidden hydrophobic motif that determines the subcellular localisation of recombinant RHDV RdRp in transfected cells. This novel motif, 189LLWGCDVGVAVCAAAVFHNICY210, is located within the F homomorph, between the conserved F3 and A motifs of the core RdRp domain. Amino acid substitutions that decrease the hydrophobicity of this motif reduced the ability of the protein to accumulate in multiple subcellular foci and to induce a rearrangement of the Golgi network. Furthermore, preliminary molecular dynamics simulations suggest that the RHDV RdRp could align with the negatively charged surfaces of biological membranes and undergo a conformational change involving the F homomorph. These changes would expose the newly identified hydrophobic motif so it could immerse itself into the outer leaflet of intracellular membranes.

  15. The Atmospheric Fate of Organic Nitrogen Compounds

    Science.gov (United States)

    Borduas, Nadine

    Organic nitrogen compounds are present in our atmosphere from biogenic and anthropogenic sources and have impacts on air quality and climate. Due to recent advances in instrumentation, these compounds are being detected in the gas and particle phases, raising questions as to their source, processing and sinks in the environment. With their recently identified role as contributors to aerosol formation and growth, their novel large scale use as solvents in carbon capture and storage (CCS) technology and their emissions from cigarette smoke, it is now important to address the gaps in our understanding of the fate of organic nitrogen. Experimentally and theoretically, I studied the chemical atmospheric fate of specific organic nitrogen compounds in the amine, amide and isocyanate families, yielding information that can be used in chemical transport models to assess the fate of this emerging class of atmospheric molecules. I performed kinetic laboratory studies in a smog chamber to measure the room temperature rate coefficient for reaction with the hydroxyl radical of monoethanolamine, nicotine, and five different amides. I employed online-mass spectrometry techniques to quantify the oxidation products. I found that amines react quickly with OH radicals with lifetimes of a few hours under sunlit conditions, producing amides as oxidation products. My studies on amides revealed that they have much longer lifetimes in the atmosphere, ranging from a few hours to a week. Photo-oxidation of amides produces isocyanates and I investigated these mechanisms in detail using ab initio calculations. Furthermore, I experimentally measured isocyanic acid's Henry's Law constant as well as its hydrolysis rate constants to better understand its sinks in the atmosphere. Finally, I re-examined the structure-activity relationship (SAR) of organic nitrogen molecules for improved model parameterizations.

  16. A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue

    Directory of Open Access Journals (Sweden)

    Dimauro Ivan

    2012-09-01

    Full Text Available Abstract Background We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing. Findings The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment. Conclusion This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  18. Substrates with patterned extracellular matrix and subcellular stiffness gradients reveal local biomechanical responses.

    Science.gov (United States)

    Tseng, Peter; Di Carlo, Dino

    2014-02-26

    A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

  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

    , 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca(2+)]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution...

  20. 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.

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

    Science.gov (United States)

    2013-08-15

    coined the term POLKADOTS or punctate and oligomeric killing or activating domains transducing signals to describe these structures (92...signalsome: Three decades of discovery. Cell Cycle 2011;10:2485-96. 94. Rudner DZ, Losick R. Protein Subcellular localization in bacteria . Cold

  2. Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes.

    Science.gov (United States)

    1985-09-01

    development, the use of anti-FPM antibodies as a means of targeting liposome encapsulated trypanocidal drugs would seem to be an additional area worthy of...Diagnostics) either free (5) or complexed to 6- cyclodextrin (6,19). Relative fluorescence was then determined in the various subcellular fractions

  3. Methods of biomedical optical imaging: from subcellular structures to tissues and organs

    Science.gov (United States)

    Turchin, I. V.

    2016-05-01

    Optical bioimaging methods have a wide range of applications in the life sciences, most notably including the molecular resolution study of subcellular structures, small animal molecular imaging, and structural and functional clinical diagnostics of tissue layers and organs. We review fluorescent microscopy, fluorescent macroscopy, optical coherence tomography, optoacoustic tomography, and optical diffuse spectroscopy and tomography from the standpoint of physical fundamentals, applications, and progress.

  4. Subcellular location of the helper component-proteinase of Cowpea Aphid-Borne Mosaic Virus

    NARCIS (Netherlands)

    Mlotshwa, S.; Verver, J.; Sithole-Niang, I.; Gopinath, K.; Carette, J.; Kammen, van A.; Wellink, J.

    2002-01-01

    The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of H

  5. 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...

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

    Directory of Open Access Journals (Sweden)

    Huanhuan eXu

    2016-05-01

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

  7. Determining the Sub-Cellular Localization of Proteins within Caenorhabditis elegans Body Wall Muscle

    Science.gov (United States)

    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. PMID:21611156

  8. Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution

    NARCIS (Netherlands)

    Bader, A.N.; Hofman, E.G.; Voortman, J.; Henegouwen, P.; Gerritsen, H.C.

    2009-01-01

    Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and

  9. LocateP : genome-scale subcellular-location predictor for bacterial proteins

    NARCIS (Netherlands)

    Zhou, M.; Boekhorst, J.; Francke, C.; Siezen, R.J.

    2008-01-01

    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 class

  10. Development of a Charged Particle Microbeam for Targeted and Single Particle Subcellular Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yanch, Jacquelyn C.

    2004-03-12

    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.

  11. Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions.

    Science.gov (United States)

    Wiśniewski, Jacek R; Wegler, Christine; Artursson, Per

    2016-09-15

    The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap-frozen human liver by differential centrifugation and performed quantitative mass spectrometry-based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP-glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the subcellular distribution of proteins and can be used as a guide for development of fractionation procedures. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2013-12-01

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

  13. Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells

    Energy Technology Data Exchange (ETDEWEB)

    Reinhard, J.F. Jr.; Diliberto, E.J. Jr.; Viveros, O.H.; Daniels, A.J.

    1987-11-01

    Cultures of bovine adrenomedullary chromaffin cells accumulated 1-methyl-4-phenylpyridinium (MPP/sup +/) in a time- and concentration-dependent manner by a process that was prevented by desmethylimipramine. The subcellular localization of the incorporated (methyl-/sup 3/H)MPP/sup +/ was examined by differential centrifugation and sucrose density gradient fractionation and was found to be predominantly colocalized with catecholamines in chromaffin vesicles, and negligible amounts were detected within the mitochondrial fraction. When chromaffin cell membranes were made permeable with the detergent digitonin the absence of calcium, there was no increase in the release of (/sup 3/H)MPP/sup +/, indicating that there is negligible accumulation of the neurotoxin in the cytosol. Simultaneous exposure to digitonin and calcium induced cosecretion of MPP/sup +/ and catecholamines. Stimulation of the cells with nicotine released both catecholamines and MPP/sup +/ at identical rates and percentages of cellular content in a calcium-dependent manner. Last, when cells were incubated with MPP/sup +/ in the presence of tetrabenazine (an inhibitor of vesicular uptake), the chromaffin cell toxicity of MPP/sup +/ was potentiated. The authors submit that the ability of the chromaffin cells to take up and store MPP/sup +/ in the chromaffin vesicle prevents the toxin's interaction with other structures and, thus, prevents cell damage. As an extension of this hypothesis, the relative resistance of some brain monoaminergic neurons to the toxic actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may result from the subcellular sequestration of MPP/sup +/ in the storage vesicle.

  14. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Michael I Koukourakis

    Full Text Available LC3s (MAP1-LC3A, B and C are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli, where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

  15. Prediction of the environmental fate of chemicals.

    Science.gov (United States)

    Vighi, M; Calamari, D

    1993-01-01

    An overview is presented of the possibilities of applying multimedia compartmental evaluative models, and in particular the fugacity approach, to predict the environmental distribution and fate of organic chemicals. The use of this predictive approach for the evaluation of exposure to pollutants in the aquatic system is described, with reference to different environments or discharge patterns (surface and groundwaters, point and diffuse sources of pollution). The value and limitations of this approach are noted and the need for more research to improve predictive capability and practical usefulness is indicated. Finally some practical applications of evaluative models in the proposal of quantitative indices for ecotoxicological evaluation of risk from chemicals are described.

  16. Fate of Trace Metals in Anaerobic Digestion.

    Science.gov (United States)

    Fermoso, F G; van Hullebusch, E D; Guibaud, G; Collins, G; Svensson, B H; Carliell-Marquet, C; Vink, J P M; Esposito, G; Frunzo, L

    2015-01-01

    A challenging, and largely uncharted, area of research in the field of anaerobic digestion science and technology is in understanding the roles of trace metals in enabling biogas production. This is a major knowledge gap and a multifaceted problem involving metal chemistry; physical interactions of metal and solids; microbiology; and technology optimization. Moreover, the fate of trace metals, and the chemical speciation and transport of trace metals in environments--often agricultural lands receiving discharge waters from anaerobic digestion processes--simultaneously represents challenges for environmental protection and opportunities to close process loops in anaerobic digestion.

  17. Predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic K-Nearest Neighbor classifiers.

    Science.gov (United States)

    Chou, Kuo-Chen; Shen, Hong-Bin

    2006-08-01

    Facing the explosion of newly generated protein sequences in the post genomic era, we are challenged to develop an automated method for fast and reliably annotating their subcellular locations. Knowledge of subcellular locations of proteins can provide useful hints for revealing their functions and understanding how they interact with each other in cellular networking. Unfortunately, it is both expensive and time-consuming to determine the localization of an uncharacterized protein in a living cell purely based on experiments. To tackle the challenge, a novel hybridization classifier was developed by fusing many basic individual classifiers through a voting system. The "engine" of these basic classifiers was operated by the OET-KNN (Optimized Evidence-Theoretic K-Nearest Neighbor) rule. As a demonstration, predictions were performed with the fusion classifier for proteins among the following 16 localizations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cyanelle, (5) cytoplasm, (6) cytoskeleton, (7) endoplasmic reticulum, (8) extracell, (9) Golgi apparatus, (10) lysosome, (11) mitochondria, (12) nucleus, (13) peroxisome, (14) plasma membrane, (15) plastid, and (16) vacuole. To get rid of redundancy and homology bias, none of the proteins investigated here had >/=25% sequence identity to any other in a same subcellular location. The overall success rates thus obtained via the jack-knife cross-validation test and independent dataset test were 81.6% and 83.7%, respectively, which were 46 approximately 63% higher than those performed by the other existing methods on the same benchmark datasets. Also, it is clearly elucidated that the overwhelmingly high success rates obtained by the fusion classifier is by no means a trivial utilization of the GO annotations as prone to be misinterpreted because there is a huge number of proteins with given accession numbers and the corresponding GO numbers, but their subcellular locations are still unknown, and that the

  18. X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.

    Science.gov (United States)

    Grubman, A; James, S A; James, J; Duncan, C; Volitakis, I; Hickey, J L; Crouch, P J; Donnelly, P S; Kanninen, K M; Liddell, J R; Cotman, S L; de Jonge; White, A R

    2014-06-01

    Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6(nclf) (CbCln6(nclf) ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in CbCln6(nclf) cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6(nclf) cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6(nclf) cells. Importantly, a zinc-complex, Zn(II)(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6(nclf) cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. Zn(II)(atsm) treatment also resulted in a

  19. Variations in phthalate ester (PAE) accumulation and their formation mechanism in Chinese flowering cabbage (Brassica parachinensis L.) cultivars grown on PAE-contaminated soils.

    Science.gov (United States)

    Zhao, Hai-Ming; Du, Huan; Xiang, Lei; Chen, Yi-Liang; Lu, Lei-An; Li, Yan-Wen; Li, Hui; Cai, Quan-Ying; Mo, Ce-Hui

    2015-11-01

    Phthalate ester (PAE) accumulation in crops poses great risks to human health and has aroused great concern. Here, we investigated variations in di-n-butylphthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) accumulation by various Chinese flowering cabbage cultivars and revealed their variation mechanism. There were significant differences (p PAE concentrations of 28 cultivars. Moreover, significant positive correlations between DBP and DEHP concentrations in shoots of all cultivars indicated that they could be taken up simultaneously by various cultivars. Due to the lower translocation factor of low-PAE accumulator, its shoot PAEs concentrations were much lower than root compared to high-PAE accumulator. Further, subcellular distribution showed that PAE concentrations of root cell walls and organelles were much higher than those of shoots in low-PAE accumulator. Therefore, lower translocation from root to shoot and more PAEs accumulating in cell walls and organelles of root might act as main formation mechanism of low-PAE accumulator.

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

    Science.gov (United States)

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

    2016-07-01

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

  1. 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.

  2. 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.

  3. Investigating Student Ideas About the Fate of the Universe

    Science.gov (United States)

    Conlon, Mallory; Coble, Kimberly A.; Bailey, Janelle M.; Cominsky, Lynn R.

    2015-01-01

    Data from recent surveys have enabled astronomers to precisely quantify the composition of the Universe, though the nature of its primary component, dark energy, remains a mystery. The evolution of dark energy and how it might impact the Universe in the future is an area of intense study. As astronomers further develop an understanding of the fate of the Universe, it is essential to study student ideas on this fate so that instructors can communicate the field's current status and its underpinnings more effectively to their students. In this study, we examine undergraduate students' pre-instruction ideas of the fate of the Universe in twelve semester-long courses at four institutions. We also examine ideas about the fate of the Universe as undergraduate students progress through an introductory or advanced astronomy course at two institutions. The data include pre-course surveys given during the first week of instruction [N=291], midterm and final exam questions [N=58], post-course surveys [N=26], and student interviews [N=7]. We find that, though the term was not necessarily used, students that respond tend to describe a 'big freeze' scenario in the pre-course surveys. Students mention the Universe's expansion when describing how we know the fate of the Universe but do not discuss how we know the Universe is expanding or the relationship between expansion and the fate of the Universe. We also find that students discuss the fate of the solar system or the galaxy in the pre-course surveys instead of the fate of the Universe, suggesting conflation of the Universe with the solar system or the galaxy. At the end of the course, we find that students continue to describe a 'big freeze' scenario and fail to explain how we determine the fate of the Universe. We also find that student tendency to discuss the fate of the solar system or galaxy instead of the fate of the Universe is diminished by the end of the course.

  4. Ice slurry accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Christensen, K.G.; Kauffeld, M.

    1998-06-01

    More and more refrigeration systems are designed with secondary loops, thus reducing the refrigerant charge of the primary refrigeration plant. In order not to increase energy consumption by introducing a secondary refrigerant, alternatives to the well established single phase coolants (brines) and different concepts of the cooling plant have to be evaluated. Combining the use of ice-slurry - mixture of water, a freezing point depressing agent (antifreeze) and ice particles - as melting secondary refrigerant and the use of a cool storage makes it possible to build plants with secondary loops without increasing the energy consumption and investment. At the same time the operating costs can be kept at a lower level. The accumulation of ice-slurry is compared with other and more traditional storage systems. The method is evaluated and the potential in different applications is estimated. Aspects of practically use of ice-slurry has been examined in the laboratory at the Danish Technological Institute (DTI). This paper will include the final conclusions from this work concerning tank construction, agitator system, inlet, outlet and control. The work at DTI indicates that in some applications systems with ice-slurry and accumulation tanks have a great future. These applications are described by a varying load profile and a process temperature suiting the temperature of ice-slurry (-3 - -8/deg. C). (au)

  5. High resolution imaging of subcellular glutathione concentrations by quantitative immunoelectron microscopy in different leaf areas of Arabidopsis

    Science.gov (United States)

    Koffler, Barbara E.; Bloem, Elke; Zellnig, Günther; Zechmann, Bernd

    2013-01-01

    Glutathione is an important antioxidant and redox buffer in plants. It fulfills many important roles during plant development, defense and is essential for plant metabolism. Even though the compartment specific roles of glutathione during abiotic and biotic stress situations have been studied in detail there is still great lack of knowledge about subcellular glutathione concentrations within the different leaf areas at different stages of development. In this study a method is described that allows the calculation of compartment specific glutathione concentrations in all cell compartments simultaneously in one experiment by using quantitative immunogold electron microscopy combined with biochemical methods in different leaf areas of Arabidopsis thaliana Col-0 (center of the leaf, leaf apex, leaf base and leaf edge). The volume of subcellular compartments in the mesophyll of Arabidopsis was found to be similar to other plants. Vacuoles covered the largest volume within a mesophyll cell and increased with leaf age (up to 80% in the leaf apex of older leaves). Behind vacuoles, chloroplasts covered the second largest volume (up to 20% in the leaf edge of the younger leaves) followed by nuclei (up to 2.3% in the leaf edge of the younger leaves), mitochondria (up to 1.6% in the leaf apex of the younger leaves), and peroxisomes (up to 0.3% in the leaf apex of the younger leaves). These values together with volumes of the mesophyll determined by stereological methods from light and electron micrographs and global glutathione contents measured with biochemical methods enabled the determination of subcellular glutathione contents in mM. Even though biochemical investigations did not reveal differences in global glutathione contents, compartment specific differences could be observed in some cell compartments within the different leaf areas. Highest concentrations of glutathione were always found in mitochondria, where values in a range between 8.7 mM (in the apex of younger

  6. Fate in the religion of the Lepchas

    Directory of Open Access Journals (Sweden)

    Halfdan Siiger

    1967-02-01

    Full Text Available The Lepchas are mountainous agriculturalists who live in the State of Sikkim in the Himalayas and in some adjacent Indian districts. To the Lepchas the supernatural world is divided into two groups, the rum, or the mainly benevolent supernatural beings, and the mung, or the malignant supernatural beings. Any evil occurrence is in the first instance ascribed to the malignant activities of the mung, but it may, under certain conditions, also be due to temporary on the part of some or other rum. If it is obvious that the evil occurrence is caused by a human being, this person is considered to be governed by some mung, or he may, which is much worse, be a mung in human disguise. At all events, any evil occurrence is experienced as the result of the evil will-power of some or other malignant supernatural being. Consequently, we cannot apply our technical term "Fate" to such occurrences, and Fate as an abstract concept cannot be used, when we speak of the Lepchas.

  7. Connecting Mitochondria, Metabolism, and Stem Cell Fate.

    Science.gov (United States)

    Wanet, Anaïs; Arnould, Thierry; Najimi, Mustapha; Renard, Patricia

    2015-09-01

    As sites of cellular respiration and energy production, mitochondria play a central role in cell metabolism. Cell differentiation is associated with an increase in mitochondrial content and activity and with a metabolic shift toward increased oxidative phosphorylation activity. The opposite occurs during reprogramming of somatic cells into induced pluripotent stem cells. Studies have provided evidence of mitochondrial and metabolic changes during the differentiation of both embryonic and somatic (or adult) stem cells (SSCs), such as hematopoietic stem cells, mesenchymal stem cells, and tissue-specific progenitor cells. We thus propose to consider those mitochondrial and metabolic changes as hallmarks of differentiation processes. We review how mitochondrial biogenesis, dynamics, and function are directly involved in embryonic and SSC differentiation and how metabolic and sensing pathways connect mitochondria and metabolism with cell fate and pluripotency. Understanding the basis of the crosstalk between mitochondria and cell fate is of critical importance, given the promising application of stem cells in regenerative medicine. In addition to the development of novel strategies to improve the in vitro lineage-directed differentiation of stem cells, understanding the molecular basis of this interplay could lead to the identification of novel targets to improve the treatment of degenerative diseases.

  8. Fate of Fusarium Toxins during Brewing.

    Science.gov (United States)

    Habler, Katharina; Geissinger, Cajetan; Hofer, Katharina; Schüler, Jan; Moghari, Sarah; Hess, Michael; Gastl, Martina; Rychlik, Michael

    2017-01-11

    Some information is available about the fate of Fusarium toxins during the brewing process, but only little is known about the single processing steps in detail. In our study we produced beer from two different barley cultivars inoculated with three different Fusarium species, namely, Fusarium culmorum, Fusarium sporotrichioides, and Fusarium avenaceum, producing a wide range of mycotoxins such as type B trichothecenes, type A trichothecenes, and enniatins. By the use of multi-mycotoxin LC-MS/MS stable isotope dilution methods we were able to follow the fate of Fusarium toxins during the entire brewing process. In particular, the type B trichothecenes deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol showed similar behaviors. Between 35 and 52% of those toxins remained in the beer after filtration. The contents of the potentially hazardous deoxynivalenol-3-glucoside and the type A trichothecenes increased during mashing, but a rapid decrease of deoxynivalenol-3-glucoside content was found during the following steps of lautering and wort boiling. The concentration of enniatins greatly decreased with the discarding of spent grains or finally with the hot break. The results of our study show the retention of diverse Fusarium toxins during the brewing process and allow for assessing the food safety of beer regarding the monitored Fusarium mycotoxins.

  9. Glucocorticoid regulation of astrocytic fate and function.

    Directory of Open Access Journals (Sweden)

    Shuang Yu

    Full Text Available Glial loss in the hippocampus has been suggested as a factor in the pathogenesis of stress-related brain disorders that are characterized by dysregulated glucocorticoid (GC secretion. However, little is known about the regulation of astrocytic fate by GC. Here, we show that astrocytes derived from the rat hippocampus undergo growth inhibition and display moderate activation of caspase 3 after exposure to GC. Importantly, the latter event, observed both in situ and in primary astrocytic cultures is not followed by either early- or late-stage apoptosis, as monitored by stage I or stage II DNA fragmentation. Thus, unlike hippocampal granule neurons, astrocytes are resistant to GC-induced apoptosis; this resistance is due to lower production of reactive oxygen species (ROS and a greater buffering capacity against the cytotoxic actions of ROS. We also show that GC influence hippocampal cell fate by inducing the expression of astrocyte-derived growth factors implicated in the control of neural precursor cell proliferation. Together, our results suggest that GC instigate a hitherto unknown dialog between astrocytes and neural progenitors, adding a new facet to understanding how GC influence the cytoarchitecture of the hippocampus.

  10. Cell fate regulation in early mammalian development

    Science.gov (United States)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  11. Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation

    Science.gov (United States)

    Robison, Gregory; Sullivan, Brendan; Cannon, Jason R.; Pushkar, Yulia

    2015-01-01

    Manganese serves as a cofactor to a variety of proteins necessary for proper bodily development and function. However, an overabundance of Mn in the brain can result in manganism, a neurological condition resembling Parkinson’s disease (PD). Bulk sample measurement techniques have identified the globus pallidus and thalamus as targets of Mn accumulation in the brain, however smaller structures/cells cannot be measured. Here, X-ray fluorescence microscopy determined the metal content and distribution in the substantia nigra (SN) of the rodent brain. In vivo retrograde labeling of dopaminergic cells (via FluoroGold™) of the SN pars compacta (SNc) subsequently allowed for XRF imaging of dopaminergic cells in situ at subcellular resolution. Chronic Mn exposure resulted in a significant Mn increase in both the SN pars reticulata (>163%) and the SNc (>170%) as compared to control; no other metal concentrations were significantly changed. Subcellular imaging of dopaminergic cells demonstrated that Mn is located adjacent to the nucleus. Measured intracellular manganese concentrations range between 40–200 μM; concentrations as low as 100 μM have been observed to cause cell death in cell cultures. Direct observation of Mn accumulation in the SNc could establish a biological basis for movement disorders associated with manganism, specifically Mn caused insult to the SNc. Accumulation of Mn in dopaminergic cells of the SNc may help clarify the relationship between Mn and the loss of motor skills associated with manganism. PMID:25695229

  12. High resolution imaging of temporal and spatial changes of subcellular ascorbate, glutathione and H₂O₂ distribution during Botrytis cinerea infection in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Uwe K Simon

    Full Text Available In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H₂O₂ was monitored over a time frame of 96 h post inoculation (hpi in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS and the area around the IS which was defined as area adjacent to the inoculation site (AIS. H₂O₂ accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H₂O₂ in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size, chlorosis and necrosis of the leaves.

  13. Cell fate and cell differentiation status in the Arabidopsis root

    NARCIS (Netherlands)

    Scheres, B.J.G.; Berg, C. van den; Weisbeek, P.

    1998-01-01

    Post-embryonic development in plants is mainly achieved by its meristems. Within the Arabidopsis root meristem, both the fate and origin of its cells can be predicted with high accuracy. Mutants defective in the determination of root cell fates show that the corresponding genes are first required

  14. Cell fate determination in the Caenorhabditis elegans epidermal lineages

    NARCIS (Netherlands)

    Soete, G.A.J.

    2007-01-01

    The starting point for this work was to use the hypodermal seam of C. elegans as a model system to study cell fate determination. Even though the seam is a relatively simple developmental system, the mechanisms that control cell fate determination in the seam lineages are connected in a highly compl

  15. Analysis, fate and effects of the antibiotic sulfadiazine in soil.

    NARCIS (Netherlands)

    Schauss, K.; Focks, A.; Heuer, H.; Kotzerke, A.; Schmitt, H.; Thiele-Bruhn, S.; Smalla, K.; Wilke, B.M.; Matthies, M.

    2009-01-01

    This review summarizes current knowledge about the interplay between fate and effects of the antibiotic sulfadiazine in soil ecosystems. In applying manure from antibiotic-treated animals to arable soils, sulfadiazine can reach the environment, but fate and transformation processes and the consequen

  16. Spatially explicit fate modelling of nanomaterials in natural waters

    NARCIS (Netherlands)

    Quik, J.T.K.; Klein, de J.J.M.; Koelmans, A.A.

    2015-01-01

    Site specific exposure assessments for engineered nanoparticles (ENPs) require spatially explicit fate models, which however are not yet available. Here we present an ENP fate model (NanoDUFLOW) that links ENP specific process descriptions to a spatially explicit hydrological model. The link enables

  17. Cell fate determination in the Caenorhabditis elegans epidermal lineages

    NARCIS (Netherlands)

    Soete, G.A.J.

    2007-01-01

    The starting point for this work was to use the hypodermal seam of C. elegans as a model system to study cell fate determination. Even though the seam is a relatively simple developmental system, the mechanisms that control cell fate determination in the seam lineages are connected in a highly compl

  18. Analysis, fate and effects of the antibiotic sulfadiazine in soil.

    NARCIS (Netherlands)

    Schauss, K.; Focks, A.; Heuer, H.; Kotzerke, A.; Schmitt, H.|info:eu-repo/dai/nl/304831042; Thiele-Bruhn, S.; Smalla, K.; Wilke, B.M.; Matthies, M.

    2009-01-01

    This review summarizes current knowledge about the interplay between fate and effects of the antibiotic sulfadiazine in soil ecosystems. In applying manure from antibiotic-treated animals to arable soils, sulfadiazine can reach the environment, but fate and transformation processes and the

  19. Fate and Bioavailability of Engineered Nanoparticles in Soils: A Review

    NARCIS (Netherlands)

    Cornelis, G.; Hund-Rinke, K.; Kuhlbusch, T.; Brink, van den N.W.; Nickel, C.

    2014-01-01

    Interactions within natural soils have often been neglected when assessing fate and bioavailability of engineered nanomaterials (ENM) in soils. This review combines patchwise ENM research using natural soils with the much wider literature on ENM performed in standard tests or on the fate of colloids

  20. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

    Section 06 - 08*) of the AA where the dispersion (and hence the horizontal beam size) is large. One can distinguish (left to right): A vacuum-tank, two bending magnets (BST06 and BST07 in blue) with a quadrupole (QDN07, in red) in between, another vacuum-tank, a wide quadrupole (QFW08) and a further tank . The tanks are covered with heating tape for bake-out. The tank left of BST06 contained the stack core pickup for stochastic cooling (see 7906193, 7906190, 8005051), the two other tanks served mainly as vacuum chambers in the region where the beam was large. Peter Zettwoch works on BST06. *) see: H. Koziol, Antiproton Accumulator Parameter List, PS/AA/Note 84-2 (1984)

  1. Solids Accumulation Scouting Studies

    Energy Technology Data Exchange (ETDEWEB)

    Duignan, M. R.; Steeper, T. J.; Steimke, J. L.

    2012-09-26

    The objective of Solids Accumulation activities was to perform scaled testing to understand the behavior of remaining solids in a Double Shell Tank (DST), specifically AW-105, at Hanford during multiple fill, mix, and transfer operations. It is important to know if fissionable materials can concentrate when waste is transferred from staging tanks prior to feeding waste treatment plants. Specifically, there is a concern that large, dense particles containing plutonium could accumulate in poorly mixed regions of a blend tank heel for tanks that employ mixing jet pumps. At the request of the DOE Hanford Tank Operations Contractor, Washington River Protection Solutions, the Engineering Development Laboratory of the Savannah River National Laboratory performed a scouting study in a 1/22-scale model of a waste staging tank to investigate this concern and to develop measurement techniques that could be applied in a more extensive study at a larger scale. Simulated waste tank solids: Gibbsite, Zirconia, Sand, and Stainless Steel, with stainless steel particles representing the heavier particles, e.g., plutonium, and supernatant were charged to the test tank and rotating liquid jets were used to mix most of the solids while the simulant was pumped out. Subsequently, the volume and shape of the mounds of residual solids and the spatial concentration profiles for the surrogate for heavier particles were measured. Several techniques were developed and equipment designed to accomplish the measurements needed and they included: 1. Magnetic particle separator to remove simulant stainless steel solids. A device was designed and built to capture these solids, which represent the heavier solids during a waste transfer from a staging tank. 2. Photographic equipment to determine the volume of the solids mounds. The mounds were photographed as they were exposed at different tank waste levels to develop a composite of topographical areas. 3. Laser rangefinders to determine the volume of

  2. Asymmetric cell division during T cell development controls downstream fate

    Science.gov (United States)

    Pham, Kim; Shimoni, Raz; Charnley, Mirren; Ludford-Menting, Mandy J.; Hawkins, Edwin D.; Ramsbottom, Kelly; Oliaro, Jane; Izon, David; Ting, Stephen B.; Reynolds, Joseph; Lythe, Grant; Molina-Paris, Carmen; Melichar, Heather; Robey, Ellen; Humbert, Patrick O.; Gu, Min

    2015-01-01

    During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal. PMID:26370500

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

    Directory of Open Access Journals (Sweden)

    María Rodríguez-Escudero

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  5. BasinBox: a generic multimedia fate model for predicting the fate of chemicals in river catchments

    NARCIS (Netherlands)

    Hollander, A; Huijbregts, M A J; Ragas, A M J; Meent, D van de

    2006-01-01

    Multimedia fate models have proven to be very useful tools in chemical risk assessment and management. This paper presents BasinBox, a newly developed steady-state generic multimedia fate model for evaluating risks of new and existing chemicals in river basins. The model concepts, as well as the int

  6. C. Linnaeus' ideas concerning retribution and fate

    Directory of Open Access Journals (Sweden)

    K. Rob. V. Wikman

    1967-02-01

    Full Text Available Linnæus' Nemesis divina has been interpreted in different ways. Crucial is its central problem: the ideas of fate and retribution, but these are, in turn, dependent on Linnæus' conception of God and nature and not least on his opinions concerning the unity and coherence of the natural and ethical order of the world. From whatever sources Linnæus may have derived his religious ideas and whatever changes they may have undergone, his religious attitude in face of the works of nature remained unshaken. But Linnæus' religion, as we find it fragmentarily in these literary sources, was entirely undogmatic, untheological and, from a Christian point of view, even heterodox. Partly, this was in accord with his belief in the necessary immanent coherence in the processes of nature and the concomitant idea of the righteous divine order of the world.

  7. Fate of pesticides during beer brewing.

    Science.gov (United States)

    Inoue, Tomonori; Nagatomi, Yasushi; Suga, Keiko; Uyama, Atsuo; Mochizuki, Naoki

    2011-04-27

    The fates of more than 300 pesticide residues were investigated in the course of beer brewing. Ground malt artificially contaminated with pesticides was brewed via steps such as mashing, boiling, and fermentation. Analytical samples were taken from wort, spent grain, and beer produced at certain key points in the brewing process. The samples were extracted and purified with the QuEChERS (Quick Easy Cheap Effective Rugged and Safe) method and were then analyzed by LC-MS/MS using a multiresidue method. In the results, a majority of pesticides showed a reduction in the unhopped wort and were adsorbed onto the spent grain after mashing. In addition, some pesticides diminished during the boiling and fermentation. This suggests that the reduction was caused mainly by adsorption, pyrolysis, and hydrolysis. After the entire process of brewing, the risks of contaminating beer with pesticides were reduced remarkably, and only a few pesticides remained without being removed or resolved.

  8. Fate of selected pharmaceuticals in river waters.

    Science.gov (United States)

    Calza, P; Medana, C; Padovano, E; Giancotti, V; Minero, C

    2013-04-01

    The aqueous environmental fate of two antibiotics, lincomycin and clarithromycin, and an antiepileptic drug, carbamazepine, was investigated by monitoring drugs decomposition and identifying intermediates in Po river water (North Italy). Initially, control experiments in the dark and under illumination were performed on river water spiked with drugs to simulate all possible transformation processes occurring in the aquatic system. Under illumination, these pharmaceuticals were degraded and transformed into numerous organic intermediate compounds. Several species were formed and characterised by analysing MS and MS(n) spectra and by comparison with parent molecule fragmentation pathways. River water was sampled at three sampling points in an urban area. The selected pharmaceuticals were detected in all samples. Eight transformation products identified in the laboratory simulation were found in natural river water from carbamazepine degradation, three from clarithromycin and two from lincomycin. Their transformation occurring in aquatic system mainly involved mono- and poly-hydroxylation followed by oxidation of the hydroxyl groups.

  9. Binary cell fate decisions and fate transformation in the Drosophila larval eye.

    Directory of Open Access Journals (Sweden)

    Abhishek Kumar Mishra

    Full Text Available The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5 or the green-sensitive Rhodopsin 6 (Rh6. Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.

  10. The Fate of Neutron Star Binary Mergers

    Science.gov (United States)

    Piro, Anthony L.; Giacomazzo, Bruno; Perna, Rosalba

    2017-08-01

    Following merger, a neutron star (NS) binary can produce roughly one of three different outcomes: (1) a stable NS, (2) a black hole (BH), or (3) a supramassive, rotationally supported NS, which then collapses to a BH following angular momentum losses. Which of these fates occur and in what proportion has important implications for the electromagnetic transient associated with the mergers and the expected gravitational wave (GW) signatures, which in turn depend on the high density equation of state (EOS). Here we combine relativistic calculations of NS masses using realistic EOSs with Monte Carlo population synthesis based on the mass distribution of NS binaries in our Galaxy to predict the distribution of fates expected. For many EOSs, a significant fraction of the remnants are NSs or supramassive NSs. This lends support to scenarios in which a quickly spinning, highly magnetized NS may be powering an electromagnetic transient. This also indicates that it will be important for future GW observatories to focus on high frequencies to study the post-merger GW emission. Even in cases where individual GW events are too low in signal to noise to study the post merger signature in detail, the statistics of how many mergers produce NSs versus BHs can be compared with our work to constrain the EOS. To match short gamma-ray-burst (SGRB) X-ray afterglow statistics, we find that the stiffest EOSs are ruled out. Furthermore, many popular EOSs require a significant fraction of ˜60%-70% of SGRBs to be from NS-BH mergers rather than just binary NSs.

  11. Distribution and fate of legacy and emerging contaminants along the Adriatic Sea: A comparative study.

    Science.gov (United States)

    Combi, Tatiane; Pintado-Herrera, Marina G; Lara-Martin, Pablo A; Miserocchi, Stefano; Langone, Leonardo; Guerra, Roberta

    2016-11-01

    The spatial distributions and fates of selected legacy and emerging compounds were investigated and compared in surface sediments sampled along the Adriatic mud-wedge and in deep-sea regions from the southern Adriatic basin. Results indicated that the concentrations of legacy contaminants (PAHs, PCBs and DDTs) and emerging contaminants (tonalide, galaxolide, EHMC, octocrylene, BP3 and NP) ranged from 0.1 to 572 ng g(-1) and from emerging contaminants to sediments in the Adriatic Sea. Nevertheless, the prevalence of some UV filters and fragrances in the central and southern Adriatic indicates that the proximity to tourist areas and WWTPs discharges seems to affect the distribution of those compounds. The accumulation of contaminants in the deep-sea areas supports the inference that this region may act as an important repository for contaminants within the Adriatic Sea. Estimated annual contaminant accumulation reveals that both, legacy and emerging contaminants accumulate preferentially in the northern Adriatic (40-60% of the total annual contaminant accumulation), where the presence of legacy, and to a lesser extent emerging contaminants, are likely to pose an immediate or long-term hazard to resident biota.

  12. Aquatic pathways model to predict the fate of phenolic compounds

    Energy Technology Data Exchange (ETDEWEB)

    Aaberg, R.L.; Peloquin, R.A.; Strenge, D.L.; Mellinger, P.J.

    1983-04-01

    Organic materials released from energy-related activities could affect human health and the environment. To better assess possible impacts, we developed a model to predict the fate of spills or discharges of pollutants into flowing or static bodies of fresh water. A computer code, Aquatic Pathways Model (APM), was written to implement the model. The computer programs use compartmental analysis to simulate aquatic ecosystems. The APM estimates the concentrations of chemicals in fish tissue, water and sediment, and is therefore useful for assessing exposure to humans through aquatic pathways. The APM will consider any aquatic pathway for which the user has transport data. Additionally, APM will estimate transport rates from physical and chemical properties of chemicals between several key compartments. The major pathways considered are biodegradation, fish and sediment uptake, photolysis, and evaporation. The model has been implemented with parameters for distribution of phenols, an important class of compounds found in the water-soluble fractions of coal liquids. Current modeling efforts show that, in comparison with many pesticides and polyaromatic hydrocarbons (PAH), the lighter phenolics (the cresols) are not persistent in the environment. The properties of heavier molecular weight phenolics (indanols, naphthols) are not well enough understood at this time to make similar judgements. For the twelve phenolics studied, biodegradation appears to be the major pathway for elimination from aquatic environments. A pond system simulation (using APM) of a spill of solvent refined coal (SRC-II) materials indicates that phenol, cresols, and other single cyclic phenolics are degraded to 16 to 25 percent of their original concentrations within 30 hours. Adsorption of these compounds into sediments and accumulation by fish was minor.

  13. Fate of ingested Clostridium difficile spores in mice.

    Directory of Open Access Journals (Sweden)

    Amber Howerton

    Full Text Available Clostridium difficile infection (CDI is a leading cause of antibiotic-associated diarrhea, a major nosocomial complication. The infective form of C. difficile is the spore, a dormant and resistant structure that forms under stress. Although spore germination is the first committed step in CDI onset, the temporal and spatial distribution of ingested C. difficile spores is not clearly understood. We recently reported that CamSA, a synthetic bile salt analog, inhibits C. difficile spore germination in vitro and in vivo. In this study, we took advantage of the anti-germination activity of bile salts to determine the fate of ingested C. difficile spores. We tested four different bile salts for efficacy in preventing CDI. Since CamSA was the only anti-germinant tested able to prevent signs of CDI, we characterized CamSa's in vitro stability, distribution, and cytotoxicity. We report that CamSA is stable to simulated gastrointestinal (GI environments, but will be degraded by members of the natural microbiota found in a healthy gut. Our data suggest that CamSA will not be systemically available, but instead will be localized to the GI tract. Since in vitro pharmacological parameters were acceptable, CamSA was used to probe the mouse model of CDI. By varying the timing of CamSA dosage, we estimated that C. difficile spores germinated and established infection less than 10 hours after ingestion. We also showed that ingested C. difficile spores rapidly transited through the GI tract and accumulated in the colon and cecum of CamSA-treated mice. From there, C. difficile spores were slowly shed over a 96-hour period. To our knowledge, this is the first report of using molecular probes to obtain disease progression information for C. difficile infection.

  14. Fate of microplastics in the marine isopod Idotea emarginata.

    Science.gov (United States)

    Hämer, Julia; Gutow, Lars; Köhler, Angela; Saborowski, Reinhard

    2014-11-18

    Plastic pollution is an emerging global threat for marine wildlife. Many species of birds, reptiles, and fishes are directly impaired by plastics as they can get entangled in ropes and drown or they can ingest plastic fragments which, in turn, may clog their stomachs and guts. Microplastics of less than 1 mm can be ingested by small invertebrates, but their fate in the digestive organs and their effects on the animals are yet not well understood. We embedded fluorescent microplastics in artificial agarose-based food and offered the food to marine isopods, Idotea emarginata. The isopods did not distinguish between food with and food without microplastics. Upon ingestion, the microplastics were present in the stomach and in the gut but not in the tubules of the midgut gland which is the principal organ of enzyme-secretion and nutrient resorption. The feces contained the same concentration of microplastics as the food which indicates that no accumulation of microplastics happens during the gut passage. Long-term bioassays of 6 weeks showed no distinct effects of continuous microplastic consumption on mortality, growth, and intermolt duration. I. emarginata are able to prevent intrusion of particles even smaller than 1 μm into the midgut gland which is facilitated by the complex structure of the stomach including a fine filter system. It separates the midgut gland tubules from the stomach and allows only the passage of fluids and chyme. Our results indicate that microplastics, as administered in the experiments, do not clog the digestive organs of isopods and do not have adverse effects on their life history parameters.

  15. Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.

    Science.gov (United States)

    Yang, Helen H; St-Pierre, François; Sun, Xulu; Ding, Xiaozhe; Lin, Michael Z; Clandinin, Thomas R

    2016-06-30

    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

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

    Science.gov (United States)

    Chang, Yulei; Li, Xiaodan; Zhang, Li; Xia, Lu; Liu, Xiaomin; Li, Cuixia; Zhang, Youlin; Tu, Langping; Xue, Bin; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

    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 efficacy. It is of vital importance for these nanophotosensitizers to reach specifically the organelles and to perform PDT with precise time control. To do so, we have in this work traced the dynamic subcellular distribution, especially in organelles such as lysosomes and mitochondria, of the poly(allylamine)-modified and dual-loaded nanophotosensitizers. The apoptosis of the cancer cells induced by PDT with the dependence of the distribution status of the nanophotosensitizers in organelles was obtained, which has provided an in-depth picture of intracellular trafficking of organelle-targeted nanophotosensitizers. Our results shall facilitate the improvement of nanotechnology assisted photodynamic therapy of cancers. PMID:28361967

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  19. Predicting apoptosis protein subcellular location with PseAAC by incorporating tripeptide composition.

    Science.gov (United States)

    Liao, Bo; Jiang, Jun-Bao; Zeng, Qing-Guang; Zhu, Wen

    2011-11-01

    The function of the protein is closely correlated with its subcellular localization. Probing into the mechanism of protein sorting and predicting protein subcellular location can provide important clues or insights for understanding the function of proteins. In this paper, we introduce a new PseAAC approach to encode the protein sequence based on the physicochemical properties of amino acid residues. Each of the protein samples was defined as a 146D (dimensional) vector including the 20 amino acid composition components and 126 adjacent triune residues contents. To evaluate the effectiveness of this encoding scheme, we did jackknife tests on three datasets using the support vector machine algorithm. The total prediction accuracies are 84.9%, 91.2%, and 92.6%, respectively. The satisfactory results indicate that our method could be a useful tool in the area of bioinformatics and proteomics.

  20. 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.

  1. Fast Fourier Transform-based Support Vector Machine for Subcellular Localization Prediction Using Different Substitution Models

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    There are approximately 109 proteins in a cell. A hotspot in bioinformatics is how to identify a protein's subcellular localization, if its sequence is known. In this paper, a method using fast Fourier transform-based support vector machine is developed to predict the subcellular localization of proteins from their physicochemical properties and structural parameters. The prediction accuracies reached 83% in prokaryotic organisms and 84% in eukaryotic organisms with the substitution model of the c-p-v matrix (c, composition; p, polarity; and v, molecular volume). The overall prediction accuracy was also evaluated using the "leave-one-out" jackknife procedure. The influence of the substitution model on prediction accuracy has also been discussed in the work. The source code of the new program is available on request from the authors.

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

    Science.gov (United States)

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

    2012-11-01

    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.

  3. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

    A section of the AA where the dispersion (and hence the horizontal beam size) is large. One can distinguish (left to right): A large vacuum-tank, a quadrupole (QDN09*), a bending magnet (BST08), another vacuum-tank, a wide quadrupole (QFW08) and (in the background) a further bending magnet (BST08). The tanks are covered with heating tape for bake-out. The tank left of QDN09 contained the kickers for stochastic pre-cooling (see 790621, 8002234, 8002637X), the other one served mainly as vacuum chamber in the region where the beam was large. Peter Zettwoch works on QFW08. * see: H. Koziol, Antiproton Accumulator Parameter List, PS/AA/Note 84-2 (1984) See under 7911303, 7911597X, 8004261 and 8202324. For photos of the AA in different phases of completion (between 1979 and 1982) see: 7911303, 7911597X, 8004261, 8004608X, 8005563X, 8005565X, 8006716X, 8006722X, 8010939X, 8010941X, 8202324, 8202658X, 8203628X .

  4. ITER helium ash accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Hogan, J.T.; Hillis, D.L.; Galambos, J.; Uckan, N.A. (Oak Ridge National Lab., TN (USA)); Dippel, K.H.; Finken, K.H. (Forschungszentrum Juelich GmbH (Germany, F.R.). Inst. fuer Plasmaphysik); Hulse, R.A.; Budny, R.V. (Princeton Univ., NJ (USA). Plasma Physics Lab.)

    1990-01-01

    Many studies have shown the importance of the ratio {upsilon}{sub He}/{upsilon}{sub E} in determining the level of He ash accumulation in future reactor systems. Results of the first tokamak He removal experiments have been analysed, and a first estimate of the ratio {upsilon}{sub He}/{upsilon}{sub E} to be expected for future reactor systems has been made. The experiments were carried out for neutral beam heated plasmas in the TEXTOR tokamak, at KFA/Julich. Helium was injected both as a short puff and continuously, and subsequently extracted with the Advanced Limiter Test-II pump limiter. The rate at which the He density decays has been determined with absolutely calibrated charge exchange spectroscopy, and compared with theoretical models, using the Multiple Impurity Species Transport (MIST) code. An analysis of energy confinement has been made with PPPL TRANSP code, to distinguish beam from thermal confinement, especially for low density cases. The ALT-II pump limiter system is found to exhaust the He with maximum exhaust efficiency (8 pumps) of {approximately}8%. We find 1<{upsilon}{sub He}/{upsilon}{sub E}<3.3 for the database of cases analysed to date. Analysis with the ITER TETRA systems code shows that these values would be adequate to achieve the required He concentration with the present ITER divertor He extraction system.

  5. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

    Directory of Open Access Journals (Sweden)

    Salih Maysoon

    2009-06-01

    Full Text Available Abstract Background Tail anchored (TA membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP possesses two alternatively spliced tail anchors (TA1 or TA2 but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER, whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  6. Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

    Science.gov (United States)

    Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

    2010-11-01

    The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

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

    OpenAIRE

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

    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-Isothiocyanob...

  8. PlantLoc: an accurate web server for predicting plant protein subcellular localization by substantiality motif

    OpenAIRE

    Tang, Shengnan; Li, Tonghua; Cong, Peisheng; Xiong, Wenwei; Wang, Zhiheng; Sun, Jiangming

    2013-01-01

    Knowledge of subcellular localizations (SCLs) of plant proteins relates to their functions and aids in understanding the regulation of biological processes at the cellular level. We present PlantLoc, a highly accurate and fast webserver for predicting the multi-label SCLs of plant proteins. The PlantLoc server has two innovative characters: building localization motif libraries by a recursive method without alignment and Gene Ontology information; and establishing simple architecture for rapi...

  9. Subcellular localization of the Vif protein of human immunodeficiency virus type 1.

    OpenAIRE

    Goncalves, J.; Jallepalli, P; Gabuzda, D H

    1994-01-01

    The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of lo...

  10. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

    OpenAIRE

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physio...

  11. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

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

    OpenAIRE

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

    2016-01-01

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

  13. Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization

    OpenAIRE

    2008-01-01

    For many microRNAs, in many normal tissues and in cancer cells, the cellular levels of mature microRNAs are not simply determined by transcription of microRNA genes. This mini-review will discuss how microRNA biogenesis and function can be regulated by various nuclear and cytoplasmic processing events, including emerging evidence that microRNA pathway components can be selectively regulated by control of their subcellular localization and by modifications that occur during dynamic cellular si...

  14. PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

    Directory of Open Access Journals (Sweden)

    Suzuki Harukazu

    2006-02-01

    Full Text Available Abstract Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse http://phosphoreg.imb.uq.edu.au that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered.

  15. 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.

  16. Environmental fate and effects of the lampricide Bayluscide: A review

    Science.gov (United States)

    Dawson, V.K.

    2003-01-01

    Bayluscide is an additive to TFM that increases the effectiveness of TFM as a lampricide. A review of the literature was undertaken to determine the environmental fate and effects of Bayluscide. Niclosamide (2???, 5-dichloro-4???-nitrosalicylanilide), the active ingredient of Bayluscide, degrades rapidly in natural water and sediment systems, however, the rate of degradation is very slow in autoclaved samples. This difference suggests that degradation under laboratory conditions is dependent on microbial activity and hydrolysis plays a minor role in degradation of niclosamide. The major degradation product of niclosamide has been reported to be aminoniclosamide (2???,5-dichloro-4???-aminosalicylanilide), which represented more than 50% of the residues extractable from sediments. Significantly more of the chemical is adsorbed to sediments with higher organic content and at lower pH's. The mobility of niclosamide in soil can be characterized as slight to medium; the estimated leaching distance would range from 0 to > 25 cm depending on the soil type and pH. The active ingredient of Bayluscide (niclosamide) is decomposed by ultra-violet light depending on the intensity and duration of the exposure. The uptake of residues by most invertebrates exposed to 14C-niclosamide is fairly rapid and equilibrium is reached within 24 h. About 90% of the accumulated residues were lost within 48 h after the organisms were transferred to clean flowing water. As with invertebrates, fish rapidly accumulate and eliminate residues of niclosamide. Three distinct residues were isolated from the extracts of edible fillet tissue; parent niclosamide, the glucuronide conjugate of niclosamide, and the sulfate ester of niclosamide. Aquatic plants and agricultural crops do not appear to be adversely affected at concentrations of Bayluscide used for lamprey or snail control. Mayflies (Hexagenia sp.). tend to be susceptible to TFM, but are relatively resistant to the effects of exposure to

  17. Origin and evolution of metabolic sub-cellular compartmentalization in eukaryotes.

    Science.gov (United States)

    Gabaldón, Toni; Pittis, Alexandros A

    2015-12-01

    A high level of subcellular compartmentalization is a hallmark of eukaryotic cells. This intricate internal organization was present already in the common ancestor of all extant eukaryotes, and the determination of the origins and early evolution of the different organelles remains largely elusive. Organellar proteomes are determined through regulated pathways that target proteins produced in the cytosol to their final subcellular destinations. This internal sorting of proteins can vary across different physiological conditions, cell types and lineages. Evolutionary retargeting - the alteration of a subcellular localization of a protein in the course of evolution - has been rampant in eukaryotes and involves any possible combination of organelles. This fact adds another layer of difficulty to the reconstruction of the origins and evolution of organelles. In this review we discuss current themes in relation to the origin and evolution of organellar proteomes. Throughout the text, a special focus is set on the evolution of mitochondrial and peroxisomal proteomes, which are two organelles for which extensive proteomic and evolutionary studies have been performed. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  18. 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.

  19. 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.

  20. Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

    Directory of Open Access Journals (Sweden)

    David M Doughty

    Full Text Available The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

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

    Directory of Open Access Journals (Sweden)

    Roslyn N. Brown

    2012-01-01

    Full Text Available Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% 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 and 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 locations for Salmonella and a framework for further investigations using computational modeling.

  2. Automated learning of generative models for subcellular location: building blocks for systems biology.

    Science.gov (United States)

    Zhao, Ting; Murphy, Robert F

    2007-12-01

    The goal of location proteomics is the systematic and comprehensive study of protein subcellular location. We have previously developed automated, quantitative methods to identify protein subcellular location families, but there have been no effective means of communicating their patterns to integrate them with other information for building cell models. We built generative models of subcellular location that are learned from a collection of images so that they not only represent the pattern, but also capture its variation from cell to cell. Our models contain three components: a nuclear model, a cell shape model and a protein-containing object model. We built models for six patterns that consist primarily of discrete structures. To validate the generated images, we showed that they are recognized with reasonable accuracy by a classifier trained on real images. We also showed that the model parameters themselves can be used as features to discriminate the classes. The models allow the synthesis of images with the expectation that they are drawn from the same underlying statistical distribution as the images used to train them. They can potentially be combined for many proteins to yield a high resolution location map in support of systems biology.

  3. Asymmetric subcellular distribution of glucose transporters in the endothelium of small contractile arteries.

    Science.gov (United States)

    Gaudreault, N; Scriven, D R L; Moore, E D W

    2006-01-01

    The authors have recently reported the presence and asymmetric distribution of the glucose transporters GLUT-1 to -5 and SGLT-1 in the endothelium of rat coronary artery (Gaudreault et al. 2004, Diabetologica, 47, 2081-2092). In the present study the authors investigate and compare the presence and subcellular distribution of the classic glucose transporter isoforms in endothelial cells of cerebral, renal, and mesenteric arteries. The GLUTs and SGLT-1 were examined with immunohistochemistry and wide-field fluorescence microscopy coupled to deconvolution in en face preparation of intact artery. We identified GLUT-1 to -5 and SGLT-1 in the endothelial cells of all three vascular beds. The relative level of expression for each isoform was found comparable amongst arteries. Clusters of the glucose transporter isoforms were found at a high density in proximity to the cell-to-cell junctions. In addition, a consistent asymmetric distribution of GLUT-1 to -5 was found, predominantly located on the abluminal side of the endothelium in all three vascular beds examined (ranging from 68% to 91%, p<.05). The authors conclude that the expression and subcellular distribution of glucose transporters are similar in endothelial cells from vascular beds of comparable diameter and suggest that their subcellular organization may facilitate transendothelial transport of glucose in small contractile arteries.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  5. Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages.

    Science.gov (United States)

    Di Raimo, Tania; Leopizzi, Martina; Mangino, Giorgio; Rocca, Carlo Della; Businaro, Rita; Longo, Lucia; Lo Vasco, Vincenza Rita

    2016-12-01

    Macrophages' phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs' expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes' mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes' expression and PLC proteins' presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

  6. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    Science.gov (United States)

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation.

    Science.gov (United States)

    Kruse, Janis; Meier, Doreen; Zenk, Fides; Rehders, Maren; Nellen, Wolfgang; Hammann, Christian

    2016-10-02

    The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

  8. Phytotoxicity, accumulation and transport of silver nanoparticles by Arabidopsis thaliana.

    Science.gov (United States)

    Geisler-Lee, Jane; Wang, Qiang; Yao, Ying; Zhang, Wen; Geisler, Matt; Li, Kungang; Huang, Ying; Chen, Yongsheng; Kolmakov, Andrei; Ma, Xingmao

    2013-05-01

    The widespread availability of nano-enabled products in the global market may lead to the release of a substantial amount of engineered nanoparticles in the environment, which frequently display drastically different physiochemical properties than their bulk counterparts. The purpose of the study was to evaluate the impact of citrate-stabilised silver nanoparticles (AgNPs) on the plant Arabidopsis thaliana at three levels, physiological phytotoxicity, cellular accumulation and subcellular transport of AgNPs. The monodisperse AgNPs of three different sizes (20, 40 and 80 nm) aggregated into much larger sizes after mixing with quarter-strength Hoagland solution and became polydisperse. Immersion in AgNP suspension inhibited seedling root elongation and demonstrated a linear dose-response relationship within the tested concentration range. The phytotoxic effect of AgNPs could not be fully explained by the released silver ions. Plants exposed to AgNP suspensions bioaccumulated higher silver content than plants exposed to AgNO3 solutions (Ag(+) representative), indicating AgNP uptake by plants. AgNP toxicity was size and concentration dependent. AgNPs accumulated progressively in this sequence: border cells, root cap, columella and columella initials. AgNPs were apoplastically transported in the cell wall and found aggregated at plasmodesmata. In all the three levels studied, AgNP impacts differed from equivalent dosages of AgNO3.

  9. SCLpredT: Ab initio and homology-based prediction of subcellular localization by N-to-1 neural networks

    OpenAIRE

    Adelfio, Alessandro; Volpato, Viola; Pollastri, Gianluca

    2013-01-01

    Abstract The prediction of protein subcellular localization is a important step towards the prediction of protein function, and considerable effort has gone over the last decade into the development of computational predictors of protein localization. In this article we design a new predictor of protein subcellular localization, based on a Machine Learning model (N-to-1 Neural Networks) which we have recently developed. This system, in three versions specialised, respectively, on Plants, Fung...

  10. Cell fate patterning during C. elegans vulval development

    OpenAIRE

    Hill, Russell J.; Sternberg, Paul W.

    1993-01-01

    Precursor cells of the vulva of the C. elegans hermaphrodite choose between two vulval cell fates (1° and 2°) and a non-vulval epidermal fate (3°) in response to three intercellular signals. An inductive signal produced by the anchor cell induces the vulval precursors to assume the 1° and 2° vulval fates. This inductive signal is an EGF-like growth factor encoded by the gene lin-3. An inhibitory signal mediated by lin-15, and which may originate from the surrounding epidermis, prevents the vu...

  11. EMT and MET as paradigms for cell fate switching

    Institute of Scientific and Technical Information of China (English)

    Jiekai Chen; Qingkai Han; Duanqing Pei

    2012-01-01

    Cell fate determination is a major unsolved problem in cell and developmental biology,The discovery of reprogramming by pluripotent factors offers a rational system to investigate the molecular mechanisms associated with cell fate decisions.The idea that reprogramming of fibroblasts starts with a mesenchymal-epithelial transition (MET) suggests that the process is perhaps a reversal of epithelial to mesenchymal transition (EMT) found frequently during early embryogenesis,As such,we believe that investigations into MET-EMT may yield detailed molecular insights into cell fate decisions,not only for the switching between epithelial and mesenchymal cells,but also other cell types.

  12. Integrated optical fiber lattice accumulators

    OpenAIRE

    Atherton, Adam F

    1997-01-01

    Approved for public release; distribution is unlimited. Sigma-delta modulators track a signal by accumulating the error between an input signal and a feedback signal. The accumulated energy is amplitude analyzed by a comparator. The comparator output signal is fed back and subtracted from the input signal. This thesis is primarily concerned with designing accumulators for inclusion in an optical sigma-delta modulator. Fiber lattice structures with optical amplifiers are used to perform the...

  13. Subcellular tension fields and mechanical resistance of the lamella front related to the direction of locomotion.

    Science.gov (United States)

    Bereiter-Hahn, J; Lüers, H

    1998-01-01

    Keratocytes derived from the epidermis of aquatic vertebrates are now widely used for investigation of the mechanism of cell locomotion. One of the main topics under discussion is the question of driving force development and concomitantly subcellular force distribution. Do cells move by actin polymerization-driven extension of the lamella, or is the lamella edge extended at regions of weakness by a flow of cytoplasm generated by hydrostatic pressure? Thus, elasticity changes were followed and the stiffness of the leading front of the lamella was manipulated by local application of phalloidin and cytochalasin D (CD). In scanning acoustic microscopy (SAM), elasticity is revealed from the propagation velocity of longitudinal sound waves (1 GHz). The lateral resolution of SAM is in the micrometer range. Using this method, subcellular tension fields with different stiffnesses (elasticity) can be determined. A typical pattern of subcellular stiffness distribution is related to the direction of migration. Cells forced to change their direction of movement by exposure to DC electric fields of varying polarity alter their pattern of subcellular stiffness in relationship to the new direction. The cells spread into the direction of low stiffness and retract at zones of high stiffness. The pattern of subcellular stiffness distribution reveals force distribution in migrating cells; i.e., if a cell moves exactly in a direction perpendicular to its long axis, then the contractile forces are largest along the long axis and decrease toward the short axis. Locomotion in any angle oblique to this axis requires an asymmetric stiffness distribution. Inhibition of actomyosin contractions by La3+ (2 mM), which inhibits Ca2+ influx, reduces cytoplasmic stiffness accompanied by an immediate cessation of locomotion and a change of cell shape. Local release of CD in front of a progressing lamella activates a cell to follow the CD gradient: The lamella thickens locally and is extended toward

  14. Fate of microbial nitrogen, carbon, hydrolysable amino acids, monosaccharides, and fatty acids in sediment

    Science.gov (United States)

    Veuger, Bart; van Oevelen, Dick; Middelburg, Jack J.

    2012-04-01

    The fate of microbial carbon, nitrogen, hydrolysable amino acids (HAAs), monosaccharides, and fatty acids in sediment was investigated experimentally. The microbial community of a tidal flat sediment was labeled with 13C-enriched glucose and 15N-enriched ammonium, and sediment was incubated for up to 371 days. Analysis of total concentrations and 13C- and 15N content of bulk sediment, hydrolysable amino acids (including D-alanine), monosaccharides, total fatty acids (TFAs), and phospholipid-derived fatty acids (PLFAs) allowed us to trace the fate of microbial biomass and -detritus and the major biochemical groups therein (proteins, carbohydrates, and lipids) over intermediate time scales (weeks-months). Moreover, the unidentified fraction of the labeled material (i.e. not analyzed as HAA, FA, or carbohydrate) provided information on the formation and fate of molecularly uncharacterizable organic matter. Loss of 13C and 15N from the sediment was slow (half live of 433 days) which may have been due to the permanently anoxic conditions in the experiment. Loss rates for the different biochemical groups were also low with the following order of loss rate constants: PLFA > TFA > HAA > monosaccharides. The unidentified 13C-pool was rapidly formed (within days) and then decreased relatively slowly, resulting in a gradual relative accumulation of this pool over time. Degradation and microbial reworking of the labeled material resulted in subtle, yet consistent, diagenetic changes within the different biochemical groups. In the HAA pool, glycine, lysine, and proline were lost relatively slowly (i.e. best preserved) while there was no accumulation of D-alanine relative to L-alanine, indicating no relative accumulation of bacterial macromolecules rich in D-alanine. In the fatty acid pool, there was very little difference between PLFAs and TFAs, indicating a very similar lability of these pools. Differences between individual fatty acids included a relatively slow loss of i15

  15. Three-dimensional ultrastructural study of oil and astaxanthin accumulation during encystment in the green alga Haematococcus pluvialis.

    Directory of Open Access Journals (Sweden)

    Marina Wayama

    Full Text Available Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%. In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%, in which the total chloroplast volume was markedly decreased (9.7%. Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.

  16. ASSESSMENT OF THE SUBSURFACE FATE OF MONOETHANOLAMINE

    Energy Technology Data Exchange (ETDEWEB)

    James A. Sorensen; John R. Gallagher; Lori G. Kays

    2000-05-01

    Burial of amine reclaimer unit sludges and system filters has resulted in contamination of soil at the CanOxy Okotoks decommissioned sour gas-processing plant with amines, amine byproducts, and salts. A three-phase research program was devised to investigate the natural attenuation process that controls the subsurface transport and fate of these contaminants and to apply the results toward the development of a strategy for the remediation of this type of contamination in soils. Phase I experimental activities examined interactions between monoethanolamine (MEA) and sediment, the biodegradability of MEA in soils at various concentrations and temperatures, and the biodegradability of MEA sludge contamination in a soil slurry bioreactor. The transport and fate of MEA in the subsurface was found to be highly dependant on the nature of the release, particularly MEA concentration and conditions of the subsurface environment, i.e., pH, temperature, and oxygen availability. Pure compound biodegradation experiments in soil demonstrated rapid biodegradation of MEA under aerobic conditions and moderate temperatures (>6 C). Phase II landfarming activities confirmed that these contaminants are readily biodegradable in soil under ideal laboratory conditions, yet considerable toxicity was observed in the remaining material. Examination of water extracts from the treated soil suggested that the toxicity is water-soluble. Phase II activities led to the conclusion that landfarming is not the most desirable bioremediation technique; however, an engineered biopile with a leachate collection system could remove the remaining toxic fraction from the soil. Phase III was initiated to conduct field-based experimental activities to examine the optimized remediation technology. A pilot-scale engineered biopile was constructed at a decommissioned gas-sweetening facility in Okotoks, Alberta, Canada. On the basis of a review of the analytical and performance data generated from soil and

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

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

  18. Fate of the fecal indicator Escherichia coli in irrigation with partially treated wastewater.

    Science.gov (United States)

    Vergine, P; Saliba, R; Salerno, C; Laera, G; Berardi, G; Pollice, A

    2015-11-15

    Treated wastewater reuse is increasing in semi-arid regions as a response to the effects of climate change and increased competition for natural water resources. Investigating the fate of bacterial indicators is relevant to assess their persistence in the environment and possible transfer to groundwater or to the food chain. A long-term field-scale experimental campaign and a soil column test were carried out to evaluate the fate of the fecal indicator Escherichia coli (E. coli) in a cultivated soil when contaminated water resources are used for irrigation. For field experiments, fecal contamination was simulated by dosing the indicator to the effluent of a membrane bioreactor, thus simulating a filtration system's failure, and irrigating a test field where grass was grown. The presence of E. coli on grass and topsoil samples was monitored under different scenarios. For evaluating the fate of the same indicator in the subsoil, a set of soil columns was installed next to the field, operated, and monitored for E. coli concentration over time and along depth. Real municipal wastewater was used in this case as source of fecal contamination. Results showed that short- and medium-term effects on topsoil were strongly dependent on the concentration of E. coli in the irrigation water. Limited persistence and no relevant accumulation of the indicator on the grass and in the topsoil were observed. Watering events performed after fecal contamination did not influence significantly the decay in the topsoil, which followed a log-linear model. The trend of the E. coli concentrations in the leaching of the soil columns followed a log-linear model as well, suggesting bacterial decay as the dominant mechanism affecting the underground indicator's concentration.

  19. Microbial degradation on glacier surface is the missing piece of environmental fate of pesticides in cold areas

    Science.gov (United States)

    Ambrosini, Roberto; Ferrario, Claudia; Pittino, Francesca; Tagliaferri, Ilario; Gandolfi, Isabella; Bestetti, Giuseppina; Azzoni, Roberto S.; Diolaiuti, Guglielmina A.; Smiraglia, Claudio; Franzetti, Andrea; Villa, Sara

    2017-04-01

    Organic contaminants deposited on glacier surfaces undergo different partition and degradation processes which determine their environmental fate and accumulation into the trophic chains. Among these processes, biodegradation by supraglacial bacteria has been neglected so far. To assess the relevance of biodegradative processes, in situ microcosm experiments were conducted simulating cryoconite hole systems on an Alpine glacier exposed to the organophosphorus insecticide chlorpyrifos (CPF) as model of xenobiotic molecule which accumulate on glaciers after medium range transports. Results showed that biodegradation is the most efficient process contributing to the removal of CPF on the glacier surface. The high concentrations of CPF in cryoconite and its half-life in the range of 35 - 69 days indicated that biodegradation process can significantly contrast the release of CPF transported on glaciers. Moreover, the metabolic versatility of cryoconite bacteria suggest that these habitats might contribute to the degradation of a wide class of pollutants with different physical-chemical properties. Metagenomics data indicated that photoheterotrophic bacteria might be involved in the biodegradation of CPF by using light to supplement their metabolic demands, thus contributing to the biological removal of CPF without the constrain of using this pesticide as sole energy source. In conclusion. cryoconite might act as a "biofilter" for organic pollutants on glaciers by accumulating them and promoting their biodegradation. Owing to its relevance, the contribution of cryoconite to the removal of organic pollutants should be included in the models predicting the environmental fate of these compounds in cold areas.

  20. Modelling the fate of organic micropollutants in stormwater ponds

    DEFF Research Database (Denmark)

    Vezzaro, Luca; Eriksson, Eva; Ledin, Anna;

    2011-01-01

    substance inherent properties to calculate MP fate but differ in their ability to represent the small physical scale and high temporal variability of stormwater treatment systems. Therefore the three models generate different results. A Global Sensitivity Analysis (GSA) highlighted that settling....../resuspension of particulate matter was themost sensitive process for the dynamic model. The uncertainty of the estimated MP fluxes can be reduced by calibrating the dynamic model against total suspended solids data. This reduction in uncertainty was more significant for the substances with strong tendency to sorb, i...... models. The fate of four different MP in a stormwater retention pond was simulated by applying two steady-state multimedia fate models (EPI Suite and SimpleBox) commonly applied in chemical risk assessment and a dynamic multimedia fate model (Stormwater Treatment Unit Model for Micro Pollutants — STUMP...

  1. Investigating the Toxicity and Environmental Fate of Graphene Nanomaterials

    Science.gov (United States)

    The Hersam Laboratory at Northwestern University works with the Center for Environmental Implications of Nanotechnology and the United States Environmental Protection Agency to study the toxicity and environmental fate of emergent nanomaterials, specifically carbon-based nanomate...

  2. Hydrologic modeling of pathogen fate and transport.

    Science.gov (United States)

    Dorner, Sarah M; Anderson, William B; Slawson, Robin M; Kouwen, Nicholas; Huck, Peter M

    2006-08-01

    A watershed-scale fate and transport model has been developed for Escherichia coli and several waterborne pathogens: Cryptosporidiumspp., Giardiaspp., Campylobacter spp, and E. coli O157:H7. The objectives were to determine the primary sources of pathogenic contamination in a watershed used for drinking water supply and to gain a greater understanding of the factors that most influence their survival and transport. To predict the levels of indicator bacteria and pathogens in surface water, an existing hydrologic model, WATFLOOD, was augmented for pathogen transport and tested on a watershed in Southwestern Ontario, Canada. The pathogen model considered transport as a result of overland flow, subsurface flow to tile drainage systems, and in-stream routing. The model predicted that most microorganisms entering the stream from land-based sources enter the stream from tile drainage systems rather than overland transport. Although the model predicted overland transport to be rare, when it occurred, it corresponded to the highest observed and modeled microbial concentrations. Furthermore, rapid increases in measured E. coli concentrations during storm events suggested that the resuspension of microorganisms from stream sediments may be of equal or greater importance than land-based sources of pathogens.

  3. The Fate of Unstable Gauge Flux Compactifications

    CERN Document Server

    Burgess, C P; Zavala, I

    2009-01-01

    Fluxes are widely used to stabilise extra dimensions, but if they arise within a non-abelian gauge sector they are often unstable. We seek the fate of this instability, focussing on the simplest examples: sphere-monopole compactifications in six dimensions. Without gravity most non-abelian monopoles are unstable, decaying into the unique stable monopole in the same topological class. We show that the same is true in Einstein-YM systems, with the geometry adjusting accordingly: a Mink(d)xS2 geometry supported by an unstable monopole relaxes to an AdS(d)xS2. For 6D supergravity, the dilaton obstructs this simple evolution, acquiring a gradient and thus breaking some of the spacetime symmetries. We argue that it is the 4D symmetries that break, and examine several endpoint candidates. Oxidising the supergravity system into a higher-dimensional Einstein-YM monopole, we use the latter to guide us to the corresponding endpoint. The result is a singular Kasner-like geometry conformal to Mink(4)xS2. The solution has ...

  4. The fate of Earth’s ocean

    Directory of Open Access Journals (Sweden)

    C. Bounama

    2001-01-01

    Full Text Available Questions of how water arrived on the Earth’s surface, how much water is contained in the Earth system as a whole, and how much water will be available in the future in the surface reservoirs are of central importance to our understanding of the Earth. To answer the question about the fate of the Earth’s ocean, one has to study the global water cycle under conditions of internal and external forcing processes. Modern estimates suggest that the transport of water to the surface is five times smaller than water movement to the mantle, so that the Earth will lose all its sea-water in one billion years from now. This straightforward extrapolation of subduction-zone fluxes into the future seems doubtful. Using a geophysical modelling approach it was found that only 27% of the modern ocean will be subducted in one billion years. Internal feedbacks will not be the cause of the ocean drying out. Instead, the drying up of surface reservoirs in the future will be due to the increase in temperature caused by a maturing Sun connected to hydrogen escape to outer space. Keywords: Surface water reservoir, water fluxes, regassing, degassing, global water cycle

  5. Evolution and fate of very massive stars

    CERN Document Server

    Yusof, Norhasliza; Meynet, Georges; Crowther, Paul A; Ekstrom, Sylvia; Frischknecht, Urs; Georgy, Cyril; Kassim, Hasan Abu; Schnurr, Olivier; 10.1093/mnras/stt794

    2013-01-01

    There is observational evidence that supports the existence of Very Massive Stars in the local universe. First, very massive stars (Mini<=320 M) have been observed in the Large Magellanic Cloud . Second, there are observed SNe that bear the characteristics of Pair Creation Supernovae which have very massive stars as progenitors. The most promising candidate to date is SN2007bi. In order to investigate the evolution and fate of nearby very massive stars, we calculated a new grid of models for such objects, for solar, LMC and SMC metallicities, which covers the initial mass range from 120 to 500M. Both rotating and non-rotating models were calculated using the Geneva stellar evolution code and evolved until at least the end of helium burning and for most models until oxygen burning. Since very massive stars have very large convective cores during the Main-Sequence phase, their evolution is not so much affected by rotational mixing, but more by mass loss through stellar winds. Their evolution is never far fro...

  6. An illusion of control modulates the reluctance to tempt fate

    Directory of Open Access Journals (Sweden)

    Chloe L. Swirsky

    2011-10-01

    Full Text Available The tempting fate effect is that the probability of a fateful outcome is deemed higher following an action that ``tempts'' the outcome than in the absence of such an action. In this paper we evaluate the hypothesis that the effect is due to an illusion of control induced by a causal framing of the situation. Causal frames require that the action make a difference to an outcome and that the action precedes the outcome. If an illusion of control modulates the reluctance to tempt fate, then actions that make a difference to well-being and that occur prior to the outcome should tempt fate most strongly. In Experiments 1--3 we varied whether the action makes a difference and the temporal order of action and outcome. In Experiment 4 we tested whether an action can tempt fate if all outcomes are negative. The results of all four experiments supported our hypothesis that the tempting fate effect depends on a causal construal that gives rise to a false sense of control.

  7. Genetic fate mapping using site-specific recombinases.

    Science.gov (United States)

    Legué, Emilie; Joyner, Alexandra L

    2010-01-01

    Understanding how cells are assembled in three dimensions to generate an organ, or a whole organism, is a pivotal question in developmental biology. Similarly, it is critical to understand how adult stem cells integrate into an existing organ during regeneration or in response to injury. Key to discovering the answers to these questions is being able to study the various behaviors of distinct cell types during development or regeneration. Fate mapping techniques are fundamental to studying cell behaviors such as proliferation, movement, and lineage segregation, as the techniques allow precursor cells to be marked and their descendants followed and characterized over time. The generation of transgenic mice, combined with the use of site-specific recombinases (SSR) in the mouse genome, has provided a means to develop powerful genetic fate mapping approaches. A key advantage of genetic fate mapping is that it allows cells to be genetically marked, and therefore the mark is transmitted to all the descendants of the initially marked cells. By making modifications to the SSRs that render their enzymatic activity inducible, and the development of an assortment of reporter alleles for marking cells, increasingly sophisticated genetic fate mapping studies can be performed. In this chapter, we review the four main genetic fate mapping methods that utilize intrachromosomal recombination to mark cells (cumulative, inducible, clonal, and intersectional) and one interchromosomal method, the tools required to carry out each approach, and the practical considerations that have to be taken into account before embarking on each type of genetic fate mapping study.

  8. Essentials of recombinase-based genetic fate mapping in mice.

    Science.gov (United States)

    Jensen, Patricia; Dymecki, Susan M

    2014-01-01

    Fate maps, by defining the relationship between embryonic tissue organization and postnatal tissue structure, are one of the most important tools on hand to developmental biologists. In the past, generating such maps in mice was hindered by their in utero development limiting the physical access required for traditional methods involving tracer injection or cell transplantation. No longer is physical access a requirement. Innovations over the past decade have led to genetic techniques that offer means to "deliver" cell lineage tracers noninvasively. Such "genetic fate mapping" approaches employ transgenic strategies to express genetically encoded site-specific recombinases in a cell type-specific manner to switch on expression of a cell-heritable reporter transgene as lineage tracer. The behaviors and fate of marked cells and their progeny can then be explored and their contributions to different tissues examined. Here, we review the basic concepts of genetic fate mapping and consider the strengths and limitations for their application. We also explore two refinements of this approach that lend improved spatial and temporal resolution: (1) Intersectional and subtractive genetic fate mapping and (2) Genetic inducible fate mapping.

  9. Fate and effects of anthropogenic chemicals in mangrove ecosystems: A review

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Michael, E-mail: lewis.michael@epa.gov [U.S. Environmental Protection Agency, Office of Research and Development, 1 Sabine Island Drive, Gulf Breeze, FL 32561 (United States); Pryor, Rachel; Wilking, Lynn [U.S. Environmental Protection Agency, Office of Research and Development, 1 Sabine Island Drive, Gulf Breeze, FL 32561 (United States)

    2011-10-15

    The scientific literature for fate and effects of non-nutrient contaminant concentrations is skewed for reports describing sediment contamination and bioaccumulation for trace metals. Concentrations for at least 22 trace metals have been reported in mangrove sediments. Some concentrations exceed sediment quality guidelines suggesting adverse effects. Bioaccumulation results are available for at least 11 trace metals, 12 mangrove tissues, 33 mangrove species and 53 species of mangrove-habitat biota. Results are specific to species, tissues, life stage, and season and accumulated concentrations and bioconcentration factors are usually low. Toxicity tests have been conducted with 12 mangrove species and 8 species of mangrove-related fauna. As many as 39 effect parameters, most sublethal, have been monitored during the usual 3 to 6 month test durations. Generalizations and extrapolations for toxicity between species and chemicals are restricted by data scarcity and lack of experimental consistency. This hinders chemical risk assessments and validation of effects-based criteria. - Chemical risk assessments and resource management are restricted by the limited chemical fate and effects database for mangroves.

  10. Polycomb controls gliogenesis by regulating the transient expression of the Gcm/Glide fate determinant.

    Directory of Open Access Journals (Sweden)

    Anna Popkova

    Full Text Available The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.

  11. Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Paula Rego Bittencourt-Cunha

    2013-06-01

    Full Text Available In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL are transferred to the haemolymphatic lipophorin (Lp and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs. 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80% and NL (20%. The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

  12. The long-term fate of permafrost peatlands under rapid climate warming

    DEFF Research Database (Denmark)

    Swindles, Graeme T.; Morris, Paul J.; Mullan, Donal

    2015-01-01

    Permafrost peatlands contain globally important amounts of soil organic carbon, owing to cold conditions which suppress anaerobic decomposition. However, climate warming and permafrost thaw threaten the stability of this carbon store. The ultimate fate of permafrost peatlands and their carbon...... stores is unclear because of complex feedbacks between peat accumulation, hydrology and vegetation. Field monitoring campaigns only span the last few decades and therefore provide an incomplete picture of permafrost peatland response to recent rapid warming. Here we use a high-resolution palaeoecological...... approach to understand the longer-term response of peatlands in contrasting states of permafrost degradation to recent rapid warming. At all sites we identify a drying trend until the late-twentieth century; however, two sites subsequently experienced a rapid shift to wetter conditions as permafrost thawed...

  13. The long-term fate of permafrost peatlands under rapid climate warming

    DEFF Research Database (Denmark)

    Swindles, Graeme T.; Morris, Paul J.; Mullan, Donal;

    2015-01-01

    Permafrost peatlands contain globally important amounts of soil organic carbon, owing to cold conditions which suppress anaerobic decomposition. However, climate warming and permafrost thaw threaten the stability of this carbon store. The ultimate fate of permafrost peatlands and their carbon...... stores is unclear because of complex feedbacks between peat accumulation, hydrology and vegetation. Field monitoring campaigns only span the last few decades and therefore provide an incomplete picture of permafrost peatland response to recent rapid warming. Here we use a high-resolution palaeoecological...... approach to understand the longer-term response of peatlands in contrasting states of permafrost degradation to recent rapid warming. At all sites we identify a drying trend until the late-twentieth century; however, two sites subsequently experienced a rapid shift to wetter conditions as permafrost thawed...

  14. 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.

  15. 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.

  16. A multiresolution approach to automated classification of protein subcellular location images

    Directory of Open Access Journals (Sweden)

    Srinivasa Gowri

    2007-06-01

    Full Text Available Abstract Background Fluorescence microscopy is widely used to determine the subcellular location of proteins. Efforts to determine location on a proteome-wide basis create a need for automated methods to analyze the resulting images. Over the past ten years, the feasibility of using machine learning methods to recognize all major subcellular location patterns has been convincingly demonstrated, using diverse feature sets and classifiers. On a well-studied data set of 2D HeLa single-cell images, the best performance to date, 91.5%, was obtained by including a set of multiresolution features. This demonstrates the value of multiresolution approaches to this important problem. Results We report here a novel approach for the classification of subcellular location patterns by classifying in multiresolution subspaces. Our system is able to work with any feature set and any classifier. It consists of multiresolution (MR decomposition, followed by feature computation and classification in each MR subspace, yielding local decisions that are then combined into a global decision. With 26 texture features alone and a neural network classifier, we obtained an increase in accuracy on the 2D HeLa data set to 95.3%. Conclusion We demonstrate that the space-frequency localized information in the multiresolution subspaces adds significantly to the discriminative power of the system. Moreover, we show that a vastly reduced set of features is sufficient, consisting of our novel modified Haralick texture features. Our proposed system is general, allowing for any combinations of sets of features and any combination of classifiers.

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

    Directory of Open Access Journals (Sweden)

    Linea Frimodt Obel

    2012-03-01

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

  18. Effect heat stress on subcellular localization of Ca2+ in tomato fruits

    Directory of Open Access Journals (Sweden)

    Grażyna Garbaczewska

    2014-01-01

    Full Text Available The aim of this paper was to compare the fruit cell ultrastructure and subcellular localization of Ca2+ after heat stress with the use of the potassium antimonate method (Slocum and Roux 1982, Tretyn et al. 1992. The tomato plants Robin cv., relatively tolerant to heat stress, were grown under uncontrolled greenhouse conditions to the stage of fruiting. The plants were placed for 20h in two temperature regimes: 23oC (optimal temperature or 40oC (heat stress in darkness, under water vapour saturated atmosphere. Immediately after heat stress the fruits were harvested to estimate water soluble and insoluble calcium contents and subcellular localization of Ca2+. After heating the concentration of calcium in tomato fruits increased about twice. In both temperature treatments the water soluble fractions were lower than insoluble ones at smaller differences between insoluble and soluble fractions after heat stress. The shapes and localization of Ca2+ detected with the use of potassium antimonate method show that in fruits of control plants the precipitates were numerous, small and of oval shape. They were dispersed in cytosol or adjoined to endoplasmic reticulum or to external membrane of chloroplast. In the fruit of heated plants the precipitates were irregular in shape, amorphous and singly dispersed in the cytosol. We observed also some cytological changes in the structure of membranes and organelles of the plants of both experimental treatments. The heat induced increase of calcium content and the changes in subcellular localization of Ca2+ under heat stress suggest that calcium ions may be involved in avoiding heat injury. The problem requires more detailed further investigations.

  19. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    Science.gov (United States)

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (<0.05mM), while vacuoles at high Cd substrate (≥0.05mM). Moreover, the concentrations and proportions of Cd in inorganic and water-soluble form also reduced by AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice.

  20. Linking sub-cellular biomarkers to embryo aberrations in the benthic amphipod Monoporeia affinis.

    Science.gov (United States)

    Reutgard, Martin; Furuhagen, Sara

    2016-04-01

    To adequately assess and monitor environmental status in the aquatic environment a broad approach is needed that integrates physical variables, chemical analyses and biological effects at different levels of the biological organization. Embryo aberrations in the Baltic Sea key species Monoporeia affinis can be induced by both metals and organic substances as well as by hypoxia, increasing temperatures and malnutrition. This amphipod has therefore been used for more than three decades as a biological effect indicator in monitoring and assessment of chemical pollution and environmental stress. However, little is known about the sub-cellular mechanisms underlying embryo aberrations. An improved mechanistic understanding may open up the possibility of including sub-cellular alterations as sensitive warning signals of stress-induced embryo aberrations. In the present study, M. affinis was exposed in microcosms to 4 different sediments from the Baltic Sea. After 88-95 days of exposure, survival and fecundity were determined as well as the frequency and type of embryo aberrations. Moreover, oxygen radical absorption capacity (ORAC) was assayed as a proxy for antioxidant defense, thiobarbituric acid reactive substances (TBARS) level as a measure of lipid peroxidation and acetylcholinesterase (AChE) activity as an indicator of neurotoxicity. The results show that AChE and ORAC can be linked to the frequency of malformed embryos and arrested embryo development. The occurrence of dead broods was significantly associated with elevated TBARS levels. It can be concluded that these sub-cellular biomarkers are indicative of effects that could affect Darwinian fitness and that oxidative stress is a likely mechanism in the development of aberrant embryos in M. affinis.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  2. Pistil starch reserves at anthesis correlate with final flower fate in avocado (Persea americana.

    Directory of Open Access Journals (Sweden)

    María Librada Alcaraz

    Full Text Available A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit.

  3. Pistil starch reserves at anthesis correlate with final flower fate in avocado (Persea americana).

    Science.gov (United States)

    Alcaraz, María Librada; Hormaza, José Ignacio; Rodrigo, Javier

    2013-01-01

    A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana) is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree) was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit.

  4. Pistil Starch Reserves at Anthesis Correlate with Final Flower Fate in Avocado (Persea americana)

    Science.gov (United States)

    Alcaraz, María Librada; Hormaza, José Ignacio; Rodrigo, Javier

    2013-01-01

    A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana) is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree) was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit. PMID:24167627

  5. The Fate of Merging Neutron Stars

    Science.gov (United States)

    Kohler, Susanna

    2017-08-01

    state. They then combined this information with Monte Carlo simulations based on the mass distribution of neutron-star binaries in our galaxy. From these simulations, Piro and collaborators could predict the distribution of fates expected for merging neutron-star binaries, given different equations of state.The authors found that the fate of the merger could vary greatly depending on the equation of state you assume. Intriguingly, all equations of state resulted in a surprisingly high fraction of systems that merged to form a neutron star or a supramassive neutron star in fact, four out of the five equations of state predicted that 80100% of systems would result in a neutron star or a supermassive neutron star.Lessons from ObservationsThe frequency bands covered by various current and planned gravitational wave observatories. Advanced LIGO has the right frequency coverage to be able to explore a neutron-star remnant if the signal is loud enough. [Christopher Moore, Robert Cole and Christopher Berry]These results have important implications for our future observations. The high predicted fraction of neutron stars resulting from these mergers tells us that its especially important for gravitational-wave observatories to probe 14 kHz emission. This frequency range will enable us to study the post-merger neutron-star or supramassive-neutron-star remnants.Even if we cant observe the remnants behavior after it forms, we can still compare the distribution of remnants that we observe in the future to the predictions made by Piro and collaborators. This will potentially allow us to constrain the neutron-star equation of state, revealing the physics of neutron-star interiors even without direct observations.CitationAnthony L. Piro et al 2017 ApJL 844 L19. doi:10.3847/2041-8213/aa7f2f

  6. Fate of manufactured nanoparticles in environmental systems

    Science.gov (United States)

    Gelabert, A.; Sivry, Y.; Siron, V.; Akrout, A.; Ferrari, R.; Juillot, F.; Menguy, N.; Benedetti, M. F.

    2009-12-01

    Because of their specific physico-chemical properties, engineered nanoparticles (NPs) have become largely widespread in numerous industrial fields such as biomedicine, cosmetics, and material sciences. However, their growing use could possibly result in the release of various NPs amounts in environmental settings. Thus, an accurate understanding of their behaviour in natural systems is required, and of first importance is an estimation of their persistence and/or physico-chemical modifications since they can greatly alter their fate and bioavailability in the biogeosphere. The present study focuses on dissolution rate estimations for commercial NPs ZnO and TiO2 in natural waters (i.e. filtered Seine river water and seawater). Both NPs were used uncoated and coated with an organic polymer. Native NPs size and shape were investigated using TEM, and appeared as 20-50 nm spheroids, with an associated specific surface area of 37.5 and 57.6 m2/g for ZnO and TiO2, respectivelly. NPs dissolution rates were determined using both ultrafiltration (UF) and Donnan Membrane Techniques (DMT, [1]). The latter method allows a direct in-situ measurement of the free metal ion concentration only (here Zn2+), while the UF membrane small nominal pore size (approx. 2 nm) results in the separation of small inorganic complexes in addition to free metal ions. After a fast dissolution step reaching 1% of total zinc within the first hour for uncoated ZnO NPs in Seine water, precipitation of new mineral phases occurred with the formation of smithonite and hydrozincite as observed by XRD and TEM and confirmed by thermodynamic calculations (Visual Minteq). Interestingly, the behaviour of the coated ZnO NPs is slightly different since the initial dissolution step takes place during the first 72 hours, to reach up to 10% of the total zinc in our system. However, despite this difference in dissolution kinetics, both systems evolve similarly after 3 days, and they reach a steady state after

  7. LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro.

    Science.gov (United States)

    Balzer, Erica; Heine, Christian; Jiang, Qiang; Lee, Vivian M; Moss, Eric G

    2010-03-01

    LIN28 is an RNA-binding protein that is expressed in many developing tissues. It can block let-7 (Mirlet7) microRNA processing and help promote pluripotency. We have observed LIN28 expression in the developing mouse neural tube, colocalizing with SOX2, suggesting a role in neural development. To better understand its normal developmental function, we investigated LIN28 activity during neurogliogenesis in vitro, where the succession of neuronal to glial cell fates occurs as it does in vivo. LIN28 expression was high in undifferentiated cells, and was downregulated rapidly upon differentiation. Constitutive LIN28 expression caused a complete block of gliogenesis and an increase in neurogenesis. LIN28 expression was compatible with neuronal differentiation and did not increase proliferation. LIN28 caused significant changes in gene expression prior to any effect on let-7, notably on Igf2. Furthermore, a mutant LIN28 that permitted let-7 accumulation was still able to completely block gliogenesis. Thus, at least two biological activities of LIN28 are genetically separable and might involve distinct mechanisms. LIN28 can differentially promote and inhibit specific fates and does not function exclusively by blocking let-7 family microRNAs. Importantly, the role of LIN28 in cell fate succession in vertebrate cells is analogous to its activity as a developmental timing regulator in C. elegans.

  8. Mapping the Microscale Origins of MRI Contrast with Subcellular NV Diamond Magnetometry

    CERN Document Server

    Davis, Hunter C; Bhatnagar, Aadyot; Lee-Gosselin, Audrey; Barry, John F; Glenn, David R; Walsworth, Ronald L; Shapiro, Mikhail G

    2016-01-01

    Magnetic resonance imaging (MRI) is a widely used biomedical imaging modality that derives much of its contrast from microscale magnetic field gradients in biological tissues. However, the connection between these sub-voxel field patterns and MRI contrast has not been studied experimentally. Here, we describe a new method to map subcellular magnetic fields in mammalian cells and tissues using nitrogen vacancy diamond magnetometry and connect these maps to voxel-scale MRI contrast, providing insights for in vivo imaging and contrast agent design.

  9. Subcellular localization of the five members of the human steroid 5α-reductase family

    Directory of Open Access Journals (Sweden)

    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

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

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate;

    2012-01-01

    ,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...

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

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash [Cornell SIMS Laboratory, Department of Earth and Atmospheric Sciences, Snee Hall, Cornell University, Ithaca, NY 14853 (United States)], E-mail: sc40@cornell.edu

    2008-12-15

    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{sub 2}{sup +}) revealed local secondary ion signal enhancements correlated with the water image signals of {sup 19}(H{sub 3}O){sup +}. 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{sup +} and Na{sup +} in physiologically relevant concentrations. The high K

  12. Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

    Science.gov (United States)

    Breitkreuz, K. E.; Shelp, B. J.

    1995-05-01

    The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

  13. Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple

    OpenAIRE

    2009-01-01

    NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis re...

  14. 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...

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

    CERN Document Server

    Ammari, Habib; Shi, Cong

    2016-01-01

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

  16. Geochemical fate of arsenic in swine litter

    Science.gov (United States)

    Quazi, S.; Makris, K.; Sarkar, D.; Datta, R.; Punamiya, P.

    2007-12-01

    Swine diet is often supplemented by organoarsenicals, such as roxarsone to treat diseases and to promote growth. Recent data reported roxarsone degradation under anaerobic conditions in poultry litter, but no such data exist for swine wastes typically stored in unprotected lagoons in concentrated animal feeding operations (CAFOs). However, serious environmental health risk may arise upon significant arsenic (As) release into solution. The problem may be exacerbated under certain environmental conditions where organoarsenicals, such as roxarsone transform into the more toxic inorganic As, posing serious health risk to the surrounding ecosystem. The objective of this study were to analyze swine wastes collected from 19 randomly selected CAFOs in the USA for As concentrations, and to determine the geochemical fate of As in the swine waste suspensions. Swine wastes were analyzed for total-recoverable, total soluble, and water-extractable As, which were measured by ICP-MS. Speciation of As was performed following a well-established hyphenated technique using HPLC- ICPMS. Swine waste suspensions differed in solids contents; thus, the particulate matters with varying As concentrations were spiked with roxarsone and incubated under dark/light and aerobic/anaerobic conditions. Findings show the prevalence of inorganic As [As(V)] in swine waste suspension solutions. Roxarsone underwent degradation to both organoarsenicals, such as p-ASA, as well as inorganic arsenate and to a number of unidentified metabolites. Roxarsone degradation kinetics was influenced by the solids content and the air conditions (anaerobic/aerobic) of the swine waste suspensions. Maximum degradation rates were observed under anaerobic conditions, in suspensions which were low in solids content. Roxarsone degradation was primarily microbially-mediated, but in certain cases abiotic degradation was also observed, which were significantly slower.

  17. Manganese As a Metal Accumulator

    Science.gov (United States)

    Manganese deposits in water distribution systems accumulate metals, radionuclides and oxyanions by a combination of surface complexation, adsorption and solid substitution, as well as a combination of oxidation followed by manganese reduction and sorption of the oxidized constitu...

  18. Manganese As a Metal Accumulator

    Science.gov (United States)

    Manganese deposits in water distribution systems accumulate metals, radionuclides and oxyanions by a combination of surface complexation, adsorption and solid substitution, as well as a combination of oxidation followed by manganese reduction and sorption of the oxidized constitu...

  19. Modeling IRA Accumulation and Withdrawals

    OpenAIRE

    Sabelhaus, John

    2000-01-01

    Empirical analysis of IRA accumulation and withdrawal patterns is limited because information about IRA balances and flows is not available for a sample of taxpayers. This paper combines survey data on IRA balances with individual tax return data on IRA flows to study IRA accumulation and withdrawal patterns across cohorts. The analysis shows that IRA rules such as penalties for early withdrawals and minimum distribution requirements have predictable effects on IRA flows. The estimated propen...

  20. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    Science.gov (United States)

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

  1. 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.

  2. Multiple sequence elements facilitate Chp Rho GTPase subcellular location, membrane association, and transforming activity.

    Science.gov (United States)

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

    2006-07-01

    Cdc42 homologous protein (Chp) is a member of the Rho family of small GTPases and shares significant sequence and functional similarity with Cdc42. However, unlike classical Rho GTPases, we recently found that Chp depends on palmitoylation, rather than prenylation, for association with cellular membranes. Because palmitoylation alone is typically not sufficient to promote membrane association, we evaluated the possibility that other carboxy-terminal residues facilitate Chp subcellular association with membranes. We found that Chp membrane association and transforming activity was dependent on the integrity of a stretch of basic amino acids in the carboxy terminus of Chp and that the basic amino acids were not simply part of a palmitoyl acyltransferase recognition motif. We also determined that the 11 carboxy-terminal residues alone were sufficient to promote Chp plasma and endomembrane association. Interestingly, stimulation with tumor necrosis factor-alpha activated only endomembrane-associated Chp. Finally, we found that Chp membrane association was not disrupted by Rho guanine nucleotide dissociation inhibitory proteins, which are negative regulators of Cdc42 membrane association and biological activity. In summary, the unique carboxy-terminal sequence elements that promote Chp subcellular location and function expand the complexity of mechanisms by which the cellular functions of Rho GTPases are regulated.

  3. Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

    Institute of Scientific and Technical Information of China (English)

    Petra Talapka; Nikolett Bódi; Izabella Battonyai; (E)va Fekete; Mária Bagyánszki

    2011-01-01

    AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS (nNOS),endothelial NOS (eNOS),and inducible NOS (iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed. CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype- specific differences in the subcellular distributions of NOS isoforms.

  4. 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.

  5. 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.

  6. Subcellular distribution and chemical forms of cadmium in a dark septate endophyte (DSE), Exophiala pisciphila.

    Science.gov (United States)

    Zhan, Fangdong; He, Yongmei; Li, Yuan; Li, Tao; Yang, Yun-Ya; Toor, Gurpal S; Zhao, Zhiwei

    2015-11-01

    Our objective was to understand the cadmium (Cd) tolerance mechanisms by investigating the subcellular distribution, chemical forms of Cd and adsorptive groups in the mycelia of Exophiala pisciphila. We grew E. pisciphila in the liquid media with increasing Cd concentrations (0, 25, 50, 100, 200, and 400 mg L(-1)). Increased Cd in the media caused a proportional increase in the Cd uptake by E. pisciphila. Subcellular distribution indicated that 81 to 97% of Cd was associated with the cell walls. The largest amount and proportion (45-86%) of Cd was extracted with 2% acetic acid, and a concentration-dependent extraction was observed, both of which suggest that Cd-phosphate complexes were the major chemical form in E. pisciphila. A large distribution of phosphate and Cd on the mycelia surface was observed by scanning electron microscopy-energy dispersive spectrometer (SEM-EDS). The precipitates associated with the mycelia were observed to contain Cd by transmission electron microscopy-energy dispersive X-ray spectroscopy (TEM-EDX). Fourier transform infrared (FTIR) identified that hydroxyl, amine, carboxyl, and phosphate groups were responsible for binding Cd. We conclude that Cd associated with cell walls and integrated with phosphate might be responsible for the tolerance of E. pisciphila to Cd.

  7. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.

    Science.gov (United States)

    Cox, Brian; Emili, Andrew

    2006-01-01

    We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.

  8. 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

    2015-11-01

    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.

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

    Science.gov (United States)

    Zhang, Bailing

    2010-01-01

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

  10. EXPRESSION AND SUBCELLULAR LOCALIZATION OF P9-ZFD PROTEIN IN PATIENTS WITH MYASTHENIA GRAVIS

    Institute of Scientific and Technical Information of China (English)

    Ming-shan Ren; Chuan-zhen Lu; Jian Qiao; Hui-min Ren; Ren Xu; Ren-bao Gan

    2004-01-01

    Objective To express and purify the protein coded by the TRAF-type zinc finger domain ofmyasthenia gravis (MG)-related gene P9 ( P9-ZFD ) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG.Methods The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET-24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its fiter and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied.Results The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control.Conclusion P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

  11. Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in Hep-2 cells

    Institute of Scientific and Technical Information of China (English)

    Min CHEN; Ping JIANG

    2004-01-01

    AIM: To study the effects of actinomycin D on subcellular distribution of nucleolar protein fibrillarin in HEp-2(human esophageal epithelial type 2) cells, and molecular mechanisms for maintenance of fibrillarin in nucleolus.METHODS: Indirect immunofiuorescence assay was employed to investigate subcellular distribution of nucleolar protein fibrillarin and immunoblotting analysis was used to detect the total cellular amount of fibrillarin. RESULTS:Control cells with no drug treatment showed bright clumpy nucleolar staining, which indicated that fibrillarin decorated the nucleolus only. Treatment with actinomycin D caused dislocation of fibrillarin from nucleoli to nucleoplasm with numerous stained small nucleoplasmic entities. Immunoblotting showed that neither total cellular amount of fibrillarin nor the integrity of fibrillarin was changed upon the treatment. The dislocation of fibrillarin in cells treated at a lower concentration (0.05 mg/L) of actinomycin D, was totally reversible after removal of the drug from the medium. However, this reversion was not observed at a high drug concentration (1 mg/L). CONCLUSION:Actinomycin D induced dislocation of fibrillarin from nucleoli to nucleoplasm in HEp-2 cells. The retention of fibrillarin within the nucleolus was related to active RNA synthesis.

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

    Directory of Open Access Journals (Sweden)

    Patricia eKreis

    2014-04-01

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

  13. An Overview of Sub-Cellular Mechanisms Involved in the Action of TTFields

    Directory of Open Access Journals (Sweden)

    Jack A. Tuszynski

    2016-11-01

    Full Text Available Long-standing research on electric and electromagnetic field interactions with biological cells and their subcellular structures has mainly focused on the low- and high-frequency regimes. Biological effects at intermediate frequencies between 100 and 300 kHz have been recently discovered and applied to cancer cells as a therapeutic modality called Tumor Treating Fields (TTFields. TTFields are clinically applied to disrupt cell division, primarily for the treatment of glioblastoma multiforme (GBM. In this review, we provide an assessment of possible physical interactions between 100 kHz range alternating electric fields and biological cells in general and their nano-scale subcellular structures in particular. This is intended to mechanistically elucidate the observed strong disruptive effects in cancer cells. Computational models of isolated cells subject to TTFields predict that for intermediate frequencies the intracellular electric field strength significantly increases and that peak dielectrophoretic forces develop in dividing cells. These findings are in agreement with in vitro observations of TTFields’ disruptive effects on cellular function. We conclude that the most likely candidates to provide a quantitative explanation of these effects are ionic condensation waves around microtubules as well as dielectrophoretic effects on the dipole moments of microtubules. A less likely possibility is the involvement of actin filaments or ion channels.

  14. Regional and subcellular distribution of gold in brain of gold thioglucose obese mouse.

    Science.gov (United States)

    Kataoka, K; Danbara, H; Sunayashiki, K; Okuno, S; Sorimachi, M; Tanaka, M; Chikamori, K

    1978-01-01

    By employing neutron activation analysis, endogenous content of gold was estimated quantitatively in discrete brain areas and in subcellular fractions of the hypothalamus of gold thioglucose (GTG) induced obese mice. The highest concentration of gold was obtained in the ventromedial hypothalamus (VMH) reaching approximately 100 ng/mg wet tissue. Significantly higher concentrations were observed in other hypothalamic subareas followed by certain limbic areas and the thalamus, while in the cerebral and the cerebellar cortex the gold concentration was very low. Subcellularly, the hypothalamic gold was principally recovered in the supernatant fraction particularly after a hyposmotic shock treatment of the crude mitochondrial fraction. Contrary to GTG, treatment with gold thiomalate (GTM) did not induce obesity in the mouse, although considerable amount of gold was observed in the VMH, a finding suggesting the existence in the VMH of at least a two step mechanisms for inducing GTG obesity. To identify the satiety neuron transmitter, an analysis of certain enzyme activities involved in the synthesis of known transmitters, such as acetylcholine or gamma-aminobutyric acid (GABA), was made in the GTG-obese mice. There were no significant changes in any of the areas functionally related to the VMH.

  15. 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.

  16. Role of Glucokinase in the Subcellular Localization of Glucokinase Regulatory Protein

    Directory of Open Access Journals (Sweden)

    Ling Jin

    2015-04-01

    Full Text Available Glucokinase (GCK is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein, GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

  17. Slit2 expression and its correlation with subcellular localization of β-catenin in gastric cancer.

    Science.gov (United States)

    Shi, Rongliang; Liu, Weiyan; Liu, Bingya; Xu, Ziping; Chen, Liping; Zhang, Ziping

    2013-10-01

    Gastric cancer is the fourth most common cancer worldwide. Several signaling pathways are involved in gastric cancer development and progression. Slit2 was recently found to be involved in cancer; however, its expression pattern in gastric cancer has not been discovered yet. In the present study, we investigated the expression of Slit2 in human gastric cancer and its correlation with the expression and subcellular localization of β-catenin. Immunohistochemistry (IHC) staining revealed that Slit2 was highly expressed in human gastric cancer tissues, while it was low or weakly expressed in normal gastric tissues. The differences in clinicopathological features between different groups were determined using Pearson's χ2 test. Slit2 levels were significantly associated with differentiation, Lauren's classification, lymph node metastasis and TNM staging. Slit2 levels were positively correlated with β-catenin level in gastric cancer tissues and cell lines. High levels of Slit2 were correlated with the membrane localization of β-catenin, and low levels of Slit2 were correlated with nuclear translocation of β-catenin in both gastric cancer tissues and cell lines assayed by IHC and immunofluorescence staining, respectively. Our data suggest that Slit2 was highly expressed in gastric cancer patients with less advanced clinicopathological features. Slit2 levels were correlated with β-catenin level and subcellular localization.

  18. Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

    Science.gov (United States)

    Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

    2017-01-22

    Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

  20. Differential spatial expression and subcellular localization of CtBP family members in rodent brain.

    Directory of Open Access Journals (Sweden)

    Diana Hübler

    Full Text Available C-terminal binding proteins (CtBPs are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.

  1. Differential spatial expression and subcellular localization of CtBP family members in rodent brain.

    Science.gov (United States)

    Hübler, Diana; Rankovic, Marija; Richter, Karin; Lazarevic, Vesna; Altrock, Wilko D; Fischer, Klaus-Dieter; Gundelfinger, Eckart D; Fejtova, Anna

    2012-01-01

    C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.

  2. Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple.

    Science.gov (United States)

    Wang, Xiu-Ling; Xu, Yan-Hong; Peng, Chang-Cao; Fan, Ren-Chun; Gao, Xin-Qi

    2009-01-01

    NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

  3. Control of stem cell fate by engineering their micro andnanoenvironment

    Institute of Scientific and Technical Information of China (English)

    Michelle F Griffin; Peter E Butler; Alexander M Seifalian; Deepak M Kalaskar

    2015-01-01

    Stem cells are capable of long-term self-renewal anddifferentiation into specialised cell types, making theman ideal candidate for a cell source for regenerativemedicine. The control of stem cell fate has become amajor area of interest in the field of regenerative medicineand therapeutic intervention. Conventional methodsof chemically inducing stem cells into specific lineagesis being challenged by the advances in biomaterialtechnology, with evidence highlighting that materialproperties are capable of driving stem cell fate. Materialsare being designed to mimic the clues stem cells receivein their in vivo stem cell niche including topographicaland chemical instructions. Nanotopographical clues thatmimic the extracellular matrix (ECM) in vivo have shownto regulate stem cell differentiation. The delivery of ECMcomponents on biomaterials in the form of short peptidessequences has also proved successful in directing stem celllineage. Growth factors responsible for controlling stemcell fate in vivo have also been delivered via biomaterialsto provide clues to determine stem cell differentiation. Analternative approach to guide stem cells fate is to providegenetic clues including delivering DNA plasmids andsmall interfering RNAs via scaffolds. This review, aims toprovide an overview of the topographical, chemical andmolecular clues that biomaterials can provide to guidestem cell fate. The promising features and challenges ofsuch approaches will be highlighted, to provide directionsfor future advancements in this exciting area of stem celltranslation for regenerative medicine.

  4. Grass meristems II: inflorescence architecture, flower development and meristem fate.

    Science.gov (United States)

    Tanaka, Wakana; Pautler, Michael; Jackson, David; Hirano, Hiro-Yuki

    2013-03-01

    Plant development depends on the activity of various types of meristems that generate organs such as leaves and floral organs throughout the life cycle. Grass species produce complex inflorescences and unique flowers. The grass inflorescence is composed of different types of branches, including a specialized branch called a spikelet. The spikelet is a special unit of the inflorescence and forms one to several florets, depending on the species. In the floret, floral organs such as perianth organs, carpels and stamens are formed. In Arabidopsis, because the inflorescence meristem (IM) forms the floral meristems (FMs) directly on its flanks, the change of meristem fate is relatively simple. In contrast, in grasses, different types of meristem, such as the IM, the branch meristem (BM), the spikelet pair meristem (SPM) in some grasses, the spikelet meristem (SM) and the FM, are responsible for the elaboration of their complex inflorescences and flowers. Therefore, sequential changes of meristem fate are required, and a number of genes involved in the specification of the fate of each meristem have been identified. In this review, we focus on the following issues concerning the fate of the reproductive meristems in two grass species, maize (Zea mays) and rice (Oryza sativa): (i) meristem regulation during inflorescence development; (ii) specification and fate change of the BM and the SM; (iii) determinacy of the FM; and (iv) communication between the meristem and lateral organs.

  5. Environmental fate of alachlor and metolachlor.

    Science.gov (United States)

    Chesters, G; Simsiman, G V; Levy, J; Alhajjar, B J; Fathulla, R N; Harkin, J M

    1989-01-01

    Decision-makers, scientists, and the interested public should be informed what future research and education is needed if a strong pesticide regulatory program is imposed. Recommendations are intended to highlight research gaps. Some may be of general concern and apply to many pesticides. A situation that calls into question the value of many of our management decisions, is the lack of good field-scale experimentation and of logical mechanisms for translating and extrapolating laboratory data to field-scale dimensions. Many experiments were not designed to allow application of basic statistical criteria. High costs often preclude sufficient replication in field-scale experiments so that researchers must make the "no-win" choice between doing one investigation well or doing two or three poorly. The following observations about alachlor and metolachlor are provided: Pysicochemical properties are accurately determined. The herbicides' modes of action and plant selectivity have received a great deal of attention, but gaps remain in defining which of three modes of action are most important. Geographic distribution and extent of residue contamination of surface waters is documented, but groundwater contamination is poorly defined. Any groundwater monitoring protocol should limit the investigation based on sound scientific judgment since a nationwide monitoring network cannot be economically justified. Enough data are needed, however, to allow mathematical model development, verification and validation for a diversity of soil, geographic, climatic, and agricultural management conditions. In view of the importance of adsorption in determining the fate of pesticides, improved methods of determining adsorption coefficients (KD) are needed particularly for very low concentrations. The impact of soil aggregation on adsorption/desorption needs to be examined. The role of temperature and water content in adsorption/desorption processes needs clearer definition. Although

  6. Fate of Organic Carbon Deposited in Reservoirs

    Science.gov (United States)

    Huntington, T. G.; Rhoton, F. E.; Bennett, S. J.; Hudnall, W. H.

    2002-05-01

    , sediment texture, and mineralogy, allows estimation of the autochthonous fraction. These sediment properties evaluated at different positions in the reservoir relative to inlet points can also be used to understand the fate of the SOC. Points furthest from the reservoir inlet should have higher fractions of finer textured sediments and more anoxic conditions. Sediments deposited closer to the inlet points experience periodic draining because of fluctuating water levels in the reservoir. These differences suggest that SOC is likely to be more highly conserved at positions furthest from the inlet points.

  7. Fate of Organic Micropollutants during Hydrothermal Carbonization

    Science.gov (United States)

    Weiner, B.; Baskyr, I.; Pörschmann, J.; Kopinke, F.-D.

    2012-04-01

    contaminated biomass. Chlorinated aromatic compounds are not fully degraded during HTC. Therefore, the addition of catalysts and reagents for a possible reduction has been studied. Zero-valent environmentally acceptable metals, such as Fe or Si, are presented as potential additives for the dechlorination of chloronaphthalene as a representative of chloroaromatics. Furthermore, when using municipal household waste, such as the 'organic' bin, or gardening greens as biomass educts, these materials often contain traces of synthetic plastics, which can lead to problems during waste incineration. Initial studies on the fate of synthetic polymers will also be presented.

  8. The final fate of planetary systems

    Science.gov (United States)

    Gaensicke, Boris

    2015-12-01

    The discovery of the first extra-solar planet around a main-sequence star in 1995 has changed the way we think about the Universe: our solar system is not unique. Twenty years later, we know that planetary systems are ubiquitous, orbit stars spanning a wide range in mass, and form in an astonishing variety of architectures. Yet, one fascinating aspect of planetary systems has received relatively little attention so far: their ultimate fate.Most planet hosts will eventually evolve into white dwarfs, Earth-sized stellar embers, and the outer parts of their planetary systems (in the solar system, Mars and beyond) can survive largely intact for billions of years. While scattered and tidally disrupted planetesimals are directly detected at a small number of white dwarfs in the form infrared excess, the most powerful probe for detecting evolved planetary systems is metal pollution of the otherwise pristine H/He atmospheres.I will present the results of a multi-cycle HST survey that has obtained COS observations of 136 white dwarfs. These ultraviolet spectra are exquisitely sensitive to the presence of metals contaminating the white atmosphere. Our sophisticated model atmosphere analysis demonstrates that at least 27% of all targets are currently accreting planetary debris, and an additional 29% have very likely done so in the past. These numbers suggest that planet formation around A-stars (the dominant progenitors of today's white dwarf population) is similarly efficient as around FGK stars.In addition to post-main sequence planetary system demographics, spectroscopy of the debris-polluted white dwarf atmospheres provides a direct window into the bulk composition of exo-planetesimals, analogous to the way we use of meteorites to determine solar-system abundances. Our ultraviolet spectroscopy is particularly sensitive to the detection of Si, a dominant rock-forming species, and we identify up to ten additional volatile and refractory elements in the most strongly

  9. Environmental fate data base (ENVIROFATE): environmental fate data (DATALOG), June 1988. Data file

    Energy Technology Data Exchange (ETDEWEB)

    Boethling, R.

    1988-06-01

    DATALOG is a bibliographic pointer file of environmental fate data and each record contains a chemical CAS registry number (9 bytes), one of 18 data types (9 bytes), and a reference number (6 bytes) to the full citation in the XREF file. The data types identify physical properties, degradation and transport studies, and food, occupational and ambient monitoring data. The file should be used with CASLST (in order to link the full reference citation). A list of possible data types and their abbreviations are: Adsorption (ADSORP), Bioconcentration (BIOCON), Biodegradation (BIODEG), Dissociation constant (DISS CON), Ecosystem (ECOS), Effluent concentrations (EFFL), Evaporation from water (EVAP), Food and crop concentrations (FOOD), Field studies (FIELD), Henry's Law constant (HENRY CON), Hydrolysis (HYDROL), Monitoring (MONIT), Occupational concentrations (OCCUP), Octanol/water partition coefficient (O/W PART), Photoxidation (PHOTOOXID), UV Spectra (UV), Vapor pressure (VP) and Water solubility (WATER SOL).

  10. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    Science.gov (United States)

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

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

    Directory of Open Access Journals (Sweden)

    Liqi Li

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

  12. Organelle-Targeted H2S Probes Enable Visualization of the Subcellular Distribution of H2S Donors.

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2016-06-07

    Hydrogen sulfide (H2S) is an essential biological signaling molecule in diverse biological regulatory pathways. To provide new chemical tools for H2S imaging, we report here a fluorescent H2S detection platform (HSN2-BG) that is compatible with subcellular localization SNAP-tag fusion protein methodologies and use appropriate fusion protein constructs to demonstrate mitochondrial and lysosomal localization. We also demonstrate the efficacy of this detection platform to image endogenous H2S in Chinese hamster ovary (CHO) cells and use the developed constructs to report on the subcellular H2S distributions provided by common H2S donor molecules AP39, ADT-OH, GYY4137, and diallyltrisulfide (DATS). The developed constructs provide a platform poised to provide new insights into the subcellular distribution of common H2S donors and a useful tool for investigating H2S biochemistry.

  13. In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure

    OpenAIRE

    2010-01-01

    Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discret...

  14. Modelling accumulation of marine plastics in the coastal zone; what are the dominant physical processes?

    Science.gov (United States)

    Critchell, Kay; Lambrechts, Jonathan

    2016-03-01

    Anthropogenic marine debris, mainly of plastic origin, is accumulating in estuarine and coastal environments around the world causing damage to fauna, flora and habitats. Plastics also have the potential to accumulate in the food web, as well as causing economic losses to tourism and sea-going industries. If we are to manage this increasing threat, we must first understand where debris is accumulating and why these locations are different to others that do not accumulate large amounts of marine debris. This paper demonstrates an advection-diffusion model that includes beaching, settling, resuspension/re-floating, degradation and topographic effects on the wind in nearshore waters to quantify the relative importance of these physical processes governing plastic debris accumulation. The aim of this paper is to prioritise research that will improve modelling outputs in the future. We have found that the physical characteristic of the source location has by far the largest effect on the fate of the debris. The diffusivity, used to parameterise the sub-grid scale movements, and the relationship between debris resuspension/re-floating from beaches and the wind shadow created by high islands also has a dramatic impact on the modelling results. The rate of degradation of macroplastics into microplastics also have a large influence in the result of the modelling. The other processes presented (settling, wind drift velocity) also help determine the fate of debris, but to a lesser degree. These findings may help prioritise research on physical processes that affect plastic accumulation, leading to more accurate modelling, and subsequently management in the future.

  15. Sub-cellular distribution of UNC-104(KIF1A) upon binding to adaptors as UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α) in C. elegans neurons.

    Science.gov (United States)

    Hsu, C-C; Moncaleano, J D; Wagner, O I

    2011-03-10

    The accumulation of cargo (tau, amyloid precursor protein, neurofilaments etc.) in neurons is a hallmark of various neurodegenerative diseases while we have only little knowledge how axonal transport is regulated. Kinesin-3 UNC-104(KIF1A) is the major transporter of synaptic vesicles and recent reports suggest that a cargo itself can affect the motor's activity. Inspecting an interactome map, we identify three putative UNC-104 interactors, namely UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α), known to be adaptors in essential neuronal protein complexes. We then employed the novel method bimolecular fluorescence complementation (BiFC) assay to visualize motor-adaptor complexes in the nervous system of living C. elegans. Interestingly, the binding of UNC-104 to each adaptor protein results in different sub-cellular distributions and has distinctive effects on the motor's motility. Specifically, if UNC-104 bound to UNC-16, the motor is primarily localized in the soma of neurons while bound to DNC-1, the motor is basically found in axonal termini. On the other hand, if UNC-104 is bound to SYD-2 we identify motor populations mostly along axons. Therefore, these three adaptors inherit different functions in steering the motor to specific sub-cellular locations in the neuron.

  16. Cell fate control in the developing central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Guérout, Nicolas; Li, Xiaofei; Barnabé-Heider, Fanie, E-mail: Fanie.Barnabe-Heider@ki.se

    2014-02-01

    The principal neural cell types forming the mature central nervous system (CNS) are now understood to be diverse. This cellular subtype diversity originates to a large extent from the specification of the earlier proliferating progenitor populations during development. Here, we review the processes governing the differentiation of a common neuroepithelial cell progenitor pool into mature neurons, astrocytes, oligodendrocytes, ependymal cells and adult stem cells. We focus on studies performed in mice and involving two distinct CNS structures: the spinal cord and the cerebral cortex. Understanding the origin, specification and developmental regulators of neural cells will ultimately impact comprehension and treatments of neurological disorders and diseases. - Highlights: • Similar mechanisms regulate cell fate in different CNS cell types and structures. • Cell fate regulators operate in a spatial–temporal manner. • Different neural cell types rely on the generation of a diversity of progenitor cells. • Cell fate decision is dictated by the integration of intrinsic and extrinsic signals.

  17. Epigenetic memory and cell fate reprogramming in plants.

    Science.gov (United States)

    Birnbaum, Kenneth D; Roudier, François

    2017-02-01

    Plants have a high intrinsic capacity to regenerate from adult tissues, with the ability to reprogram adult cell fates. In contrast, epigenetic mechanisms have the potential to stabilize cell identity and maintain tissue organization. The question is whether epigenetic memory creates a barrier to reprogramming that needs to be erased or circumvented in plant regeneration. Early evidence suggests that, while chromatin dynamics impact gene expression in the meristem, a lasting constraint on cell fate is not established until late stages of plant cell differentiation. It is not yet clear whether the plasticity of plant cells arises from the ability of cells to erase identity memory or to deploy cells that may exhibit cellular specialization but still lack an epigenetic restriction on cell fate alteration.

  18. Towards a statistical mechanics of cell fate decisions.

    Science.gov (United States)

    Garcia-Ojalvo, Jordi; Martinez Arias, Alfonso

    2012-12-01

    The spatiotemporal organization of a developing organism requires carefully orchestrated sequences of cellular differentiation events. These events are triggered by decisions made by individual cells about their fate, which are in turn controlled by gene and protein regulation processes. While these cell fate decisions are subject to stochasticity and are not reproducible at the single-cell level, they result in highly consistent, almost deterministic patterns at the level of the whole cell population. The question of how this macroscopic order arises from a disordered microscopic behaviour is still outstanding, and is reminiscent of problems in physical systems that are readily addressed by statistical mechanics. Here we review recent studies that are beginning to provide the data needed to address this question and discuss conceptual ideas that might be used in a theoretical understanding of cell fate decision processes, emphasizing the challenges that biology poses to the application of statistical mechanics approaches to developmental biology.

  19. Cell fate determination by ubiquitin-dependent regulation of translation

    Science.gov (United States)

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  20. Fate-restricted neural progenitors in the mammalian cerebral cortex.

    Science.gov (United States)

    Franco, Santos J; Gil-Sanz, Cristina; Martinez-Garay, Isabel; Espinosa, Ana; Harkins-Perry, Sarah R; Ramos, Cynthia; Müller, Ulrich

    2012-08-10

    During development of the mammalian cerebral cortex, radial glial cells (RGCs) generate layer-specific subtypes of excitatory neurons in a defined temporal sequence, in which lower-layer neurons are formed before upper-layer neurons. It has been proposed that neuronal subtype fate is determined by birthdate through progressive restriction of the neurogenic potential of a common RGC progenitor. Here, we demonstrate that the murine cerebral cortex contains RGC sublineages with distinct fate potentials. Using in vivo genetic fate mapping and in vitro clonal analysis, we identified an RGC lineage that is intrinsically specified to generate only upper-layer neurons, independently of niche and birthdate. Because upper cortical layers were expanded during primate evolution, amplification of this RGC pool may have facilitated human brain evolution.

  1. Accumulation and elimination of cadmium and zinc in the Asian periwinkle Littorina brevicula.

    Science.gov (United States)

    Han, Soo-Jeong; Park, Jin-Sung; Lee, In-Sook

    2003-05-01

    To elucidate the differences between the detoxification mechanisms of essential metal (Zn) and non-essential metal (Cd) in Littorina brevicula that is highly resistant to a wide range of heavy metal concentrations, Asian periwinkles were exposed to Cd (400 microg/L), Zn (3000 microg/L) and a mixture of both metals. We examined metal accumulation, elimination and subcellular distribution for binding to proteins. The metal concentration in L. brevicula increased gradually with exposure time (up to 70 days), following which accumulated levels reached saturation point. The accumulated Zn content was increased in the presence of Cd, while Cd uptake was decreased when Zn was present. During the depuration period (42 days), Cd was not removed from periwinkles, while Zn was eliminated in a rate of 2.19 microg Zn g(-1) day(-1). This elimination rate was particularly high on exposure to the metal mixture (3.80 microg Zn g(-1) day(-1)). Subcellular distribution studies on Cd and Zn revealed that most Cd (80%) was bound to cytosolic ligand, while more than 75% Zn was distributed in the membrane (insoluble) fraction. An additional difference in sequestering of metal in the cytosol was noted between Cd and Zn; most Cd in the cytosol was bound to metallothionein-like cadmium binding protein, MBP-1 (9.8 kDa), while the profile for Zn distribution revealed the presence of four Zn-binding ligand peaks, specifically, HMW (60 kDa), MBP-1 (9.8 kDa), MBP-2 (5 kDa) and LMW (<1 kDa). Our data confirm that metallothionein-like cadmium binding protein, MBP-1, has the same affinity for Zn, while MBP-2 displays comparatively higher affinity for Zn than Cd.

  2. How a (subcellular coincidence detection mechanism featuring layer-5 pyramidal cells may help produce various visual phenomena

    Directory of Open Access Journals (Sweden)

    Talis eBachmann

    2015-12-01

    Full Text Available Perceptual phenomena such as spatio-temporal illusions and masking are typically explained by psychological (cognitive processing theories or large-scale neural theories involving inter-areal connectivity and neural circuits comprising of hundreds or more interconnected single cells. Subcellular mechanisms are hardly used for such purpose. Here a mechanistic theoretical view is presented on how a subcellular brain mechanism of integration of presynaptic signals that arrive at different compartments of layer-5 pyramidal neurons could explain a couple of spatiotemporal visual-phenomenal effects unfolding along very brief time intervals within the range of sub-second temporal scale.

  3. Fate of mercury in the Arctic (FOMA)

    DEFF Research Database (Denmark)

    Skov, H.; Christensen, J.; Asmund, G.

    This report is the final reporting of the project FONA, funded by the Danish Environmental Protection Agency with means from the MIKA/DANCEA funds for Environmental Support to the Arctic Region. The aim of the project is to study the intercompartment mercury transport chain in the arctic area. From...... atmospheric deposition of mercury on sea surfaces to uptake in marine organisms, bio-accumulation, and finally mercury levels in mammals. The studies in the project are focused on the behaviour of mercury during the spring period where special phenomena lead to an enhanced deposition of mercury in the Arctic...... environment, at a time where the marine ecosystem is particularly active. The studies also include a comprehensive time trend study of mercury in top carnivore species. Each of these studies contributes towards establishing the knowledge necessary to develop a general model for transport and uptake of mercury...

  4. Fate of mercury in the Arctic (FOMA)

    DEFF Research Database (Denmark)

    Skov, H.; Christensen, J.; Asmund, G.

    This report is the final reporting of the project FONA, funded by the Danish Environmental Protection Agency with means from the MIKA/DANCEA funds for Environmental Support to the Arctic Region. The aim of the project is to study the intercompartment mercury transport chain in the arctic area. From...... atmospheric deposition of mercury on sea surfaces to uptake in marine organisms, bio-accumulation, and finally mercury levels in mammals. The studies in the project are focused on the behaviour of mercury during the spring period where special phenomena lead to an enhanced deposition of mercury in the Arctic...... environment, at a time where the marine ecosystem is particularly active. The studies also include a comprehensive time trend study of mercury in top carnivore species. Each of these studies contributes towards establishing the knowledge necessary to develop a general model for transport and uptake of mercury...

  5. Fate of platinum metals in the environment.

    Science.gov (United States)

    Pawlak, Justyna; Łodyga-Chruścińska, Elżbieta; Chrustowicz, Jakub

    2014-07-01

    For many years now automotive exhaust catalysts have been used to reduce the significant amounts of harmful chemical substances generated by car engines, such as carbon monoxide, nitrogen oxides, and aromatic hydrocarbons. Although they considerably decrease environmental contamination with the above-mentioned compounds, it is known that catalysts contribute to the environmental load of platinum metals (essential components of catalysts), which are released with exhaust fumes. Contamination with platinum metals stems mainly from automotive exhaust converters, but other major sources also exist. Since platinum group elements (PGEs): platinum (Pt), palladium (Pd), rhodium (Rh), ruthenium (Ru) and iridium (Ir) seem to spread in the environment and accumulate in living organisms, they may pose a threat to animals and humans. This paper discusses the modes and forms of PGE emission as well as their impact on the environment and living organisms.

  6. BTG interacts with retinoblastoma to control cell fate in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Daniele Conte

    Full Text Available BACKGROUND: In the genesis of many tissues, a phase of cell proliferation is followed by cell cycle exit and terminal differentiation. The latter two processes overlap: genes involved in the cessation of growth may also be important in triggering differentiation. Though conceptually distinct, they are often causally related and functional interactions between the cell cycle machinery and cell fate control networks are fundamental to coordinate growth and differentiation. A switch from proliferation to differentiation may also be important in the life cycle of single-celled organisms, and genes which arose as regulators of microbial differentiation may be conserved in higher organisms. Studies in microorganisms may thus contribute to understanding the molecular links between cell cycle machinery and the determination of cell fate choice networks. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that in the amoebozoan D. discoideum, an ortholog of the metazoan antiproliferative gene btg controls cell fate, and that this function is dependent on the presence of a second tumor suppressor ortholog, the retinoblastoma-like gene product. Specifically, we find that btg-overexpressing cells preferentially adopt a stalk cell (and, more particularly, an Anterior-Like Cell fate. No btg-dependent preference for ALC fate is observed in cells in which the retinoblastoma-like gene has been genetically inactivated. Dictyostelium btg is the only example of non-metazoan member of the BTG family characterized so far, suggesting that a genetic interaction between btg and Rb predated the divergence between dictyostelids and metazoa. CONCLUSIONS/SIGNIFICANCE: While the requirement for retinoblastoma function for BTG antiproliferative activity in metazoans is known, an interaction of these genes in the control of cell fate has not been previously documented. Involvement of a single pathway in the control of mutually exclusive processes may have relevant implication in the

  7. Cell Fate Switch during In Vitro Plant Organogenesis

    Institute of Scientific and Technical Information of China (English)

    Xiang Yu Zhao; Ying Hua Su; Zhi Juan Cheng; Xian Sheng Zhang

    2008-01-01

    Plant mature cells have the capability to reverse their state of differenUation and produce new organs under cultured conditions. Two phases, dedifferentiation and redifferentiation, are commonly characterized during in vitro organogenesis.In these processes, cells undergo fate switch several times regulated by both extrinsic and intrinsic factors, which are associated with reentry to the cell cycle, the balance between euchromatin and heterochromatin, reprogramming of gene expression, and so forth. This short article reviews the advances in the mechanism of organ regeneration from plant somatic cells in molecular, genomic and epigenetic aspects, aiming to provide important information on the mechanism underlying cell fate switch during in vitro plant organogenesis.

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

    Science.gov (United States)

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

    2015-07-01

    The sediment-dwelling ragworm, Nereis diversicolor was exposed to sediment spiked with aqueous Cu (CuAq, CuCl2), CuO nanoparticles (CuONP) or CuO microparticles (CuOMicro) at 150 μg Cu g(-1) dw sediment for 10d. Exposures to CuAq and CuOMicro caused mortality (62.5 and 37.5%, respectively), whereas mean burrowing time increased during exposure to CuAq and CuONP from 0.12 h (controls) to 19.3 and 12.2 h, respectively. All Cu treatments bioaccumulated, especially CuAq (up to 4 times more than the other treatments). Cu was roughly equally distributed among the five subcellular fractions in controls and worms exposed to CuONP or CuOMicro. In contrast, ≈50% of accumulated Cu in CuAq exposed worms was found in metal rich granules and significantly more Cu was present in heat-denatured proteins and organelles than in worms exposed to CuOMicro or in controls. Our results suggest that Cu form affects its bioaccumulation and subsequent toxicity and detoxification in a polychaete like N. diversicolor. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Subcellular partitioning of non-essential trace metals (Ag, As, Cd, Ni, Pb, and Tl) in livers of American (Anguilla rostrata) and European (Anguilla anguilla) yellow eels.

    Science.gov (United States)

    Rosabal, Maikel; Pierron, Fabien; Couture, Patrice; Baudrimont, Magalie; Hare, Landis; Campbell, Peter G C

    2015-03-01

    We determined the intracellular compartmentalization of the trace metals Ag, As, Cd, Ni, Pb, and Tl in the livers of yellow eels collected from the Saint Lawrence River system in Canada (Anguilla rostrata) and in the area of the Gironde estuary in France (Anguilla anguilla). Differential centrifugation, NaOH digestion and thermal shock were used to separate eel livers into putative "sensitive" fractions (heat-denatured proteins, mitochondria and microsomes+lysosomes) and detoxified metal fractions (heat-stable peptides/proteins and granules). The cytosolic heat-stable fraction (HSP) was consistently involved in the detoxification of all trace metals. In addition, granule-like structures played a complementary role in the detoxification of Ni, Pb, and Tl in both eel species. However, these detoxification mechanisms were not completely effective because increasing trace metal concentrations in whole livers were accompanied by significant increases in the concentrations of most trace metals in "sensitive" subcellular fractions, that is, mitochondria, heat-denatured cytosolic proteins and microsomes+lysosomes. Among these "sensitive" fractions, mitochondria were the major binding sites for As, Cd, Pb, and Tl. This accumulation of non-essential metals in "sensitive" fractions likely represents a health risk for eels inhabiting the Saint Lawrence and Gironde environments. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Paris-Palacios, Severine [Universite de Reims Champagne-Ardenne (URCA), UFR Sciences Exactes et Naturelles, Laboratoire d' Eco-Toxicologie, Institut International de Recherche sur les Ions Metalliques, B.P. 1039-51687 Reims cedex 2 (France)]. E-mail: severine.paris@univ-reims.fr; Biagianti-Risbourg, Sylvie [Universite de Reims Champagne-Ardenne (URCA), UFR Sciences Exactes et Naturelles, Laboratoire d' Eco-Toxicologie, Institut International de Recherche sur les Ions Metalliques, B.P. 1039-51687 Reims cedex 2 (France)]. E-mail: sylvie.biagianti@univ-reims.fr

    2006-05-10

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

  11. Kinetics and subcellular localization of 5-ALA-induced PpIX in DHL cells via two-photon excitation fluorescence microscopy.

    Science.gov (United States)

    Chen, Rong; Huang, Zufang; Chen, Guannan; Li, Yongzeng; Chen, Xianlian; Chen, Jianxin; Zeng, Haishan

    2008-04-01

    Two-photon excitation fluorescence (TPEF) microscopy was used to measure the 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence in follicular lymphoma DHL cells. Kinetics of 5-ALA-induced PpIX accumulation in DHL cells under various 5-ALA concentrations was studied. We found that during the course of continuous incubation with 5-ALA, the relationship between the DHL cell fluorescence signal and the incubation time showed a biphasic variation. Initially the PpIX signal increased with the incubation time and reached the maximal value at about 3 h, and then it decreased with time during the subsequent incubation period. By labeling the 5-ALA incubated DHL cells with different organelle-specific fluorescence probes: Rhodamine 123 (for mitochondria), DioC6(3) (for endoplasmic reticulum) and LysoTracker Green (for lysosomes) respectively, we found that 5-ALA-induced PpIX was primarily localized in endoplasmic reticulum and mitochondria; its concentration in the lysosome was much lower. The results suggested that 5-ALA could potentially be an effective photosensitizer in photodynamic purging of DHL cells. Two-photon excitation fluorescence microscope is a useful tool for studying 5-ALA-induced PpIX subcellular localization.

  12. Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

    Science.gov (United States)

    Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J

    2015-05-01

    Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

  13. Metabolism of the environmental toxicant benzo(a)pyrene by subcellular fractions of human ovary.

    Science.gov (United States)

    Rekhadevi, P V; Diggs, D L; Huderson, A C; Harris, K L; Archibong, A E; Ramesh, A

    2014-02-01

    Knowledge of the ability of the female reproductive system to metabolize environmental chemicals is critical not only from the standpoint of toxicity but also from infertility risk assessment. Benzo(a)pyrene (BaP) is a toxicant that is released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, BaP becomes activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. Studies on animal models conducted in our laboratories and those of others have shown that BaP possess endocrine disrupting properties. Thus, this chemical has the potential to cause infertility and cancers in the female genital tract. An understanding of BaP metabolism in the female reproductive system will be of importance in the diagnosis and management of female fertility as well as cancers in the reproductive tissues. Therefore, the objective of our study was to examine the metabolism of BaP by human ovarian subcellular fractions. Human ovary samples (eight individuals) were obtained from postoperative tissue removed from subjects with uterine tumors. Subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were prepared by differential centrifugation. BaP (1 μM and 3 μM) was individually incubated with individual subcellular fractions for 15 min and the products were analyzed by high-performance liquid chromatography. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions. The BaP metabolites identified were as follows: BaP-9,10-diol, BaP-4,5-diol, BaP-7,8-diol, 9(OH) BaP, 3(OH) BaP, BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione. Of interest was the presence of DNA-reactive metabolites such as BaP-3,6-dione, BaP-6,12-dione, and BaP 7,8-diol, which have been implicated in the causation of infertility and cancer. Our results indicate that women who are exposed to BaP via

  14. Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane.

    Science.gov (United States)

    Harrison, Mark D; Geijskes, Jason; Coleman, Heather D; Shand, Kylie; Kinkema, Mark; Palupe, Anthony; Hassall, Rachael; Sainz, Manuel; Lloyd, Robyn; Miles, Stacy; Dale, James L

    2011-10-01

    A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

  15. Accumulation and chemical states of radiocesium by fungus Saccharomyces cerevisiae

    Science.gov (United States)

    Ohnuki, Toshihiko; Sakamoto, Fuminori; Kozai, Naofumi; Yamasaki, Shinya; Yu, Qianqian

    2014-05-01

    mineral for 137Cs in the medium solution. The concentration of 137Cs in the cells lowered in the medium containing higher mineral content. These results indicate that radiocesium was competively accumulated in the cells with minerals in the soil. Higher concentration of stable Cs was accumulated in the cells in the metabolically active condition than in the resting cells condition. XAFS analyses showed that the k3-weighted extended-XAFS functions and the radial structural function of Cs accumulated by the cells in the metabolically active condition were similar to those in the resting condition, indicating that chemical states of the accumulated Cs were nearly the same between both conditions. These results indicate that the fungus accumulates radiocesium by competitively with minerals in the soils, and performs higher retardation of the migration of Cs in the metabolically active condition than the resting one. A part of this study is the results of "Multidisciplinary investigation on radiocesium fate and transport for safety assessment for interim storage and disposal of heterogeneous waste" carried out under the Initiatives for Atomic Energy Basic and Generic Strategic Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

  16. Occurrence and fate of acrylamide in water-recycling systems and sludge in aggregate industries.

    Science.gov (United States)

    Junqua, Guillaume; Spinelli, Sylvie; Gonzalez, Catherine

    2015-05-01

    Acrylamide is a hazardous substance having irritant and toxic properties as well as carcinogen, mutagen, and impaired fertility possible effects. Acrylamide might be found in the environment as a consequence of the use of polyacrylamides (PAMs) widely added as a flocculant for water treatment. Acrylamide is a monomer used to produce polyacrylamide (PAM) polymers. This reaction of polymerization can be incomplete, and acrylamide molecules can be present as traces in the commercial polymer. Thus, the use of PAMs may generate a release of acrylamide in the environment. In aggregate industries, PAM is widely involved in recycling process and water reuse (aggregate washing). Indeed, these industries consume large quantities of water. Thus, European and French regulations have favored loops of recycling of water in order to reduce water withdrawals. The main goal of this article is to study the occurrence and fate of acrylamide in water-recycling process as well as in the sludge produced by the flocculation treatment process in aggregate production plants. Moreover, to strengthen the relevance of this article, the objective is also to demonstrate if the recycling system leads to an accumulation effect in waters and sludge and if free acrylamide could be released by sludge during their storage. To reach this objective, water sampled at different steps of recycling water process has been analyzed as well as different sludge corresponding to various storage times. The obtained results reveal no accumulation effect in the water of the water-recycling system nor in the sludge.

  17. Taenia taeniaeformis: fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats.

    Science.gov (United States)

    Lagapa, J T; Oku, Y; Nonaka, N; Kamiya, M

    2008-04-01

    Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode's ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions.

  18. Completeness of the Accumulation Calculus

    Institute of Scientific and Technical Information of China (English)

    虞慧群; 孙永强; 等

    1998-01-01

    The accumulation calculs(AC for short)is an interval based temporal logic to specify and reason about hybrid real-time systems.This paper presents a formal proof system for AC,and proves that the system is complete relative to that of Interval Temporal Logic(ITL for short)on real domain.

  19. Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility

    Science.gov (United States)

    Fan, Zhenzhen; Sun, Yubing; di Chen; Tay, Donald; Chen, Weiqiang; Deng, Cheri X.; Fu, Jianping

    2013-07-01

    Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN - nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells.

  20. Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

    Science.gov (United States)

    Odrzykoski, I J

    2001-01-01

    Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. Immunogold quantification of amino acids and proteins in complex subcellular compartments.

    Science.gov (United States)

    Bergersen, Linda H; Storm-Mathisen, Jon; Gundersen, Vidar

    2008-01-01

    An increasing number of imaging techniques are in use to study the localization of molecules involved in cell-to-cell signaling. Here we describe the use of immunogold procedures to detect and quantify molecules on electron micrographs. To measure the areas of the subcellular compartments under investigation, the protocol uses an overlay screen with an array of regularly spaced points. On the basis of this, the densities of the gold-labeled molecules can be calculated. Despite the limited lateral resolution of the immunogold method as used by many investigators ( approximately 30 nm), it is possible to measure the content of molecules associated with tiny tissue compartments, e.g., synaptic vesicles and different types of membrane, such as plasma membranes and vesicle membranes. The quantification protocol can be carried out without using computer programs. The entire protocol can be completed in approximately 15 d.

  3. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    Science.gov (United States)

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  4. Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells

    Science.gov (United States)

    Etoc, F.; Lisse, D.; Bellaiche, Y.; Piehler, J.; Coppey, M.; Dahan, M.

    2013-03-01

    Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.

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

    Institute of Scientific and Technical Information of China (English)

    ZHANG BingBing; XIAN ChengYu; LUO YanFeng; WANG YuanLiang

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

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

  7. Exploring Nanostructure Arrays for Single-Cell and Subcellular Manipulation and Detection

    DEFF Research Database (Denmark)

    Buch-Månson, Nina

    these, arrays of vertical nanostructures (NSs) with submicron diametersand microscale lengths are particularly promising and are currently being established as bothhighly sensitive protein arrays and as platforms for manipulations and investigations at thesingle-cell or even subcellular level.To date...... on different NSarray geometries and materials, both through theoretical modeling and experiments.We first seek to improve the fundamental understanding of the cell-NS interface bytheoretical considerations of the energy balance between the cost of membrane deformationaround the NSs and the favorable gain...... in adhesive contact as a cell settles into a NS array. Cellshave both been reported to deform completely into NS arrays or to stay suspended at the NS tipslike tiny fakirs, which have important implications for the possible applications. We show that,in addition to NS density, the energy difference between...

  8. Sub-cellular structure studied by combined atomic force-fluorescence microscopy

    Science.gov (United States)

    Trache, Andreea

    2009-03-01

    A novel experimental technique that integrates atomic force microscopy (AFM) with fluorescence imaging was used to study the role of extracellular matrix proteins in cellular organization. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, we developed a new technology able to investigate cellular behavior at sub-cellular level that integrates an AFM with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. Live smooth muscle cells exhibited differences in focal adhesions and actin pattern depending on the extracellular matrix used for substrate coating. Data obtained by using the AFM-optical imaging integrated technique offer novel quantitative information that allows understanding the fundamental processes of cellular reorganization in response to extracellular matrix modulation. The integrated microscope presented here is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells.

  9. Analytical model of ionization and energy deposition by proton beams in subcellular compartments

    Science.gov (United States)

    de Vera, Pablo; Surdutovich, Eugene; Abril, Isabel; Garcia-Molina, Rafael; Solov'yov, Andrey V.

    2014-04-01

    We present an analytical model to evaluate in a fast, simple and effective manner the energy delivered by proton beams moving through a cell model made of nucleus and cytoplasm, taking into account the energy carried by the secondary electrons generated along the proton tracks. The electronic excitation spectra of these subcellular compartments have been modelled by means of an empirical parameterization of their dielectric properties. The energy loss rate and target ionization probability induced by swift protons are evaluated by means of the dielectric formalism. With the present model we have quantified the energy delivered, the specific energy, and the number of ionizations produced per incoming ion in a typical human cell by a typical hadrontherapy proton beam having energies usually reached around the Bragg peak (below 20 MeV). We find that the specific energy per incoming ion delivered in the nucleus and in the cytoplasm are rather similar for all the proton energy range analyzed.

  10. 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...... of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal...... 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...

  11. The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex.

    Science.gov (United States)

    Bachelard, H S

    1967-07-01

    1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.

  12. 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.

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

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    Kunavisarut Tada

    2012-11-01

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

  14. Human cell structure-driven model construction for predicting protein subcellular location from biological images.

    Science.gov (United States)

    Shao, Wei; Liu, Mingxia; Zhang, Daoqiang

    2016-01-01

    The systematic study of subcellular location pattern is very important for fully characterizing the human proteome. Nowadays, with the great advances in automated microscopic imaging, accurate bioimage-based classification methods to predict protein subcellular locations are highly desired. All existing models were constructed on the independent parallel hypothesis, where the cellular component classes are positioned independently in a multi-class classification engine. The important structural information of cellular compartments is missed. To deal with this problem for developing more accurate models, we proposed a novel cell structure-driven classifier construction approach (SC-PSorter) by employing the prior biological structural information in the learning model. Specifically, the structural relationship among the cellular components is reflected by a new codeword matrix under the error correcting output coding framework. Then, we construct multiple SC-PSorter-based classifiers corresponding to the columns of the error correcting output coding codeword matrix using a multi-kernel support vector machine classification approach. Finally, we perform the classifier ensemble by combining those multiple SC-PSorter-based classifiers via majority voting. We evaluate our method on a collection of 1636 immunohistochemistry images from the Human Protein Atlas database. The experimental results show that our method achieves an overall accuracy of 89.0%, which is 6.4% higher than the state-of-the-art method. The dataset and code can be downloaded from https://github.com/shaoweinuaa/. dqzhang@nuaa.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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    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

  16. 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

    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.

  17. Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

    Science.gov (United States)

    Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K; Moore, Dirk F; Sleat, David E; Lobel, Peter

    2017-02-01

    Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease

  18. Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants12

    Science.gov (United States)

    Corpas, Francisco J.; Barroso, Juan B.; Carreras, Alfonso; Quirós, Miguel; León, Ana M.; Romero-Puertas, María C.; Esteban, Francisco J.; Valderrama, Raquel; Palma, José M.; Sandalio, Luisa M.; Gómez, Manuel; del Río, Luis A.

    2004-01-01

    The cellular and subcellular localization of endogenous nitric oxide (NO˙) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO˙ was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO˙, with g = 2.05 and aN = 12.8 G, was detected in peroxisomes. By fluorometry, NO˙ was also found in these organelles, and the level measured of NO˙ was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO˙ from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO˙ mg−1 protein min−1; was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO˙ could be involved in the process of senescence of pea leaves. PMID:15347796

  19. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    Science.gov (United States)

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  20. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Zdenka Drastichova; Jiri Novotny

    2012-01-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors.At least some types of Gprotein o subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists.However,the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol.Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of Gq/1 1α protein and to determine their localization in isolated fractions and stability in naive and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G11α protein.We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the Gq/11 protein,which were found to be membrane-bound.Both of these complexes dissociated after prolonged treatment with TRH.Still other Gq/11α protein complexes of lower molecular weight were determined in the cytosol.These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h)hormone treatment.These results support the notion that a portion of Gq/11α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor.At the same time,these findings indicate that the redistribution of Gq/11α is broughtabout by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the Gq/11α protein.

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

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    Arieh Riskin

    2015-10-01

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

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

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    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 affect