Science.gov

Sample records for subcellular distribution

  1. Subcellular distribution of potassium in striated muscles

    SciTech Connect

    Edelmann, L.

    1984-01-01

    Microanalytical experiments have been performed to answer the question whether the main cellular cation, K+, follows the water distribution in the striated muscle cell or whether K+ follows the distribution of negative fixed charges (beta- and gamma-carboxyl groups of aspartic and glutamic acid residues). Subcellular localization of K and/or of the K surrogates Rb, Cs, and Tl has been investigated by the following methods: Chemical precipitation of K with tetraphenylborate. Autoradiography of alkali-metals and Tl in air-dried and frozen-hydrated preparations. TEM visualization of electron dense Cs and Tl in sections of freeze-dried and plastic embedded muscle. X-ray microanalysis of air-dried myofibrils and muscle cryosections. The experiments consistently show that K, Rb, Cs, and Tl do not follow the water distribution but are mainly accumulated in the A band, especially in the marginal regions, and at Z lines. The same sites preferentially accumulate Cs or uranyl cations when sections of freeze-dried, embedded muscle are exposed to these electron microscopic stains. It is concluded that the detected uneven distribution of K, Rb, Cs, and Tl in muscle is neither a freeze-drying artifact nor an embedding artifact and may result from a weak ion binding to the beta- and gamma-carboxyl groups of cellular proteins.

  2. Functional platform for controlled subcellular distribution of carbon nanotubes.

    PubMed

    Serag, Maged F; Kaji, Noritada; Venturelli, Enrica; Okamoto, Yukihiro; Terasaka, Kazuyoshi; Tokeshi, Manabu; Mizukami, Hajime; Braeckmans, Kevin; Bianco, Alberto; Baba, Yoshinobu

    2011-11-22

    As nanoparticles can cross different cellular barriers and access different tissues, control of their uptake and cellular fate presents a functional approach that will be broadly applicable to nanoscale technologies in cell biology. Here we show that the trafficking of single-walled carbon nanotubes (SWCNTs) through various subcellular membranes of the plant cell is facilitated or inhibited by attaching a suitable functional tag and controlling medium components. This enables a unique control over the uptake and the subcellular distribution of SWCNTs and provides a key strategy to promote their cellular elimination to minimize toxicity. Our results also demonstrate that SWCNTs are involved in a carrier-mediated transport (CMT) inside cells; this is a phenomenon that scientists could use to obtain novel molecular insights into CMT, with the potential translation to advances in subcellular nanobiology. PMID:21981659

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26911251

  5. Subcellular distribution of lead in cultured rat hepatocytes

    SciTech Connect

    Mittelstaedt, R.A.; Pounds, J.G.

    1984-10-01

    A clear understanding of the sequence and molecular mechanism of the events involved in lead toxicity is hampered by a lack of information about lead compartmentation within the cell. As part of a continuing effort to identify the mechanism by which lead affects cellular functions, we examined the subcellular distribution of /sup 210/Pb in cultured hepatocytes. The cells were isolated, labeled, homogenized in sucrose-N-((2-hydroxyethyl)piperazine)-N'-2-ethanesulfonic acid buffer, and fractionated into mitochondrial, microsomal, and cytosolic components by differential centrifugation. Complete fractionation of the cells revealed that 71% of the cellular /sup 210/Pb was associated with the mitochondria, 5% with the microsomes, and 24% with the cytosol. A modified, rapid fractionation procedure indicated that 45% of the cellular lead was associated with both the mitochondria and the cytosol and 10% with the microsomes. When the cells were separated into total particulates and cytosol with a single centrifugation, 22% of the /sup 210/Pb was associated with the soluble fraction. The process of homogenization and fractionation of the isolated hepatocytes altered the intracellular distribution of /sup 210/Pb. This experimental approach to studying the localization of lead may be compromised by the redistribution of /sup 210/Pb during the extensive centrifugations and resuspensions required for subcellular fractionation and suggests that the subcellular distribution patterns of /sup 210/Pb obtained by the fractionation of cells reflects the distribution of lead in the homogenate rather than the distribution of /sup 210/Pb in the intact cell.

  6. Tissue and subcellular distribution of CLIC1

    PubMed Central

    Ulmasov, Barbara; Bruno, Jonathan; Woost, Philip G; Edwards, John C

    2007-01-01

    Background CLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides. Results New antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of

  7. Hepatic subcellular distribution of (tritium)T-2 toxin

    SciTech Connect

    Pace, J.G.; Watts, M.R.

    1989-01-01

    Hepatic subcellular distribution of ({sup 3}H)T-2 toxin. The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with ({sup 3}H)T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%, perfusate 38% and liver 7%. Livers were fractionated into mitochondria, endoplasmic reticulum (smooth and rough), plasma membrane and nuclei. Plasma membrane fractions contained 38% of the radiolabel within 5 min, decreasing to <1% at the end of the 120-min perfusion. Smooth endoplasmic reticulum contained 27% of the radiolabel by 5 min and increased to 43% over the 120-min perfusion. The mitochondrial fraction contained 3% of the radiolabel by 30 min and increased to 10% after 120-min perfusion. Label in the nuclear fraction remained constant at 7% from 30 to 120 min. By 15 min, only the parent toxin was detected in the mitochondrial fraction. In the other fractions, radiolabel was associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distribution of radiolabel in liver suggested an immediate association of ({sup 3}H)T-2 with plasma membranes and a subsequent association of toxin and metabolites with endoplasmic reticulum, mitochondria and nuclei, the known sites of action of this toxin.

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

    DOE PAGESBeta

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

    2016-02-25

    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 Cdmore » (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.We find 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.« less

  9. Rabbit β-glucuronidase. Subcellular distribution and immunochemical properties

    PubMed Central

    Dean, Roger T.

    1974-01-01

    1. The subcellular distribution of β-glucuronidase and other hydrolases in rabbit liver was investigated. β-Glucuronidase was found in both microsomal and lysosomal fractions. 2. Multiple forms of β-glucuronidase were present in extracts of microsomal and lysosomal fractions. All forms were common to both fractions. 3. A specific antiserum against β-glucuronidase was raised, and characterized by immunoprecipitation and affinity-chromatography procedures. 4. The immunological identity of the multiple forms in the pure β-glucuronidase preparation, and the immunological identity of the β-glucuronidase complement of lysosomal extracts with that of microsomal extracts, were demonstrated by means of the antiserum. The presence of inactive enzyme in various enzyme preparations was shown. ImagesPLATE 1 PMID:4215419

  10. Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

    PubMed

    Middlebrook, J L; Wong, M D; Ishii, D N; Aronow, L

    1975-01-14

    Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system. PMID:162830

  11. Subcellular distribution and translocation of radionuclides in plants

    SciTech Connect

    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.

  12. Characterization and subcellular distribution of somatogenic receptor in rat liver

    SciTech Connect

    Husman, B.; Andersson, G.; Norstedt, G.; Gustafsson, J.A.

    1985-06-01

    Binding of (/sup 125/I)iodobovine GH ((/sup 125/I)iodo-bGH) to rat liver microsomes and Golgi/endosomal fractions isolated from male and female rats has been characterized. Binding of bGH to a pure somatogenic site was suggested by the finding that 50% inhibition of (/sup 125/I)iodo-bGH binding required 5-130 ng bGH, rGH, or hGH/incubation, while around 500 ng rat PRL/incubation were needed to obtain the same effect. Binding of (/sup 125/I)iodo-bGH to microsomes and Golgi/endosomes was time, temperature, and protein dependent. Maximal specific binding occurred at 15-16 and 15-20 h at 22 C in Golgi and microsomal membranes, respectively. Subcellular distribution studies demonstrated in the Golgi/endosomal fractions compared to the total particulate fraction, while residual microsomes devoid of Golgi/endosomal-derived components were approximately 2-fold enriched. Low levels of somatogenic receptors were detected in lysosome-enriched fractions. Removal of endogenous ligand by treating Golgi/endosomal membranes with 3M MgCl/sub 2/ increased specific binding of bGH about 2- to 3-fold. These results indicate that approximately 50% of specific somatogenic binding sites in the low density fractions represent internalized ligand-receptor complexes. The level of rat liver somatogenic receptors did not show a pronounced sex differentiation; however, an endocrine dependence of somatogenic receptor levels is suggested by the finding that livers from rats in the late stages of pregnancy had a level of somatogenic receptors exceeding that of nonpregnant rats.

  13. Subcellular Distribution of NTL Transcription Factors in Arabidopsis thaliana.

    PubMed

    Liang, Mingwei; Li, Hongjuan; Zhou, Fang; Li, Huiyong; Liu, Jin; Hao, Yi; Wang, Yingdian; Zhao, Heping; Han, Shengcheng

    2015-10-01

    NAC with a transmembrane (TM) motif1-like (NTL) transcription factors, containing three regions: the N-terminal NAC domain (ND), the middle regulation region (RR), and the C-terminal TM domain, belong to the tail-anchored proteins. Although these NTLs play numerous essential roles in plants, their subcellular distribution and the mechanism of translocation into the nucleus (NU) remain unclear. In this study, we found that most of the full-length NTLs were localized in the endoplasmic reticulum (ER), with the exception of NTL11 and NTL5, which were restricted to the NU. Furthermore, we found that NTL11 contains a TM domain, whereas NTL5 does not. The ND of all of the NTLs was responsible for nuclear localization in plants. After truncation of the TM domain, NTL8_NR, NTL10_NR and NTL13_NR localized in the cytoplasm (CT) and NU, and other NTL_NRs were only localized in the NU, suggesting that the RR of NTL8, NTL10 and NTL13 contains some inhibitory region to mask the nuclear localization signal sequence in the ND domain and permit their diffusion between CT and NU. Furthermore, the N-terminus of NTL11 was translocated to the NU, but the C-terminus was degraded in Arabidopsis mesophyll protoplasts. The chimeric construct of NTL11_ND with NTL10_RR and TM domain (11ND-10RT) was localized exclusively in the ER, and not in the NU. However, 10ND-11RT was found mainly in the NU. Our results indicated that the TM domain is essential for NTL targeting the ER and the N-terminal fragment, including ND and RR, is translocated into the NU after activation through proteolytic cleavage events upon stimulation by internal and external environmental signals. PMID:26201836

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

    PubMed

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

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected. PMID:27424751

  15. How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

    PubMed Central

    Swiecicki, Jean-Marie; Thiebaut, Frédéric; Di Pisa, Margherita; Gourdin -Bertin, Simon; Tailhades, Julien; Mansuy, Christelle; Burlina, Fabienne; Chwetzoff, Serge; Trugnan, Germain; Chassaing, Gérard; Lavielle, Solange

    2016-01-01

    Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules. PMID:26839211

  16. Changing expression and subcellular distribution of karyopherins during murine oogenesis.

    PubMed

    Mihalas, Bettina P; Western, Patrick S; Loveland, Kate L; McLaughlin, Eileen A; Holt, Janet E

    2015-12-01

    Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined (Kpna1, Kpna2, Kpna3, Kpna4, Kpna6, Kpna7, Kpnb1, Ipo5 and Xpo1), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1, Kpna2, Kpna4, Kpna6 and Ipo5 and down-regulation of Kpnb1, Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs--KPNA1, KPNA2 and IPO5--displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development. PMID:26399853

  17. Calcium: Some aspects of subcellular accumulation and distribution in milk

    SciTech Connect

    Shappell, N.W.

    1989-01-01

    Distribution and bioavailability of {sup 47}calcium in milk labeled by extrinsic and intrinsic methods was investigated. Milk from Sprague Dawley rats was labeled by both methods, and milk from a cow was labeled by the extrinsic method. Retention of {sup 47}Ca from milks administered to young male Sprague Dawley rats was determined through whole body counting for 6 days after administration of milk. Percent of {sup 47}Ca dose retained was 72% for extrinsically labeled cow milk, 62% for extrinsically labeled rat milk, and 55% for intrinsically labeled rat milk. Samples were fractionated by ultracentrifugation and by gel exclusion chromatography. {sup 47}Calcium distributions in rat milk labeled intrinsically or extrinsically were similar. The majority of {sup 47}Ca was found in a particulate, >30,000 molecular weight fraction. The amount of milk calcium retained by rats appeared to be related to the amount of noncasein micelle-associated calcium. When administered by intraperitoneal injection into rats, {sup 45}Ca specific activity of milk peaked in 60 to 90 minutes. In vitro {sup 45}Ca accumulation was compared in Golgi apparatus and endoplasmic reticulum from liver and mammary gland of lactating Dunkin Hartley guinea pigs. In the presence of ATP, highest accumulation per unit total fraction protein was found in Golgi apparatus (mammary gland 28% of available {sup 45}Ca, liver 11%) while 8% was accumulated by endoplasmic reticulum fractions.

  18. Pendrin gene ablation alters ENaC subcellular distribution and open probability

    PubMed Central

    Pech, Vladimir; Nanami, Masayoshi; Bao, Hui-Fang; Kim, Young Hee; Lazo-Fernandez, Yoskaly; Yue, Qiang; Pham, Truyen D.; Eaton, Douglas C.; Verlander, Jill W.

    2015-01-01

    The present study explored whether the intercalated cell Cl−/HCO3− exchanger pendrin modulates epithelial Na+ channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na+ absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na+ absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability. PMID:25972513

  19. Phosphoenolpyruvate carboxykinase of kidney. Subcellular distribution and response to acid–base changes

    PubMed Central

    Flores, H.; Alleyne, G. A. O.

    1971-01-01

    1. A method for the assay of phosphoenolpyruvate carboxykinase is presented, based on the enzymic determination of the phosphoenolpyruvate produced by the enzyme reaction. 2. The subcellular distribution of phosphoenolpyruvate carboxykinase in the kidney of several animal species resembled the distribution in the liver. 3. The rise in enzyme activity in the kidney cortex of rats made acidotic by feeding with ammonium chloride was not prevented by administration of ethionine or actinomycin. 4. The possibility is suggested that in the kidney acidosis causes activation of an inactive form of the enzyme already present. PMID:5128664

  20. Bioaccumulation, subcellular distribution, and acute effects of chromium in Japanese medaka (Oryzias latipes).

    PubMed

    Li, Lixia; Chen, Hongxing; Bi, Ran; Xie, Lingtian

    2015-11-01

    Chromium (Cr) is an essential element but is toxic to aquatic organisms at elevated concentrations. In the present study, adult Japanese medaka (Oryzias latipes) were exposed to a sublethal hexavalent chromium (Cr(VI)) concentration via dissolved and dietary exposures for 6 d. Various measurements of Cr were made: bioaccumulation in different tissues, subcellular distribution in the liver, effects on antioxidants and acetylcholinesterase (AChE), and Cr-induced lipid peroxidation. The results showed that bioaccumulation increased dramatically in all tested tissues from dissolved exposure but only significantly in the intestine from dietary treatment, implying that dissolved exposure may be predominant for Cr accumulation in medaka. Subcellular distribution revealed that Cr accumulated in the liver was mainly (46%) associated with the heat-stable protein fraction. Among the antioxidants examined, catalase (CAT) responded to dissolved Cr exposure in most tissues whereas superoxide dismutase (SOD) was less responsive. Malondialdehyde concentrations were significantly elevated in most tissues examined in the dissolved Cr-exposed fish, but were only elevated in the liver and intestine in the dietary Cr-exposed fish. The AChE activity in the brain was stimulated by 49% in the dissolved Cr-exposed fish. Reductions in condition factor and gonadosomatic index were also observed. These data help in an understanding of Cr tissue distribution and the acute effects of Cr in Japanese medaka. PMID:26096885

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

    PubMed

    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. PMID:26165995

  2. Comparison of subcellular distribution and chemical forms of cadmium among four soybean cultivars at young seedlings.

    PubMed

    Wang, Peng; Deng, Xiaojuan; Huang, Yian; Fang, Xiaolong; Zhang, Jie; Wan, Haibo; Yang, Cunyi

    2015-12-01

    The hydroponic experiment was carried out to investigate the Cd subcellular distribution and chemical forms in roots and shoots among four soybean seedling cultivars with two Cd treatments. HX3 and GC8, two tolerant and low-grain-Cd-accumulating cultivars, had the lowest Cd concentration in roots and high Cd concentration in shoots, while BX10 and ZH24, two sensitive and high-grain-Cd-accumulating cultivars, had the highest Cd concentration in roots and the lowest Cd concentration in shoots at young seedling stage. Furthermore, the sequence of Cd subcellular distribution in roots at two Cd levels was cell wall (53.4-75.5 %) > soluble fraction (15.8-40.4 %) > organelle fraction (2.0-14.7 %), but in shoots, was soluble fraction (39.3-74.8 %) > cell wall (16.0-52.0 %) > organelle (4.8-19.5 %). BX10 and ZH24 had higher Cd concentration in all subcellular fractions in roots, but HX3 and GC8 had higher Cd concentration of soluble fraction in shoots. The sequence of Cd chemical forms in roots was FNacl (64.1-79.5 %) > FHAC (3.4-21.5 %) > Fd-H2O (3.6-13.0 %) > Fethanol (1.4-21.8) > FHCl (0.3-1.6 %) > Fother (0.2-1.4 %) at two Cd levels but, in shoots, was FNacl (19.7-51.4 %) ≥ FHAC (10.2-31.4 %) ≥ Fd-H2O (8.8-28.2 %) ≥ Fethanol (8.9-38.6 %) > FHCl (0.2-9.6 %) > Fother (2.5-11.2 %). BX10 and ZH24 had higher Cd concentrations in each extracted solutions from roots, but from shoots for GC8 and HX3. Taken together, the results uncover that root cell walls and leaf vacuoles might play important roles in Cd detoxification and limiting the symplastic movement of Cd. PMID:26272289

  3. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    PubMed Central

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels; Westerblad, Håkan

    2014-01-01

    In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz, 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca2+]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P < 0.01, n = 9) and LIF (P < 0.01, n = 5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60–75% lower than in rested control fibres (P < 0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca2+]i and the reduction in intermyofibrillar (P = 0.051) and intramyofibrillar (P = 0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca2+]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool. PMID:24591577

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

    PubMed

    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. PMID:27450963

  5. Tissue, cellular, and subcellular distribution of /sup 241/Pu in the rat testes

    SciTech Connect

    Miller, S.C.; Bowman, B.M.

    1983-05-01

    The distribution and localization of /sup 241/Pu in rat testes were determined by quantitative autoradiography. Rats were given intravenous injection of /sup 241/Pu citrate and tissues were collected 1 week later. The tissue distribution of /sup 241/Pu was determined by light microscope autoradiography. Significant concentrations of /sup 241/Pu were observed in the interstitial tissue but not in seminiferous tubules. The cellular distribution and subcellular localization of /sup 241/Pu were determined by electron microscope autoradiography. Within the interstitial tissue, /sup 241/Pu was concentrated in microphages. There was no preferential localization of /sup 241/Pu in any other interstitial tissue components. Within interstitial tissue macrophages, /sup 241/Pu was concentrated in the lysosomal system of the cell. Other organellar compartments of the cell did not preferentially incorporate /sup 241/Pu. The association of /sup 241/Pu with the macrophage lysosomal system may explain the long retention times of Pu in testes as observed in experimental studies.

  6. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

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

    PubMed Central

    2009-01-01

    Background Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct. PMID:19948032

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

    SciTech Connect

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

    1983-05-01

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

  9. SorLA in glia: shared subcellular distribution patterns with caveolin-1.

    PubMed

    Salgado, Iris K; Serrano, Melissa; García, José O; Martínez, Namyr A; Maldonado, Héctor M; Báez-Pagán, Carlos A; Lasalde-Dominicci, José A; Silva, Walter I

    2012-04-01

    SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer's disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA's expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA's message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA's mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD. PMID:22127416

  10. Subcellular distribution of technetium-99m-N-NOEt in rat myocardium

    SciTech Connect

    Uccelli, L.; Giganti, M.; Duatti, A. ||

    1995-11-01

    The aim of this study was to determine the subcellular distribution of bis(N-ethoxy N-ethyl)dithiocarbamato, nitrido technetium(V) ({sup 99m}TcN-NOEt) in rat heart by differential centrifugation techniques. Extraction of the activity from homogenized rat heart tissue was also performed to assess whether myocardial retention might induce changes in the chemical identity of the complex. Anesthetized rats were intravenously injected with {sup 99m}TcN-NOEt, the heart tissue was extracted and homogenized and tissue fractions were obtained by differential centrifugation. The efficiency of organelle separation was determined by assay of each centrifugal fraction using enzyme markers. Lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and 5{prime}-nucleotidase (5{prime}ND) activities were assayed using standard spectrophotometric methods. Succinic dehydrogenase (SDH) activity was determined using a p-iodo-nitrotetrazolium-linked assay. Severe cell membrane and organelle disruption were induced by prolonging the homogenization time and their effect on the subcellular distribution of {sup 99m}TcN-NOEt was studied. The activity from homogenized heart tissue was extracted using the Folch technique and analyzed by TLC and HPLC. Most of the {sup 99}TcN-NOEt activity was found to be associated with the hydrophobic components of the cell. No evidence of specific association of activity with the cytosolic and mitochondrial components was observed. Organelle and membrane cleavage did not cause release of activity into the cytosol. Approximately 90% of {sup 99m}TcN-NOEt activity was extracted from ventricular tissue and the chemical nature of {sup 99m}TcN-NOEt was not altered by uptake by myocardium. Cell membranes are the most apparent site of localization of {sup 99m}TcN-NOEt in heart tissue. 21 refs., 4 figs., 1 tab.

  11. Subcellular Distribution of Organically Bound Tritium in the Rat Liver After Ingestion of Tritiated Water and Some Tritiated Organic Compounds

    SciTech Connect

    Takeda, H.; Miyamoto, K.; Fuma, S.; Ishii, N.; Yanagisawa, K

    2005-07-15

    Tritiated water and some tritiated organic compounds (leucine, glucose and thymidine) were administered to rats by oral ingestion and the content of organically bound tritium (OBT) in subcellular fractions (cold PCA soluble, ethanol-ether soluble, hot PCA soluble and alkali soluble) of the liver were determined at various time points after ingestion. In the case of tritiated water, the initial OBT content was high in the cold PCA soluble fraction, which contains low molecular weight components, but as the time proceed the OBT was distributed to other fractions, which contains relatively high molecular weight components. Significant time variation in the OBT content was observed in the hot PCA soluble fraction containing nucleic acids, in which the OBT content, expressed as percentage of OBT content in all fractions, changed from 1 % at 12 hours to 15 % at 50 days. In the cases of tritiated organic compounds, the subcellular distribution of OBT was widely changed owing to their biochemical and metabolic characteristics. Thus, the OBT distribution among subcellular fractions was changed depending on the chemical form at ingestion and on the time after ingestion. The OBT distribution among four subcellular fractions after 22 day' continuous ingestion was also dependent on the chemical form of ingested tritium. Present results should be taken into account for internal dose estimation of tritium in different chemical forms.

  12. CHARACTERIZATION OF AMINOPEPTIDASE IN THE FREE-LIVING NEMATODE PANAGRELLUS REDIVIVUS: SUBCELLULAR DISTRIBUTION AND POSSIBLE ROLE IN NEUROPEPTIDE METABOLISM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide (Ala-4-NA). Subcellular distribution of the enzyme was unequal, with approximately 80 percent of total aminopeptidase in the soluble fraction and the rem...

  13. Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants

    PubMed Central

    Vaculík, Marek; Landberg, Tommy; Greger, Maria; Luxová, Miroslava; Stoláriková, Miroslava; Lux, Alexander

    2012-01-01

    Background and Aims Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues. Methods Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of 109Cd applied at 34 nm was investigated between root and shoot. Key Results Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots. Conclusions Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots. PMID:22455991

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

    PubMed

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

    2016-06-01

    Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th. PMID:27010411

  15. Subcellular distributions of metals and metal induced stress: A field study

    SciTech Connect

    Jenkins, K.D.; Howe, S.; Sanders, B.M. )

    1988-09-01

    This paper reports the results of a field study which took place around an exploratory well located in the Santa Barbara Channel. This study was designed to test for significant temporal and spatial differences in the concentrations of a number of drilling fluid-associated metals in both the sediments and biota. Temporal changes in the distribution of Ba, Cd, Cr, Cu, Hg, Ni, Pb, and Zn were examined in the sediments, and the bioaccumulation and subcellular distribution of these metals were examined in three benthic invertebrate species before and after drilling. Statistically significant increases in the accumulation of several of the metals were found in the surface sediments down current from the site after drilling with Ba showing the most pronounced increase. Statistically significant increases in the bioaccumulation of Ba were also observed in two of the three species examined, Cyclocardia ventricosa and Pactinaria californiensis. Within these organisms the majority of the Ba was localized in the granular pellets (>97%) and less than 0.1% accumulated in the cytosol. These data indicate that although bioaccumulation of Ba occurs in some species immediately down current from the well, most of it remains in an insoluble for, presumably as BaSO{sub 4}.

  16. Enhancing near IR luminescence of thiolate Au nanoclusters by thermo treatments and heterogeneous subcellular distributions.

    PubMed

    Conroy, Cecil V; Jiang, Jie; Zhang, Chen; Ahuja, Tarushee; Tang, Zhenghua; Prickett, Cherish A; Yang, Jenny J; Wang, Gangli

    2014-07-01

    A five-to-ten fold enhancement, up to ca. 5-10% quantum efficiency, of near IR luminescence from monothiolate protected gold nanoclusters was achieved by heating in the presence of excess ligand thiols. An emission maximum in the 700-900 nm range makes these Au nanoclusters superior for bioimaging applications over other emissions centered below 650 nm due to reduced background interference, albeit visible emissions could have higher quantum efficiency. The heating procedure is shown to be effective to improve the luminescence of Au nanoclusters synthesized under a variety of conditions using two types of monothiols: mercaptosuccinic acid and tiopronin. Therefore, this heating method is believed to be a generalizable approach to improve the near IR luminescence of aqueous soluble Au nanoclusters, which enables better bioimaging applications. The high quantum yield is found relatively stable over a wide pH range. PEGylation of the Au nanoclusters reduces their quantum efficiency but improves their permeation into the cytoplasm. Interestingly, z-stack confocal analysis clearly reveals the presence of Au nanoclusters inside the cell nucleus in single cell imaging. The finding addresses controversial literature reports and demonstrates the internalization and heterogeneous subcellular distributions, particularly inside the nucleus. The high luminescence intensity, small overall dimension, cell and nuclear distribution, chemical stability and low-to-non toxicity make these Au nanoclusters promising probes for broad cell dynamics and imaging applications. PMID:24879334

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

    PubMed

    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

  18. The subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils.

    PubMed Central

    Segal, A W; Jones, O T

    1979-01-01

    A novel cytochrome b has recently been described in human neutrophils. The subcellular distribution of this cytochrome was investigated by analytical fractionation on continuous sucrose gradients and it was found to have a dual localization, the major component having a similar distribution to the plasma-membrane marker [3H]concanavalin A, and a denser peak located with the specific granules. The two components were separated on discontinuous gradients and studied independently. PMID:496906

  19. Zn Subcellular Distribution in Liver of Goldfish (Carassius Auratus) with Exposure to Zinc Oxide Nanoparticles and Mechanism of Hepatic Detoxification

    PubMed Central

    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 Zn2+. 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 Zn2+, 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 Zn2+ exposure, it increased significantly in the organelles. Metallothionein-like proteins (MTLP) were the main target for Zn2+, 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 Zn2+ were different. Overall, subcellular partitioning provided an interesting start to better understanding of the toxicity of nano- and conventional materials. PMID:24223767

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

    PubMed

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

    2015-02-01

    The subcellular distribution and chemical forms of Cd were investigated in the edible seaweed, Porphyra yezoensis. The seaweed was exposed to different Cd concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 5.0mgl(-1)) for up to 96h. In both the controls (no Cd added) and treatment groups, 41.2-79.2% of Cd was localised in the cell wall, and the proportion of Cd in the cell wall increased with increasing concentrations of Cd and exposure time. In the control groups, 74.8% of Cd was extracted by 1M NaCl, followed by 2% acetic acid, HAC (18.9%). In the treatment groups, most Cd was extracted by 2% HAC. The proportion of Cd extracted by 2% HAC increased with exposure to increasing concentrations of Cd and over time. Cell wall deposition and forming of precipitates with phosphate may be a key strategy to reduce Cd toxicity in P. yezoensis. PMID:25172682

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

    PubMed

    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. PMID:26133699

  2. LRP6 expression promotes cancer cell proliferation and tumorigenesis by altering beta-catenin subcellular distribution.

    PubMed

    Li, Yonghe; Lu, Wenyan; He, Xi; Schwartz, Alan L; Bu, Guojun

    2004-12-01

    The Wnt signaling pathway plays key roles in both embryogenesis and tumorigenesis. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6), a novel member of the expanding LDL receptor family, functions as an indispensable co-receptor for the Wnt signaling pathway. Although the role of LRP6 in embryonic development is now well established, its role in tumorigenesis is unclear. We report that LRP6 is readily expressed at the transcript level in several human cancer cell lines and human malignant tissues. Furthermore, using a retroviral gene transfer system, we find that stable expression of LRP6 in human fibrosarcoma HT1080 cells alters subcellular beta-catenin distribution such that the cytosolic beta-catenin level is significantly increased. This is accompanied by a significant increase in Wnt/beta-catenin signaling and cell proliferation. Finally, we demonstrate that LRP6 expression promotes tumorigenesis in vivo. These results thus indicate that LRP6 may function as a potential oncogenic protein by modulating Wnt/beta-catenin signaling. PMID:15516984

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

    NASA Astrophysics Data System (ADS)

    Nie, Xiaoqin; Dong, Faqin; Liu, Ning; Liu, Mingxue; Zhang, Dong; Kang, Wu; Sun, Shiyong; Zhang, Wei; Yang, Jie

    2015-08-01

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

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

    PubMed

    Nakayama, T; Yaoi, T; Kuwajima, G

    1999-01-01

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

  5. Enhancing near IR luminescence of thiolate Au nanoclusters by thermo treatments and heterogeneous subcellular distributions

    NASA Astrophysics Data System (ADS)

    Conroy, Cecil V.; Jiang, Jie; Zhang, Chen; Ahuja, Tarushee; Tang, Zhenghua; Prickett, Cherish A.; Yang, Jenny J.; Wang, Gangli

    2014-06-01

    A five-to-ten fold enhancement, up to ca. 5-10% quantum efficiency, of near IR luminescence from monothiolate protected gold nanoclusters was achieved by heating in the presence of excess ligand thiols. An emission maximum in the 700-900 nm range makes these Au nanoclusters superior for bioimaging applications over other emissions centered below 650 nm due to reduced background interference, albeit visible emissions could have higher quantum efficiency. The heating procedure is shown to be effective to improve the luminescence of Au nanoclusters synthesized under a variety of conditions using two types of monothiols: mercaptosuccinic acid and tiopronin. Therefore, this heating method is believed to be a generalizable approach to improve the near IR luminescence of aqueous soluble Au nanoclusters, which enables better bioimaging applications. The high quantum yield is found relatively stable over a wide pH range. PEGylation of the Au nanoclusters reduces their quantum efficiency but improves their permeation into the cytoplasm. Interestingly, z-stack confocal analysis clearly reveals the presence of Au nanoclusters inside the cell nucleus in single cell imaging. The finding addresses controversial literature reports and demonstrates the internalization and heterogeneous subcellular distributions, particularly inside the nucleus. The high luminescence intensity, small overall dimension, cell and nuclear distribution, chemical stability and low-to-non toxicity make these Au nanoclusters promising probes for broad cell dynamics and imaging applications.A five-to-ten fold enhancement, up to ca. 5-10% quantum efficiency, of near IR luminescence from monothiolate protected gold nanoclusters was achieved by heating in the presence of excess ligand thiols. An emission maximum in the 700-900 nm range makes these Au nanoclusters superior for bioimaging applications over other emissions centered below 650 nm due to reduced background interference, albeit visible emissions

  6. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

    PubMed Central

    Wilk, Ronit; Hu, Jack; Blotsky, Dmitry; Krause, Henry M.

    2016-01-01

    In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided. PMID:26944682

  7. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.

    PubMed

    Wilk, Ronit; Hu, Jack; Blotsky, Dmitry; Krause, Henry M

    2016-03-01

    In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided. PMID:26944682

  8. Disrupted TSH Receptor Expression in Female Mouse Lung Fibroblasts Alters Subcellular IGF-1 Receptor Distribution.

    PubMed

    Atkins, Stephen J; Lentz, Stephen I; Fernando, Roshini; Smith, Terry J

    2015-12-01

    A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors. PMID:26389690

  9. Subcellular distribution of the prion protein in sickness and in health.

    PubMed

    Godsave, Susan F; Peters, Peter J; Wille, Holger

    2015-09-01

    The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus. PMID:25683509

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

    PubMed

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

    2010-11-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2009-11-15

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

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

    SciTech Connect

    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.

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

    PubMed

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

    2016-06-01

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

  15. Characterizations of bio-accumulations, subcellular distribution and chemical forms of cesium in Brassica juncea, and Vicia faba.

    PubMed

    Fu, Qian; Lai, Jin-long; Tao, Zong-ya; Han, Na; Wu, Guo

    2016-04-01

    We aim to investigate the tolerance and enrichment mechanism of cesium (Cs) in hyperaccumulation plants. In this study, Brassica juncea and Vicia faba were subjected to varying doses of Cs for 21 days to investigate the differences in bio-accumulations, subcellular distribution and chemical forms of Cs in two cultivars by differential centrifugation, and extraction of Cs in different chemical forms, respectively. The results showed that 49.87%-61.08% of the Cs were in the leaf of B. juncea, while in V. faba, 1.58%-79.29% of the Cs was in the root. The translocation factor (TF) arrived 2.79 to 3.71 in B. juncea, while it only reached 0.26 to 0.62 in V. faba. Cs subcellular distribution of the two plants was in sequence as follows: soluble fraction > cell wall > organelles. Cs was more easily distributed to metal-sensitive fractions of V. faba. The inorganic Cs (F-ethanol), and water-soluble Cs (F-dH2O) are the main existing types of Cs in the two plants. In B. juncea, the relative content of inorganic Cs, and organic acids/CsH2PO4 (F-dH2O) were higher than that of V. faba in the stem. This suggests that Cs may induce related transporter gene expression (such as phosphate transporter, organic cation, high affinity nitrate transporter, amino acid permease, etc.) to help the transport of Cs between root to shoot. PMID:26854554

  16. 2-([sup 125]I) iodomelatonin binding sites in rat adrenals: Pharmacological characteristics and subcellular distribution

    SciTech Connect

    Persengiev, S.P. )

    1992-01-01

    Specific binding sites for 2-[[sup 125]I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[[sup 125]I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin > 2-iodomelatonin > melatonin > 5-methoxytryptamine > 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear and mitochondrial subcellular fractions.

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

    PubMed

    Lai, Hung-Yu; Cai, Ming-Cyuan

    2016-01-01

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

  18. Chronic alcohol exposure differentially affects activation of female locus coeruleus neurons and the subcellular distribution of corticotropin releasing factor receptors

    PubMed Central

    Retson, T. A.; Reyes, B.A.; Van Bockstaele, E. J.

    2014-01-01

    Understanding the neurobiological bases for sex differences in alcohol dependence is needed to help guide the development of individualized therapies for alcohol abuse disorders. In the present study, alcohol-induced adaptations in (1) anxiety-like behavior, (2) patterns of c-Fos activation and (3) subcellular distribution of corticotropin releasing factor receptor in locus coeruleus (LC) neurons was investigated in male and female Sprague-Dawley rats that were chronically exposed to ethanol using a liquid diet. Results confirm and extend reports by others showing that chronic ethanol exposure produces an anxiogenic-like response in both male and female subjects. Ethanol-induced sex differences were observed with increased c-Fos expression in LC neurons of female ethanol-treated subjects compared to controls or male subjects. Results also reveal sex differences in the subcellular distribution of the CRFr in LC-noradrenergic neurons with female subjects exposed to ethanol exhibiting a higher frequency of plasmalemmal CRFrs. These adaptations have implications for LC neuronal activity and its neural targets across the sexes. Considering the important role of the LC in ethanol-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis, the present results indicate important sex differences in feed-forward regulation of the HPA axis that may render alcohol dependent females more vulnerable to subsequent stress exposure. PMID:25149913

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

    PubMed

    Zhu, Ying; Gabbiani, Fabrizio

    2016-06-01

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

  20. Non-random subcellular distribution of variant EKLF in erythroid cells

    SciTech Connect

    Quadrini, Karen J.; Gruzglin, Eugenia; Bieker, James J.

    2008-04-15

    EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function.

  1. Non-random subcellular distribution of variant EKLF in erythroid cells.

    PubMed

    Quadrini, Karen J; Gruzglin, Eugenia; Bieker, James J

    2008-04-15

    EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function. PMID:18329016

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    PubMed

    Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity. PMID:22937104

  5. Nano-hydroxyapatite and nano-titanium dioxide exhibit different subcellular distribution and apoptotic profile in human oral epithelium.

    PubMed

    Tay, Chor Yong; Fang, Wanru; Setyawati, Magdiel Inggrid; Chia, Sing Ling; Tan, Kai Soo; Hong, Catherine Hsu Ling; Leong, David Tai

    2014-05-14

    Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2. PMID:24734929

  6. Uptake and subcellular distributions of cadmium and selenium in transplanted aquatic insect larvae.

    PubMed

    Rosabal, Maikel; Ponton, Dominic E; Campbell, Peter G C; Hare, Landis

    2014-11-01

    We transplanted larvae of the phantom midge Chaoborus punctipennis from a lake having lower concentrations of Cd and Se (Lake Dasserat) to a more contaminated lake (Lake Dufault) located near a metal smelter in Rouyn-Noranda, Quebec. Transplanted individuals were held in mesh mesocosms for up to 16 days where they were fed with indigenous contaminated zooplankton. Larval Cd and Se burdens increased over time, and came to equal those measured in indigenous C. punctipennis from contaminated Lake Dufault. Larval Se burdens increased steadily, whereas those of Cd showed an initial lag phase that we explain by a change in the efficiency with which this insect assimilated Cd from its prey. We measured Cd and Se in subcellular fractions and found that larvae sequestered the majority (60%) of the incoming Cd in a detoxified fraction containing metal-binding proteins, whereas a minority of this nonessential metal was in sensitive fractions (20%). In contrast, a much higher proportion of the essential element Se (40%) was apportioned to metabolically active sensitive fractions. Larvae took up equimolar quantities of these elements over the course of the experiment. Likewise, Cd and Se concentrations in wild larvae were equimolar, which suggests that they are exposed to equimolar bioavailable concentrations of these elements in our study lakes. PMID:25268462

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

    SciTech Connect

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

    1986-03-01

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

  8. Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

    PubMed Central

    Skaar, Karin; Korza, Henryk J.; Tarry, Michael; Sekyrova, Petra; Högbom, Martin

    2015-01-01

    Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies. PMID:26218426

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

    PubMed

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

    2016-03-01

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

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

    PubMed

    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. PMID:26452787

  11. Changes in chemical forms, subcellular distribution, and thiol compounds involved in Pb accumulation and detoxification in Athyrium wardii (Hook.).

    PubMed

    Zhao, Li; Li, Tingxuan; Yu, Haiying; Chen, Guangdeng; Zhang, Xizhou; Zheng, Zicheng; Li, Jinxing

    2015-08-01

    Athyrium wardii is one of the dominant plant species flourishing on the Pb-Zn mine tailings in Sichuan Province, China. A greenhouse pot experiment was conducted to evaluate the chemical forms, subcellular distribution, and thiol compounds in A. wardii under different Pb treatments. The results showed that plants of the mining ecotype (ME) of A. wardii were more tolerant to Pb than those of the non-mining ecotype (NME) in spite of accumulation of higher Pb concentrations. The Pb concentrations in shoots and roots of the ME were 3.2∼8.6 times and 3.0∼24.6 times higher than those of the NME, respectively. The ME was more efficient in Pb uptake than the NME. Moreover, 27.8∼39.0% of the total Pb in ME was sodium chloride (NaCl) extractable and 38.0∼48.5% was acetic acid (HAc) extractable, whereas only a minority of total Pb was in ethanol and H2O extractable. In subcellular level, 77.4∼88.8% of total Pb was stored in the cell walls of ME and 9.0∼18.9% in soluble fractions. Increasing Pb concentrations enhanced sequestration of Pb into the cell walls and soluble fractions of ME tissues to protect organelles against Pb. Synthesis of non-protein thiols (NP-SH) and phytochelatins (PCs) in roots of ME significantly enhanced in response to Pb stress, and significant increases in glutathione (GSH) were observed in shoots of ME. Higher levels of NP-SH, GSH, and PCs were observed in roots of the ME comparing with NME, especially under high Pb treatments. The results indicated that Pb was localized mainly in cell wall and soluble fraction of ME plants with low biological activity by cell wall deposition and vacuolar compartmentalization, which might be the important adapted Pb detoxification mechanisms of ME. PMID:25913310

  12. In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1.

    PubMed

    Tilly, N; Olsson, P; Hartman, T; Coderre, J; Makar, M; Malmquist, J; Sjöberg, S; Pettersson, J; Carlsson, J; Glimelius, B

    1996-01-01

    In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when

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

    PubMed

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

    2016-01-01

    Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins. PMID:26304812

  14. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  15. 3H-delta9-tetrahydrocannabinol tissue and subcellular distribution in the central nervous system and tissue distribution in peripheral organs of tolerant and nontolerant dogs.

    PubMed

    Martin, B R; Dewey, W L; Harris, L S; Beckner, J S

    1976-01-01

    Tolerant and nontolerant dogs received one i.v. administration of 0.5 mg/kg of 3H-delta9-tetrahydrocannabinol 30 minutes before they were sacrificed. Plasma, peripheral and brain tissues, as well as subcellular fractions of brain tissues from both treatment groups, were analyzed for radioactivity. Throughout the time period before sacrifice, the plasma concentrations of radioactivity in the tolerant and nontolerant dogs were not significantly different. The percentage of radioactivity in brain and plasma that was due to either unchanged delta9-tetrahydrocannabinol or a major metabolite was the same in each group. Of the radioactivity in brain, 46% was identified as delta9-tetrahydrocannabinol. Regardless of treatment, there was a specific accumulation of radioactivity in adrenals, liver, kidney, heart and pancreas. The only significant differences in radioactivity between tolerant and nontolerant peripheral tissues were found in liver, kidney cortex, heart and lymph nodes. Although all brain areas from tolerant dogs contained less radioactivity than the comparable brain areas from nontolerant animals, only pituitary and putamen were significantly less. There was a specific accumulation of radioactivity in some brain areas that could be associated with behavioral effects. The concentration in cerebellar and cerebral gray was significantly greater than that in white, and there was a marked reduction in the concentration in gray after tolerance developed. The mean percentage of radioactivity in each subcellular fraction was as follows: 23% crude nuclei, 44% mitochondria, 8% cholinergic nerve endings, 7% noncholinergic nerve endings, 2% free mitochondria and 6% synaptic vesicles. The quantity of radioactivity in homogenates of brains from tolerant dogs was 17% less than brains of nontolerant animals, which was merely a reflection of the respective plasma concentrations. The distribution of radioactivity was similar in both groups, although most of the subcellular

  16. Immunocytochemical analysis of the subcellular distribution of ferritin in Imperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant.

    PubMed

    de la Fuente, Vicenta; Rodríguez, Nuria; Amils, Ricardo

    2012-05-01

    Ferritin is of interest at the structural and functional level not only as storage for iron, a critical element, but also as a means to prevent cell damage produced by oxidative stress. The main objective of this work was to confirm by immunocytochemistry the presence and the subcellular distribution of the ferritin detected by Mösbauer spectroscopy in Imperata cylindrica, a plant which accumulates large amounts of iron. The localization of ferritin was performed in epidermal, parenchymal and vascular tissues of shoots and leaves of I. cylindrica. The highest density of immunolabeling in shoots appeared in the intracellular space of cell tissues, near the cell walls and in the cytoplasm. In leaves, ferritin was detected in the proximity of the dense network of the middle lamella of cell walls, following a similar path to that observed in shoots. Immunolabeling was also localized in chloroplasts. The abundance of immunogold labelling in mitochondria for I. cylindrica was rather low, probably because the study dealt with tissues from old plants. These results further expand the localization of ferritin in cell components other than chloroplasts and mitochondria in plants. PMID:21764425

  17. Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica

    USGS Publications Warehouse

    Cain, D.J.; Buchwalter, D.B.; Luoma, S.N.

    2006-01-01

    The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, ke, ranged from 0.20 to 0.24/d (t1/2 ??? 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 ?? 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 ?? 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 ?? 7 ng/g) than in those from the contaminated site (42 ?? 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects. ?? 2006 SETAC.

  18. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

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

    PubMed

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

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii. PMID:21211902

  20. Subcellular distribution, modulation of antioxidant and stress-related genes response to arsenic in Brassica napus L.

    PubMed

    Farooq, Muhammad A; Gill, Rafaqat A; Ali, Basharat; Wang, Jian; Islam, Faisal; Ali, Shafaqat; Zhou, Weijun

    2016-03-01

    Arsenic (As) is an environmental toxin pollutant that affects the numerous physiological processes of plants. In present study, two Brassica napus L. cultivars were subjected to various concentrations (0, 50, 100, and 200 µM) of As for 14 days, plants were examined for As subcellular distribution, photosynthesis parameters, oxidative stress, and ultrastructural changes under As-stress. Differential fraction analysis showed that significant amount of As was accumulated in the cell wall as compared to other organelles. Decline in photosynthetic efficiency under As stress was observed in term of reduced pigment contents and gas exchange parameters. Differential responses of antioxidants at both enzymatic and gene levels to higher As stress were more pronounced in cultivar ZS 758 as compared to Zheda 622. The qRT-PCR analysis showed that heat shock protein 90 (Hsp90) and metallothionein were over-expressed in As stressed B. napus plants. Disorganization of cell structure and the damages in different organelles were some of the obvious variations in cultivar Zheda 622 as compared to ZS 758. PMID:26597736

  1. Dietary toxicity of field-contaminated invertebrates to marine fish: effects of metal doses and subcellular metal distribution.

    PubMed

    Dang, Fei; Rainbow, Philip S; Wang, Wen-Xiong

    2012-09-15

    There is growing awareness of the toxicological effects of metal-contaminated invertebrate diets on the health of fish populations in metal-contaminated habitats, yet the mechanisms underlying metal bioaccumulation and toxicity are complex. In the present study, marine fish Terapon jurbua terepon were fed a commercial diet supplemented with specimens of the polychaete Nereis diversicolor or the clam Scrobicularia plana, collected from four metal-impacted estuaries (Tavy, Restronguet Creek, West Looe, Gannel) in southwest England, as environmentally realistic metal sources. A comparative toxicological evaluation of both invertebrates showed that fish fed S. plana for 21 d exhibited evident mortality compared to those fed N. diversicolor. Furthermore, a spatial effect on mortality was observed. Differences in metal doses rather than subcellular metal distributions between N. diversicolor and S. plana appeared to be the cause of such different mortalities. Partial least squares regression was used to evaluate the statistical relationship between multiple-metal doses and fish mortality, revealing that Pb, Fe, Cd and Zn in field-collected invertebrates co-varied most strongly with the observed mortality. This study provides a step toward exploring the underlying mechanism of dietary toxicity and identifying the potential causality in complex metal mixture exposures in the field. PMID:22579710

  2. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    SciTech Connect

    Czajkowski, C.M.

    1987-01-01

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of M{sub r} = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind ({sup 3}H)flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing ({sup 3}H)flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated.

  3. Computational Approaches to Analyze and Predict Small Molecule Transport and Distribution at Cellular and Subcellular Levels

    PubMed Central

    Ah Min, Kyoung; Zhang, Xinyuan; Yu, Jing-yu; Rosania, Gus R.

    2013-01-01

    Quantitative structure-activity relationship (QSAR) studies and mechanistic mathematical modeling approaches have been independently employed for analyzing and predicting the transport and distribution of small molecule chemical agents in living organisms. Both of these computational approaches have been useful to interpret experiments measuring the transport properties of small molecule chemical agents, in vitro and in vivo. Nevertheless, mechanistic cell-based pharmacokinetic models have been especially useful to guide the design of experiments probing the molecular pathways underlying small molecule transport phenomena. Unlike QSAR models, mechanistic models can be integrated from microscopic to macroscopic levels, to analyze the spatiotemporal dynamics of small molecule chemical agents from intracellular organelles to whole organs, well beyond the experiments and training data sets upon which the models are based. Based on differential equations, mechanistic models can also be integrated with other differential equations-based systems biology models of biochemical networks or signaling pathways. Although the origin and evolution of mathematical modeling approaches aimed at predicting drug transport and distribution has occurred independently from systems biology, we propose that the incorporation of mechanistic cell-based computational models of drug transport and distribution into a systems biology modeling framework is a logical next-step for the advancement of systems pharmacology research. PMID:24218242

  4. Changes in the subcellular distribution of the cytochrome b-245 on stimulation of human neutrophils.

    PubMed Central

    Garcia, R C; Segal, A W

    1984-01-01

    Cytochrome b-245 of neutrophils has a bimodal distribution in sucrose density gradients. The lighter component (d = 1.14) is shown to be associated with the plasma membrane by the similarity between its density and that of markers of this organelle, as well as a parallel increase in the density of the cytochrome and plasma membrane after treatment with digitonin or dimethyl suberimidate. The cytochrome b-245 of monocytes and cytoplasts, the latter produced by the removal of nuclei and granules from neutrophils, was located only in the plasma membrane. The denser peak of cytochrome (d = 1.19), which contained approximately half of the cytochrome b of neutrophils, had a similar density-distribution profile to the specific granules. After hypo-osmotic disruption of this denser material, the cytochrome distributed with the density of membranes, suggesting an original location within the membrane of the intracellular structure. Redistribution of the cytochrome from the granules to the membranes was observed after stimulation of respiratory activity with soluble agents or opsonized particles. This translocation is not responsible for activation of the oxidase system. There was poor agreement between the kinetics of the transfer of cytochromes from the dense component to the membranes, and degranulation of specific-granule contents, suggesting that the cytochrome may be located in another intracellular structure or that its localization becomes further modified after granule fusion. PMID:6721852

  5. Subcellular distribution of key enzymes of lipid metabolism during the euthermia-hibernation-arousal cycle

    PubMed Central

    Suozzi, Anna; Malatesta, Manuela; Zancanaro, Carlo

    2009-01-01

    Mammalian hibernation is a natural, fully reversible hypometabolic state characterized by a drastic reduction of body temperature and metabolic activity, which ensures survival to many species under adverse environmental conditions. During hibernation, many hibernators rely for energy supply almost exclusively on lipid reserves; the shift from carbohydrate to lipid metabolism implies profound rearrangement of the anabolic and catabolic pathways of energetic substrates. However, the structural counterpart of such adaptation is not known. In this study we investigated, by using immunoelectron microscopy, the fine intracellular distribution of two key enzymes involved in lipid metabolism, namely, the fatty acid synthase (FAS) and the long-chain fatty acyl-CoA synthetase (ACSL), in hepatocytes of euthermic, hibernating and arousing hazel dormice. Our results show that the two enzymes are differentially distributed in cellular compartments (cytoplasm, mitochondria and cell nuclei) of hepatocytes during euthermia. Quantitative redistribution of both enzymes among cellular compartments takes place during hibernation and arousal, in accordance with the physiological changes. Interestingly, this redistribution follows different seasonal patterns in cytoplasm, mitochondria and nuclei. In conclusion, our data represent the first quantitative morphological evidence of lipid enzyme distribution in a true hibernator throughout the year cycle, thus providing a structural framework to biochemical changes associated with the hypometabolism of hibernation. PMID:19538638

  6. Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments.

    PubMed

    Brumell, J H; Volchuk, A; Sengelov, H; Borregaard, N; Cieutat, A M; Bainton, D F; Grinstein, S; Klip, A

    1995-12-15

    Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen. PMID:7499863

  7. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes.

    PubMed

    Janski, A M; Cornell, N W

    1980-02-15

    Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1 degrees C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml. PMID:7378059

  8. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

    PubMed Central

    Janski, Alvin M.; Cornell, Neal W.

    1980-01-01

    Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml. PMID:7378059

  9. Characterization of isoform expression and subcellular distribution of MYPT1 in intestinal epithelial cells.

    PubMed

    Zha, Juan-Min; Li, Hua-Shan; Wang, Yi-Tang; Lin, Qian; Tao, Min; He, Wei-Qi

    2016-08-15

    The regulation of intestinal epithelial permeability requires phosphorylation of myosin regulatory light chain (MLC). The phosphorylation status of MLC is regulated by myosin light chain phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (PP1cδ) toward MLC depends on its regulatory subunit (MYPT1). In this study, we revealed the presence of two MYPT1 isoforms, full length and variant 2 in human intestinal (Caco-2) epithelial cells and isolated intestinal epithelial cells (IECs) from mice. In confluent Caco-2 cells, MYPT1 was distributed at cell-cell contacts and colocalized with F-actin. These results suggest that MYPT1 isoforms are expressed in intestinal epithelial cells and MYPT1 may be involved in the regulation of intestinal epithelial barrier function. PMID:27129938

  10. Intracellular Trafficking and Subcellular Distribution of a Large Array of HPMA Copolymers

    PubMed Central

    Callahan, Jon; Kopečková, Pavla; Kopeček, Jindřich

    2011-01-01

    The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel “ladders” consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import. PMID:21197960

  11. Intracellular trafficking and subcellular distribution of a large array of HPMA copolymers.

    PubMed

    Callahan, Jon; Kopečkov, Pavla; Kopeček, Jindřich

    2009-07-13

    The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel "ladders" consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import. PMID:21197960

  12. Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training

    PubMed Central

    Bradley, Helen; Shaw, Christopher S.; Worthington, Philip L.; Shepherd, Sam O.; Cocks, Matthew; Wagenmakers, Anton J. M.

    2014-01-01

    Abstract Increases in insulin‐mediated glucose uptake following endurance training (ET) and sprint interval training (SIT) have in part been attributed to concomitant increases in glucose transporter 4 (GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 μm) and smaller (<1 μm) GLUT4‐containing clusters. The large clusters likely represent trans‐Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 μm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes. PMID:25052490

  13. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  14. Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution.

    PubMed

    Jayakumar, Arumugam; Kang, Ya'an; Henderson, Ying; Mitsudo, Kenji; Liu, Xiaoling; Briggs, Katrina; Wang, Mary; Frederick, Mitchell J; El-Naggar, Adel K; Bebök, Zsuzsa; Clayman, Gary L

    2005-03-01

    The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion. PMID:15680911

  15. Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer.

    PubMed

    Orio, Julie; Bellard, Elisabeth; Baaziz, Houda; Pichon, Chantal; Mouritzen, Peter; Rols, Marie-Pierre; Teissié, Justin; Golzio, Muriel; Chabot, Sophie

    2013-01-01

    Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets. PMID:23946765

  16. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

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

    PubMed Central

    Mishra, Seema; Alfeld, Matthias; Sobotka, Roman; Andresen, Elisa; Falkenberg, Gerald; Küpper, Hendrik

    2016-01-01

    Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis. PMID:27340233

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

    PubMed

    Mishra, Seema; Alfeld, Matthias; Sobotka, Roman; Andresen, Elisa; Falkenberg, Gerald; Küpper, Hendrik

    2016-08-01

    Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis. PMID:27340233

  19. Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

    PubMed Central

    Shepherd, Malcolm; McSorley, Theresa; Olsen, Aileen E; Johnston, Lee Ann; Thomson, Neil C; Baillie, George S; Houslay, Miles D; Bolger, Graeme B

    2003-01-01

    We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types. PMID:12441002

  20. Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin.

    PubMed

    Xu, Ji; Chai, Hua; Ehinger, Konstantin; Egan, Terrance M; Srinivasan, Rahul; Frick, Manfred; Khakh, Baljit S

    2014-07-01

    P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs

  1. Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin

    PubMed Central

    Xu, Ji; Chai, Hua; Ehinger, Konstantin; Egan, Terrance M.; Srinivasan, Rahul; Frick, Manfred

    2014-01-01

    P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs

  2. Uptake of injected 125I-ricin by rat liver in vivo. Subcellular distribution and characterization of the internalized ligand.

    PubMed Central

    Frénoy, J P; Turpin, E; Janicot, M; Gehin-Fouque, F; Desbuquois, B

    1992-01-01

    Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures

  3. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin

    PubMed Central

    Lai, Hongyu; Xu, Yuan; Shiao, Frank; Huang, Nanxi; Li, Linpei; Lee, Ming-Shyue; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases. PMID:26171609

  4. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

    PubMed

    Lai, Ying-Jung J; Chang, Hsiang-Hua D; Lai, Hongyu; Xu, Yuan; Shiao, Frank; Huang, Nanxi; Li, Linpei; Lee, Ming-Shyue; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases. PMID:26171609

  5. Subcellular distribution of cadmium in two aquatic invertebrates: change over time and relationship to Cd assimilation and loss by a predatory insect.

    PubMed

    Dubois, Maï'tée; Hare, Landis

    2009-01-15

    We set out to determine if the efficiency of cadmium (Cd) assimilation and loss by a freshwater predator (the alderfly Sialis velata) differs when it is exposed, for various lengths of time, to Cd in either an insect (Chironomus riparius) or a worm (Tubifex tubifex). Prey were exposed to Cd in sediments for up to 28 days and then fractionated to measure Cd distributions in their cells. Cadmium subcellular distributions varied little over time for a given preytype but differed substantially between the two prey species; for example, the cytosol comprised a larger proportion of Cd in the insect (76%) than in the worm (34%). The predator assimilated proportionally more Cd from the insect (72%) than from the worm (46%) and these assimilation efficiencies were similar to the proportion of prey Cd that would theoretically be available to it (cytosolic Cd + organelle Cd). However, measurements of Cd in the predator's feces confirmed that to obtain an exact 1:1 relationship between predator assimilation efficiency and prey subcellular distribution we had to assume that approximately 50% of the Cd associated with the organelle fraction of T. tubifex was unavailable for digestion by the predator. Losses of Cd from the predator also varied depending on the type of prey that were the source of its Cd. PMID:19238964

  6. High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots1[C][W][OA

    PubMed Central

    Moore, Katie L.; Schröder, Markus; Wu, Zhongchang; Martin, Barry G.H.; Hawes, Chris R.; McGrath, Steve P.; Hawkesford, Malcolm J.; Feng Ma, Jian; Zhao, Fang-Jie; Grovenor, Chris R.M.

    2011-01-01

    Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters. PMID:21490163

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

    PubMed Central

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

    2015-01-01

    Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1–296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs. PMID:26359114

  8. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

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

    PubMed

    Lapointe, Dominique; Couture, Patrice

    2009-10-01

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

  10. Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells.

    PubMed

    Lentz, Michael R; Shideler, Tess

    2016-01-01

    The bovine papillomavirus E1 helicase is essential for viral replication. In dividing cells, DNA replication maintains, but does not increase, the viral genome copy number. Replication is limited by low E1 expression and an E1 nucleocytoplasmic shuttling mechanism. Shuttling is controlled in part by phosphorylation of E1 by cellular kinases. Here we investigate conserved sites for phosphorylation by kinase CK2 within the E1 nuclear localization signal. When these CK2 sites are mutated to either alanine or aspartic acid, no change in replication phenotype is observed, and there is no effect on the subcellular distribution of E1, which remains primarily nuclear. This demonstrates that phosphorylation of E1 by CK2 at these sites is not a factor in regulating viral DNA replication in dividing cells. PMID:26467928

  11. Identification and subcellular distribution of the Gi-proteins in the enterocytic-differentiated adenocarcinoma cell-line, Caco-2.

    PubMed

    Lacombe, C; Viallard, V; Schaak, S; Paris, H

    1996-01-01

    As evidenced by pertussis toxin-catalysed [32P]ADP-ribosylation, immunoblotting and Northern blot, the human adenocarcinoma intestinal cell line Caco-2 expresses Gi2 and Gi3 proteins. The localization of these two Gis within the cell was investigated by using subcellular fractionation and confocal microscopy on intact cell layer. A brush-border rich fraction and a pellet containing the remaining cellular membranes were prepared. [32P]ADP-ribosylation and immunoblotting demonstrated the presence of both alpha i2 and alpha i3 in these two preparations. Immunofluorescence studies performed on intact cells grown on Transwell filters and viewed by confocal microscopy further confirmed the localization of alpha i3-subunit on basolateral as well as on apical membranes. In contrast, alpha i2-subunit was shown to accumulate mainly in the intra-cellular compartment while only faint staining of the plasma membrane was detectable. Based upon double-labelling experiments with antibody against rough endoplasmic reticulum (RER), there is a strong possibility that intra-cellular sites of alpha i2-subunit correspond to association with RER membranes. PMID:9237368

  12. Subcellular distribution of ( sup 3 H)-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1991-01-01

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

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

    PubMed Central

    Koukourakis, Michael I.; Kalamida, Dimitra; Giatromanolaki, Alexandra; Zois, Christos E.; Sivridis, Efthimios; Pouliliou, Stamatia; Mitrakas, Achilleas; Gatter, Kevin C.; Harris, Adrian L.

    2015-01-01

    LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies. PMID:26378792

  14. Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by L-DOPA treatment

    PubMed Central

    Ahmed, M. Rafiuddin; Bychkov, Evgeny; Gurevich, Vsevolod V.; Benovic, Jeffrey L.; Gurevich, Eugenia V.

    2009-01-01

    Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson’s disease pathology and L-DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. L-DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D2/D3 agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by L-DOPA but not by DA depletion alone. Similarly, L-DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by L-DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson’s disease. Thus, these proteins may be targets for antiparkinsonian therapy. PMID:17996024

  15. A form of DISC1 enriched in nucleus: Altered subcellular distribution in orbitofrontal cortex in psychosis and substance/alcohol abuse

    PubMed Central

    Sawamura, Naoya; Sawamura-Yamamoto, Takako; Ozeki, Yuji; Ross, Christopher A.; Sawa, Akira

    2005-01-01

    Disrupted-In-Schizophrenia 1 (DISC1) was identified as the sole gene whose ORF is truncated and cosegregates with major mental illnesses in a Scottish family. DISC1 has also been suggested, by association and linkage studies, to be a susceptibility gene for schizophrenia (SZ) in independent populations. However, no analysis of DISC1 protein in human brains, especially those of patients with SZ, has yet been conducted. Here we performed a biochemical analysis of DISC1 protein in a well characterized set of autopsied brains, including brains of patients with SZ, bipolar disorder, and major depression (MD), as well as normal control brains. We identified an isoform of DISC1 by using MS and demonstrated that it is enriched in the nucleus of HeLa cells. In the orbitofrontal cortex, the subcellular distribution of this DISC1 isoform, assessed by the nuclear to cytoplasmic ratio in the immunoreactivity of the isoform, is significantly changed in brains from patients with SZ and MD. This altered distribution is also observed in those subjects with substance and alcohol abuse. The changes in MD brains are significantly influenced by substance/alcohol abuse as well as postmortem interval; however, the alteration in SZ brains is free from brain-associated confounding factors, although an interaction with substance/alcohol abuse cannot be completely ruled out. These results suggest that DISC1 may be implicated in psychiatric conditions in other populations than the unique Scottish family. PMID:15657124

  16. Subcellular and subnuclear distribution of high-light responsive serine/arginine-rich proteins, atSR45a and atSR30, in Arabidopsis thaliana.

    PubMed

    Mori, Tatsuya; Yoshimura, Kazuya; Nosaka, Ryota; Sakuyama, Harumi; Koike, Yoshiyuki; Tanabe, Noriaki; Maruta, Takanori; Tamoi, Masahiro; Shigeoka, Shigeru

    2012-01-01

    Here, we demonstrated the involvement of the domains in Arabidopsis high-light responsive serine/arginine-rich (SR) and SR-like proteins, atSR30 and atSR45a, respectively, in subcellular and subnuclear distribution using a series of structural domain-deleted mutants. Judging from the localization of the transiently expressed domain-deleted mutants in onion epidermal cells, the C terminal low complexity domain rich in arginine-serine repeats (C-RS) domain of atSR30 appeared to be necessary for the nuclear localization. On the other hand, the N-terminal RS (N-RS) domain of atSR45a was necessary for the accurate nuclear localization, although the N- or C-RS domain alone was sufficient for the nuclear speckled organization. The phosphorylation of RS domains of atSR45a is irrelevant to the regulation of its localization. atSR45a and atSR30 were co-localized in the speckles, suggesting their collaborative roles in the regulation of alternative splicing events. PMID:23132568

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

    PubMed

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

    2015-07-01

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

  18. Subcellular distributions of lipids in cultured BHK cells: evidence for the enrichment of lysobisphosphatidic acid and neutral lipids in lysosomes.

    PubMed

    Brotherus, J; Renkonen, O

    1977-03-01

    Homogenates of cultured hamster fibroblasts (BHK 21 cells) were fractionated by differential centrifugation into six main fractions: nuclear, mitochondrial, light mitochondrial, microsomal, soluble, and floating. The contents of several lipids and some marker enzymes were measured. According to the enzyme distributions, lysosomes were enriched both in the floating fraction and in the light mitochondrial fraction. Lysobisphosphatidic acid was enriched in the floating fraction more than tenfold relative to phospholipid. Cholesteryl esters and triglycerides were the main constituents of the fraction (70% of total lipids). Lysobisphosphatidic acid, triglycerides, and cholesteryl esters were enriched also in the light mitochondrial fraction. Their distribution patterns were different from those of the other lipids. Electron microscopy showed that the floating fraction contained numerous lipofuscin-like particles with darkly stained peripheries and with core regions staining like droplets of neutral lipids. Similar particles, frequently containing prominent multilamellar formations, were also common in intact cells. They contained cytochemically identified acid phosphatase. We conclude that lysobisphosphatidic acid was enriched in the lysosomes of the BHK cells and that the lysosomes also contained variable amounts of neutral lipids in the form of intralysosomal droplets. PMID:845501

  19. Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue.

    PubMed

    Zhao, Hua; Ruberu, Kalani; Li, Hongyun; Garner, Brett

    2013-07-15

    Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., age-related lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [57Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [57Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1-5), which were clearly separated from mitochondria (fractions 7-9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [57Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [57Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting. PMID:23608310

  20. Ultrastructure and subcellular distribution of Cr in Iris pseudacorus L. using TEM and X-ray microanalysis.

    PubMed

    Caldelas, Cristina; Bort, Jordi; Febrero, Anna

    2012-02-01

    Chromium pollution of freshwater is hazardous for humans and other organisms, and places a limitation on the use of polluted water sources. Phytoremediation, the use of plants to remove pollutants from the environment, is a cost-effective, environmentally friendly approach for water decontamination. To improve the efficiency of the process, it is essential to increase the current knowledge about Cr accumulation in macrophytes. Plants of Iris pseudacorus L. were treated with Cr(III) at 0.75 mM for 5 weeks to investigate Cr localization by means of transmission electron microscopy and energy dispersive X-ray analysis. Chromium induced severe ultrastructural alterations in the rhizodermis (cell wall disorganisation, thickening, plasmolysis, and electron-dense inclusions) and rhizome parenchyma (reduced cell size, cell wall detachment, vacuolation, and opaque granules). The highest Cr contents were found in the cell walls of the cortex in the roots and in the cytoplasm and intercellular spaces of the rhizome. The Cr concentration in root tissues was in the order cortex>rhizodermis>stele, whereas in the rhizome, Cr was evenly distributed. It is proposed that root and rhizome have distinct functions in the response of I. pseudacorus to Cr. The rhizodermis limits Cr uptake by means of Si deposition and cell wall thickening. The rhizome cortex generates vacuoles and granules where Cr co-occurs with S, indicating Cr sequestration by metal-binding proteins. PMID:22009188

  1. The isoenzymes of carbonic anhydrase: tissue, subcellular distribution and functional significance, with particular reference to the intestinal tract

    PubMed Central

    Carter, M. J.; Parsons, D. S.

    1971-01-01

    1. The total carbonic anhydrase activity in some guinea-pig tissues has been measured using a pH-stat procedure. Stomach, gall bladder, proximal colon and caecum all possess more carbonic anhydrase activity per unit amount of protein than does whole blood. 2. The carbonic anhydrase activity of the small intestine is low. Reasons are given for supposing that activity found there is not entirely due to contamination by whole blood, and it is suggested that in this tissue the enzyme may be localized in some cell type other than the columnar absorbing cells. 3. Evidence is presented which indicates that heavy metals interfere with the activity of the enzyme as measured in tissue homogenates. 4. The distribution and concentration of the two major isoenzymes of carbonic anhydrase have been measured in different tissues. Blood and proximal colon contain both isoenzymes in comparable concentrations, the ratio of the concentration of the `low activity' isoenzyme to that of the `high activity' being about 2. The gastric mucosa contains much `high activity' carbonic anhydrase, but only a negligible amount of the `low activity' isoenzyme. In the caecal mucosa, the `low activity' isoenzyme is predominant, the ratio of its concentration to that of the `high activity' isoenzyme being about 9. It is also found that more than 1·5% of the protein in the caecal mucosa is accounted for as carbonic anhydrase enzymes. 5. It is found that some 45% of the total carbonic anhydrase activity of sucrose homogenates of the guinea-pig colon is bound to particles. The activity is located mainly in the nuclear and microvillous fraction and in the `high-speed supernatant' fraction. The form of enzyme bound is largely of the `high activity' variety. When the tissue is homogenized in potassium chloride solutions less than 4% of the total activity is recovered in particulate fractions. The amount of activity which is bound to particulate fractions increases as the ionic strength or pH of the

  2. Subcellular Localization and Clues for the Function of the HetN Factor Influencing Heterocyst Distribution in Anabaena sp. Strain PCC 7120

    PubMed Central

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J.; Elhai, Jeff; Mullineaux, Conrad W.; Flores, Enrique

    2014-01-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide. PMID:25049089

  3. Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120.

    PubMed

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J; Elhai, Jeff; Mullineaux, Conrad W; Flores, Enrique; Herrero, Antonia

    2014-10-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide. PMID:25049089

  4. Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure.

    PubMed

    Kentala, Henriikka; Pfisterer, Simon G; Olkkonen, Vesa M; Weber-Boyvat, Marion

    2015-07-01

    Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs). We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA). Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP-VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP-VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)-VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2-VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L-VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)-VAPA localized predominantly to the plasma membrane. In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP-VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces. PMID:25681634

  5. Prenatal exposure to cocaine produces unique developmental and long-term adaptive changes in dopamine D1 receptor activity and subcellular distribution.

    PubMed

    Stanwood, Gregg D; Levitt, Pat

    2007-01-01

    Low-dose intravenous cocaine administration to pregnant rabbits causes permanent structural alterations in dopamine-rich cerebral cortical areas, substantially reduced dopamine D1 receptor coupling to G(s)-protein, and deficits in cognitive function. The developmental influences of reduced D1-G(s) coupling and the underlying cellular basis are unknown. Using primary neuronal cultures derived from the medial frontal cortex and striatum of in utero saline- and cocaine-exposed embryos, spontaneous neurite outgrowth of in utero-exposed cortical neurons was greater than in control neurons. In contrast, striatal neurons exposed to cocaine in utero exhibited an entirely opposite adaptive response, with diminished spontaneous neurite outgrowth compared with saline-exposed controls. Control neurons isolated from the two structures also exhibited opposite regulatory responses to the D1 receptor agonist SKF38393 (1-phenyl-2,3,4-5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride), inhibiting outgrowth in cortical cultures and stimulating outgrowth in striatal cultures. The agonist was ineffective in modulating neurite outgrowth of neurons from either structure isolated from cocaine-exposed fetuses, reflecting the reduced D1-Gs coupling. Total D1 receptor number was indistinguishable in neurons from the cocaine- and saline-exposed animals, but cell imaging and receptor binding of differentially isolated membranes showed that the lack of responsiveness was because of greatly reduced cell-surface localization of D1 receptors. These data suggest that prenatal exposure to cocaine causes a novel, long-lasting adaptive response in the subcellular distribution of D1 receptors, resulting in alterations in signaling capacity that have developmental and behavioral consequences. PMID:17202482

  6. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    SciTech Connect

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  7. Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver.

    PubMed

    Styles, Nathan A; Falany, Josie L; Barnes, Stephen; Falany, Charles N

    2007-06-01

    Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT. PMID:17379925

  8. Differential sub-cellular distribution and co-localization of the microsomal (mEH) and soluble epoxide hydrolases (sEH) in cultured neonatal rat brain cortical astrocytes

    PubMed Central

    Rawal, Seema; Morisseau, Christophe; Hammock, Bruce D.; Shivachar, Amruthesh C

    2013-01-01

    The microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) enzymes exist in a variety of cells and tissues, including liver, kidney and testis. However, very little is known about brain epoxide hydrolases. Here we report the expression, localization and subcellular distribution of mEH and sEH in cultured neonatal rat cortical astrocytes by immunocytochemistry, subcellular fractionation, western blotting and radiometric enzyme assays. Our results showed a diffused immunofluorescence pattern for mEH, which co-localized with the astroglial cytoskeletal marker, glial fibrillary acidic protein (GFAP). The GFAP-positive cells also expressed sEH which was mainly localized in the cytoplasm especially in and around the nucleus. Western blot analyses, revealed a distinct protein band with a molecular mass of ~50 kDa, the signal intensity of which increased about 1.5-fold in the microsomal fraction over the whole cell lysate and other subcellular fractions. The polyclonal anti-human sEH rabbit serum recognized a protein band with a molecular mass similar to that of purified sEH protein (~62 kDa), and the signal intensity increased 1.7-fold in the 105,000×g supernatant fraction over the cell lysate. Although the corresponding mEH enzyme activities generally corroborated with the immunocytochemical and western blotting data a low sEH enzyme activity was detected especially in the total cell lysate and in the soluble fractions. These results suggest that rat brain cortical astrocytes differentially co-express mEH and sEH enzymes. The differential subcellular localization of mEH and sEH may play a role in the cerebrovascular functions that are known to be affected by brain-derived vasoactive epoxides. PMID:18711743

  9. Variation in cadmium accumulation among 30 cultivars and cadmium subcellular distribution in 2 selected cultivars of water spinach (Ipomoea aquatica Forsk.).

    PubMed

    Wang, Junli; Yuan, Jiangang; Yang, Zhongyi; Huang, Baifei; Zhou, Yihui; Xin, Junliang; Gong, Yulian; Yu, Hui

    2009-10-14

    To reduce the influx of cadmium (Cd), a toxic heavy metal, into the human food chain through vegetable intake, a pot experiment for the selection of a pollution-safe cultivar (PSC) of water spinach (Ipomoea aquatica Forsk.) was carried out. The experiment with 30 tested cultivars revealed that the maximum differences in Cd concentration between the cultivars containing the highest and the lowest Cd were 3.0-3.9-fold under low-Cd treatment (soil Cd = 0.593 mg kg(-1)), 2.7-3.5-fold under middle-Cd treatment (soil Cd = 1.091 mg kg(-1)), and 2.6-2.7-fold under high-Cd treatment (soil Cd = 1.824 mg kg(-1)), large enough to define the Cd-PSCs. Concentrations of Cd in edible parts of six cultivars, cv. Daxingbaigu, Huifengqing, Qiangkunbaigu, Qiangkunqinggu, Shenniuliuye, and Xingtianqinggu, were lower than 0.2 mg kg(-1), the maximum level (ML) of Cd allowed by the Codex Alimentarius Commission (CAC) standard, even under middle-Cd treatment. Accordingly, these cultivars were treated as typical Cd-PSCs. Four cultivars, cv. Jieyangbaigeng, Xianggangdaye, Sannongbaigeng, and Taiwan 308, contained Cd in edible parts exceeding the ML even under low-Cd treatment, and they were defined as typical non-Cd-PSCs. The correlations of the Cd concentrations among the tested cultivars between the three treatments were significant at the p < 0.05 level. A conspicuous difference in Cd subcellular distribution in hydroponic plant tissues between cv. Qiangkunqinggu (a typical Cd-PSC) and cv. Taiwan 308 (a typical non-Cd-PSC) were observed. Cd absorbed by cv. Qiangkunqinggu seemed to be well-compartmentalized in root and in cell wall fragment, which may be one of the mechanisms leading to its low Cd accumulating property. The results indicated that water spinach, a leafy vegetable, could be easily polluted by soils contaminated with Cd, as 80% of the tested cultivars had exceeded the ML of Cd according to the CAC standard even under the middle-Cd treatment. Much of the evidence obtained from

  10. Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

    PubMed

    Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

    1994-08-01

    As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear

  11. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

    PubMed

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division. PMID:26654596

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

    PubMed

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

    2014-02-01

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

  13. Adaptations of proteins to cellular and subcellular pH

    PubMed Central

    2009-01-01

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

  14. Adaptations of proteins to cellular and subcellular pH.

    PubMed

    Garcia-Moreno, Bertrand

    2009-01-01

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

  15. Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips.

    PubMed

    Wang, Qiaochun; Cuellar, Wilmer J; Rajamäki, Minna-Liisa; Hirata, Yukimasa; Valkonen, Jari P T

    2008-03-01

    Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult. PMID:18705855

  16. Apical Membrane Localization of the Adenomatous Polyposis Coli Tumor Suppressor Protein and Subcellular Distribution of the β-Catenin Destruction Complex in Polarized Epithelial Cells

    PubMed Central

    Reinacher-Schick, Anke; Gumbiner, Barry M.

    2001-01-01

    The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of β-catenin as part of a high molecular weight complex known as the β-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or β-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3β and β-catenin. Therefore, it is likely to correspond to the previously characterized β-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of β-catenin, or alternatively, whether they could be involved in other functions of the protein that

  17. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  18. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-01-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised. PMID:26621372

  19. Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation.

    PubMed

    Kurusu, Mitsuhiko; Katsuki, Takeo; Zinn, Kai; Suzuki, Emiko

    2012-06-15

    Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir(2.1) indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons. PMID:22542600

  20. Alpha-adrenoceptors in dog mesenteric vessels--subcellular distribution and number of ( sup 3 H)prazosin and ( sup 3 H)rauwolscine binding sites

    SciTech Connect

    Shi, A.G.; Ahmad, S.; Kwan, C.Y.; Daniel, E.E. )

    1990-04-01

    Binding of the alpha-adrenergic antagonists ({sup 3}H)prazosin and ({sup 3}H)rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 +/- 0.1 nM for ({sup 3}H)prazosin and 5.85 +/- 0.85 nM for ({sup 3}H)rauwolscine in arteries, and 0.87 +/- 0.4 nM for ({sup 3}H)prazosin and 6.6 +/- 1.5 nM for ({sup 3}H)rauwolscine in veins. In veins, the maximum number of binding sites for ({sup 3}H)rauwolscine was higher than that for ({sup 3}H)prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for ({sup 3}H)prazosin was higher than that for ({sup 3}H)rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of ({sup 3}H)saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on alpha 2 adrenoceptors. This study thus identified two distinct populations of ({sup 3}H)prazosin and ({sup 3}H)rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of alpha 2-compared to alpha 1-adrenoceptor binding sites is required for a contractile response.

  1. Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I1- and I2-imidazoline drugs.

    PubMed

    Keller, Benjamin; García-Sevilla, Jesús A

    2015-09-01

    Various imidazoline receptor (IR) proteins have been proposed to mediate the effects of selective I1- and I2-IR drugs. However, the association of these IR-binding proteins with classic I1- and I2-radioligand binding sites remains somewhat controversial. In this study, three IR antibodies (anti-NISCH and anti-nischarin for I1-IRs; and anti-IRBP for I1/I2-IRs) were used to immunodetect, characterize and compare IR protein patterns in brain (mouse and human; total homogenate, subcellular fractionation, grey and white matter) and some cell systems (neurones, astrocytes, human platelets). Various immunoreactive IRs (specific molecular weight bands coincidently detected with the different antibodies) were related to I1-IR (167 kDa, 105/115 kDa and 85 kDa proteins) or I2-IR (66 kDa, 45 kDa and 30 kDa proteins) types. The biochemical characterization of cortical 167 kDa protein, localized in the membrane/cytosol but not in the nucleus, indicated that this I1-IR also forms part of higher order nischarin-related complexes. The contents of I1-IR (167 kDa, 105/115 kDa, and 85 kDa) proteins in mouse brain cortex were upregulated by treatment with I1-drugs (moxonidine, efaroxan) but not with I2-drugs (BU-224, LSL 61122). Conversely, the contents of I2-IR (66 kDa, 45 kDa and 30 kDa) proteins in mouse brain cortex were modulated by treatment with I2-drugs (decreases after BU-224 and LSL 61122, and increases after idazoxan) but not with I1-drugs (with the exception of moxonidine). These findings further indicate that brain immunoreactive IR proteins exist in multiple forms that can be grouped in the already known I1- and I2-IR types, which are expressed both in neurones and astrocytes. PMID:26038110

  2. Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

    PubMed

    Gallmetzer, Andreas; Silvestrini, Lucia; Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A; Scazzocchio, Claudio; Strauss, Joseph

    2015-07-01

    The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

  3. Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

    PubMed Central

    Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A.; Scazzocchio, Claudio; Strauss, Joseph

    2015-01-01

    The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

  4. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  5. Alterations in the subcellular distribution of NADPH oxidase p47(phox) in hypothalamic paraventricular neurons following slow-pressor angiotensin II hypertension in female mice with accelerated ovarian failure.

    PubMed

    Van Kempen, Tracey A; Narayan, Ankita; Waters, Elizabeth M; Marques-Lopes, Jose; Iadecola, Costantino; Glass, Michael J; Pickel, Virginia M; Milner, Teresa A

    2016-08-01

    At younger ages, women have a lower risk for hypertension than men, but this sexual dimorphism declines with the onset of menopause. These differences are paralleled in rodents following "slow-pressor" angiotensin II (AngII) administration: young male and aged female mice, but not young females, develop hypertension. There is also an established sexual dimorphism both in the cardiovascular response to the neurohypophyseal hormone arginine vasopressin (AVP) and in the expression of oxidative stress. We examined the relationship between AngII-mediated hypertension and the cellular distribution of the superoxide generating NADPH oxidase (NOX) in AVP-expressing hypothalamic paraventricular nucleus (PVN) neurons in "menopausal" female mice. Dual-labeling immunoelectron microscopy was used to determine whether the subcellular distribution of the organizer/adapter NOX p47(phox) subunit is altered in PVN dendrites following AngII administered (14 days) during the "postmenopausal" stage of accelerated ovarian failure (AOF) in young female mice treated with 4-vinylcyclohexene diepoxide. Slow-pressor AngII elevated blood pressure in AOF females and induced a significant increase in near plasmalemmal p47(phox) and a decrease in cytoplasmic p47(phox) in PVN AVP dendrites. These changes are the opposite of those observed in AngII-induced hypertensive male mice (Coleman et al. [2013] J. Neurosci. 33:4308-4316) and may be ascribed in part to baseline differences between young females and males in the near plasmalemmal p47(phox) on AVP dendrites seen in the present study. These findings highlight fundamental differences in the neural substrates of oxidative stress in the PVN associated with AngII hypertension in postmenopausal females compared with males. J. Comp. Neurol. 524:2251-2265, 2016. © 2015 Wiley Periodicals, Inc. PMID:26659944

  6. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    SciTech Connect

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.; Borrego, Belen; Brocchi, Emiliana; Armas-Portela, Rosario; Sobrino, Francisco

    2008-05-10

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. Subcellular localization of the yeast proteome

    PubMed Central

    Kumar, Anuj; Agarwal, Seema; Heyman, John A.; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G. Shirleen; Snyder, Michael

    2002-01-01

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability—a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. PMID:11914276

  10. In Cellulo Mapping of Subcellular Localized Bilirubin.

    PubMed

    Park, Jong-Seok; Nam, Eunju; Lee, Hye-Kyeong; Lim, Mi Hee; Rhee, Hyun-Woo

    2016-08-19

    Bilirubin (BR) is a de novo synthesized metabolite of human cells. However, subcellular localization of BR in the different organelles of human cells has been largely unknown. Here, utilizing UnaG as a genetically encoded fluorescent BR sensor, we report the existence of relatively BR-enriched and BR-depleted microspaces in various cellular organelles of live cells. Our studies indicate that (i) the cytoplasmic facing membrane of the endoplasmic reticulum (ER) and the nucleus are relatively BR-enriched spaces and (ii) mitochondrial intermembrane space and the ER lumen are relatively BR-depleted spaces. Thus, we demonstrate a relationship between such asymmetrical BR distribution in the ER membrane and the BR metabolic pathway. Furthermore, our results suggest plausible BR-transport and BR-regulating machineries in other cellular compartments, including the nucleus and mitochondria. PMID:27232847

  11. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  12. Study of PTEN subcellular localization

    PubMed Central

    Bononi, Angela; Pinton, Paolo

    2015-01-01

    The tumor suppressor PTEN is a key regulator of a plethora of cellular processes that are crucial in cancer development. Through its lipid phosphatase activity PTEN suppresses the PI3K/AKT pathway to govern cell proliferation, growth, migration, energy metabolism and death. The repertoire of roles fulfilled by PTEN has recently been expanded to include crucial functions in the nucleus, where it favors genomic stability and restrains cell cycle progression, as well as protein phosphatase dependent activity at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), where PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and regulates Ca2+ release from the ER and sensitivity to apoptosis. Indeed, PTEN is present in definite subcellular locations where it performs distinct functions acting on specific effectors. In this review, we summarize recent advantages in methods to study PTEN subcellular localization and the distinct biological functions of PTEN in different cellular compartments. A deeper understanding of PTEN’s compartmentalized-functions will guide the rational design of novel therapies. PMID:25312582

  13. Cellular and subcellular localization of PKMζ

    PubMed Central

    Hernández, A. Iván; Oxberry, William C.; Crary, John F.; Mirra, Suzanne S.; Sacktor, Todd Charlton

    2014-01-01

    In contrast to protein kinases that participate in long-term potentiation (LTP) induction and memory consolidation, the autonomously active atypical protein kinase C isoform, protein kinase Mzeta (PKMζ), functions in the core molecular mechanism of LTP maintenance and long-term memory storage. Here, using multiple complementary techniques for light and electron microscopic immunolocalization, we present the first detailed characterization of the cellular and subcellular distribution of PKMζ in rat hippocampus and neocortex. We find that PKMζ is widely expressed in forebrain with prominent immunostaining in hippocampal and neocortical grey matter, and weak label in white matter. In hippocampal and cortical pyramidal cells, PKMζ expression is predominantly somatodendritic, and electron microscopy highlights the kinase at postsynaptic densities and in clusters within spines. In addition, nuclear label and striking punctate immunopositive structures in a paranuclear and dendritic distribution are seen by confocal microscopy, occasionally at dendritic bifurcations. PKMζ immunoreactive granules are observed by electron microscopy in cell bodies and dendrites, including endoplasmic reticulum. The widespread distribution of PKMζ in nuclei, nucleoli and endoplasmic reticulum suggests potential roles of this kinase in cell-wide mechanisms involving gene expression, biogenesis of ribosomes and new protein synthesis. The localization of PKMζ within postsynaptic densities and spines suggests sites where the kinase stores information during LTP maintenance and long-term memory. PMID:24298142

  14. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

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

  15. Modeling biosilicification at subcellular scales.

    PubMed

    Javaheri, Narjes; Cronemberger, Carolina M; Kaandorp, Jaap A

    2013-01-01

    Biosilicification occurs in many organisms. Sponges and diatoms are major examples of them. In this chapter, we introduce a modeling approach that describes several biological mechanisms controlling silicification. Modeling biosilicification is a typical multiscale problem where processes at very different temporal and spatial scales need to be coupled: processes at the molecular level, physiological processes at the subcellular and cellular level, etc. In biosilicification morphology plays a fundamental role, and a spatiotemporal model is required. In the case of sponges, a particle simulation based on diffusion-limited aggregation is presented here. This model can describe fractal properties of silica aggregates in first steps of deposition on an organic template. In the case of diatoms, a reaction-diffusion model is introduced which can describe the concentrations of chemical components and has the possibility to include polymerization chain of reactions. PMID:24420712

  16. Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short- or long-term dietary exposure

    PubMed Central

    Brochot, Amandine; Guinot, Marine; Auchere, Daniel; Macaire, Jean-Paul; Weill, Pierre; Grynberg, Alain; Rousseau-Ralliard, Delphine

    2009-01-01

    Background Previous work showed that the functional cardiac effect of dietary alpha-linolenic acid (ALA) in rats requires a long feeding period (6 months), although a docosahexaenoic (DHA) acid-supply affects cardiac adrenergic response after 2 months. However, the total cardiac membrane n-3 polyunsaturated fatty acid (PUFA) composition remained unchanged after 2 months. This delay could be due to a specific reorganization of the different subcellular membrane PUFA profiles. This study was designed to investigate the evolution between 2 and 6 months of diet duration of the fatty acid profile in sarcolemmal (SL), mitochondrial (MI), nuclear (NU) and sarcoplasmic reticulum (SR) membrane fractions. Methods Male Wistar rats were randomly assigned to 3 dietary groups (n = 10/diet/period), either n-3 PUFA-free diet (CTL), or ALA or DHA-rich diets. After 2 or 6 months, the subcellular cardiac membrane fractions were separated by differential centrifugations and sucrose gradients. Each membrane profile was analysed by gas chromatography (GC) after lipid extraction. Results As expected the n-3 PUFA-rich diets incorporated n-3 PUFA instead of n-6 PUFA in all the subcellular fractions, which also exhibited individual specificities. The diet duration increased SFA and decreased PUFA in SL, whereas NU remained constant. The SR and MI enriched in n-3 PUFA exhibited a decreased DHA level with ageing in the DHA and CTL groups. Conversely, the n-3 PUFA level remained unchanged in the ALA group, due to a significant increase in docosapentaenoic acid (DPA). N-3 PUFA rich diets lead to a better PUFA profile in all the fractions and significantly prevent the profile modifications induced by ageing. Conclusion With the ALA diet the n-3 PUFA content, particularly in SR and SL kept increasing between 2 and 6 months, which may partly account for the delay to achieve the modification of adrenergic response. PMID:19320987

  17. The diverse functions of GAPDH: views from different subcellular compartments

    PubMed Central

    Tristan, Carlos; Shahani, Neelam; Sedlak, Thomas W.; Sawa, Akira

    2011-01-01

    Multiple roles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been recently appreciated. In addition to the cytoplasm where majority of GAPDH is located under the basal condition, GAPDH is also found in the particulate fractions, such as the nucleus, the mitochondria, and the small vesicular fractions. When cells are exposed to various stressors, dynamic subcellular re-distribution of GAPDH occurs. Here we review these multifunctional properties of GAPDH, especially linking them to its oligomerization, posttranslational modification, and subcellular localization. This includes mechanistic descriptions of how S-nitrosylation of GAPDH under oxidative stress may lead to cell death/dysfunction via nuclear translocation of GAPDH, which is counteracted by a cytosolic GOSPEL. GAPDH is also involved in various diseases, especially neurodegenerative disorders and cancers. Therapeutic strategies to these conditions based on molecular understanding of GAPDH are discussed. PMID:20727968

  18. Modeling of Protein Subcellular Localization in Bacteria

    NASA Astrophysics Data System (ADS)

    Xu, Xiaohua; Kulkarni, Rahul

    2006-03-01

    Specific subcellular localization of proteins is a vital component of important bacterial processes: e.g. the Min proteins which regulate cell division in E. coli and Spo0J-Soj system which is critical for sporulation in B. subtilis. We examine how the processes of diffusion and membrane attachment contribute to protein subcellular localization for the above systems. We use previous experimental results to suggest minimal models for these processes. For the minimal models, we derive analytic expressions which provide insight into the processes that determine protein subcellular localization. Finally, we present the results of numerical simulations for the systems studied and make connections to the observed experiemental phenomenology.

  19. The subcellular organization of neocortical excitatory connections

    PubMed Central

    Petreanu, Leopoldo; Mao, Tianyi; Sternson, Scott; Svoboda, Karel

    2009-01-01

    Understanding cortical circuits will require mapping the connections between specific populations of neurons 1, as well as determining the dendritic locations where the synapses occur 2. The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength 3-5. Here we developed an efficient Channelrhodopsin-2 (ChR2)-assisted method 6-8 to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arbors of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers (L) 3, 5A, and 5B in the mouse barrel cortex. Within the dendritic arbors of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arbors different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting a role of these signals in controlling the gain of their target neurons 9. Our experiments reveal exquisite specificity in the subcellular organization of excitatory circuits. PMID:19151697

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2014-09-01

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

  2. Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

    PubMed Central

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

    2014-01-01

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

  3. Subcellular localization of transglutaminase. Effect of collagen.

    PubMed Central

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

    PubMed Central

    Zhang, Jinyang; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xueshan

    2015-01-01

    Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures. PMID:26421138

  6. Self-calibrating viscosity probes: Design and subcellular localization

    PubMed Central

    Dakanali, Marianna; Do, Thai H.; Horn, Austin; Chongchivivat, Akaraphon; Jarusreni, Tuptim; Lichlyter, Darcy; Guizzunti, Gianni; Haidekker, Mark A.; Theodorakis, Emmanuel A.

    2012-01-01

    We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm. 2012 Elsevier Ltd. All rights reserved. PMID:22698784

  7. Cellular localization and subcellular distribution of Unc-33-like protein 6, a brain-specific protein of the collapsin response mediator protein family that interacts with the neuronal glycine transporter 2.

    PubMed

    Horiuchi, Masahisa; Loebrich, Sven; Brandstaetter, Johann Helmut; Kneussel, Matthias; Betz, Heinrich

    2005-07-01

    Unc-33-like protein (Ulip)6, a brain-specific phosphoprotein of the Ulip/collapsin response mediator protein family, was originally identified in our laboratory by yeast two-hybrid screening using the cytoplasmic N-terminal domain of the neuronal glycine transporter, glycine transporter (GlyT) 2, as a bait. Here, the interaction of Ulip6 with the N-terminal domain of GlyT2 was found to be specific for this member of the Ulip/collapsin response mediator protein family and to involve amino acids 135-184 of GlyT2. In pull-down assays and coimmunoprecipitation experiments with rat spinal cord extract, the presence of phosphatase inhibitors significantly enhanced binding of Ulip6 to GlyT2. Subcellular fractionation of spinal cord and retina homogenates at different developmental stages showed Ulip6 immunoreactivity to be associated with light vesicles that were distinct from GlyT2-containing and synaptic vesicles. Immunocytochemistry revealed punctate Ulip6 immunoreactivity in both somatic regions and processes of cultured spinal neurones; no colocalization with GlyT2 or other synaptic marker proteins was found. In retina, which expresses only GlyT1 but not GlyT2, Ulip6 was detected in the inner plexiform layer and along the somata and processes of selected bipolar, amacrine and ganglion cells. Our data support a model in which Ulip6 transiently interacts with GlyT2 in a phosphorylation-dependent manner. PMID:15998282

  8. Neuronal Computations Made Visible with Subcellular Resolution.

    PubMed

    Kaschula, Richard; Salecker, Iris

    2016-06-30

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

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

    PubMed

    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. PMID:27311553

  10. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.

    2013-01-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

  11. eSLDB: eukaryotic subcellular localization database.

    PubMed

    Pierleoni, Andea; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita

    2007-01-01

    Eukaryotic Subcellular Localization DataBase collects the annotations of subcellular localization of eukaryotic proteomes. So far five proteomes have been processed and stored: Homo sapiens, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana. For each sequence, the database lists localization obtained adopting three different approaches: (i) experimentally determined (when available); (ii) homology-based (when possible); and (iii) predicted. The latter is computed with a suite of machine learning based methods, developed in house. All the data are available at our website and can be searched by sequence, by protein code and/or by protein description. Furthermore, a more complex search can be performed combining different search fields and keys. All the data contained in the database can be freely downloaded in flat file format. The database is available at http://gpcr.biocomp.unibo.it/esldb/. PMID:17108361

  12. Recent advances in imaging subcellular processes

    PubMed Central

    Myers, Kenneth A.; Janetopoulos, Christopher

    2016-01-01

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

  13. Recent advances in imaging subcellular processes.

    PubMed

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

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

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

    PubMed Central

    Vidal, René L; Valenzuela, José I; Luján, Rafael; Couve, Andrés

    2009-01-01

    Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER) and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking. PMID:19386132

  15. Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum

    PubMed Central

    Guo, Xia; Ruan, Hangze; Li, Xia; Qin, Liming; Tao, Yi; Qi, Xianjie; Gao, Juanmei; Gan, Lin; Duan, Shumin; Shen, Wanhua

    2016-01-01

    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. PMID:26793062

  16. Subcellular taxonomy: An ultrastructural classification system with diagnostic applications

    SciTech Connect

    McLay, A.L.C.; Toner, P.G.

    1985-01-01

    Contents of this work include: Ultrastructure, Nomenclature, and Disease; Numerical Listing: YX Cellular and Subcellular Structure; Alphabetical Listing; and Appendix: Proposed Revised Listing of M-6 Codes.

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

    PubMed Central

    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.

    2014-01-01

    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 Cln6nclf (CbCln6nclf) 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 CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the

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

    PubMed

    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. Simulation of basic subcellular scattering in tissues

    NASA Astrophysics Data System (ADS)

    Spaeth, Juergen; Radina, Martin; Kessler, Manfred D.

    2001-05-01

    The main signals of light coming back from tissues result from multiple scattering interactions of the incoming light wave with the multipoles of subcellular particles. For a detailed knowledge of the systems, noninvasive optical measurement techniques in the microvolume (capillary, cellular and subcellular signals) are well established at the Institute of Physiology. Due to these methods, the scattering signals and their changes can be detected and analyzed quantitatively at the micrometers 3 volume. The scattering parameters of interest can be set separately and the results can be visualized by three dimensional imaging techniques. Mitochondria produce different scattering patterns by a change of their respiratory state due to different sizes. The algorism presented simulates this scattering. It allows fast predictions of effects like variation of particle size, variation of concentration and absorption at different geometries of lightguides. A comparison of the simulation with the measurements in microvolume shows correlation so that the algorism is reliable for qualitative and quantitative explanation of what happens in the tissue. A remarkable effect is that there are no big differences between measurements in 360- degree direction and 90 degrees because of massive multiple scattering effects.

  20. Dissecting the Subcellular Compartmentation of Proteins and Metabolites in Arabidopsis Leaves Using Non-aqueous Fractionation *

    PubMed Central

    Arrivault, Stéphanie; Guenther, Manuela; Florian, Alexandra; Encke, Beatrice; Feil, Regina; Vosloh, Daniel; Lunn, John E.; Sulpice, Ronan; Fernie, Alisdair R.; Stitt, Mark; Schulze, Waltraud X.

    2014-01-01

    Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin–Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite–protein analysis on a single tissue sample. PMID:24866124

  1. Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retards cell division

    PubMed Central

    2010-01-01

    accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13. PMID:20148114

  2. A Quantitative Proteomics Analysis of Subcellular Proteome Localization and Changes Induced by DNA Damage*

    PubMed Central

    Boisvert, François-Michel; Lam, Yun Wah; Lamont, Douglas; Lamond, Angus I.

    2010-01-01

    A major challenge in cell biology is to identify the subcellular distribution of proteins within cells and to characterize how protein localization changes under different cell growth conditions and in response to stress and other external signals. Protein localization is usually determined either by microscopy or by using cell fractionation combined with protein blotting techniques. Both these approaches are intrinsically low throughput and limited to the analysis of known components. Here we use mass spectrometry-based proteomics to provide an unbiased, quantitative, and high throughput approach for measuring the subcellular distribution of the proteome, termed “spatial proteomics.” The spatial proteomics method analyzes a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass-labeled with heavy isotopes. This was used here to measure the relative distribution between cytoplasm, nucleus, and nucleolus of over 2,000 proteins in HCT116 cells. The data show that, at steady state, the proteome is predominantly partitioned into specific subcellular locations with only a minor subset of proteins equally distributed between two or more compartments. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations, shown here by characterizing dynamic changes in protein localization elicited during the cellular response to DNA damage following treatment of HCT116 cells with etoposide. DNA damage was found to cause dissociation of the proteasome from inhibitory proteins and assembly chaperones in the cytoplasm and relocation to associate with proteasome activators in the nucleus. PMID:20026476

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

    PubMed

    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

    distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states. PMID:27409243

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

    PubMed Central

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

    2016-01-01

    distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states. PMID:27409243

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

    PubMed

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

    2012-01-01

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

  6. Controlling subcellular delivery to optimize therapeutic effect

    PubMed Central

    Mossalam, Mohanad; Dixon, Andrew S; Lim, Carol S

    2010-01-01

    This article focuses on drug targeting to specific cellular organelles for therapeutic purposes. Drugs can be delivered to all major organelles of the cell (cytosol, endosome/lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes and proteasomes) where they exert specific effects in those particular subcellular compartments. Delivery can be achieved by chemical (e.g., polymeric) or biological (e.g., signal sequences) means. Unidirectional targeting to individual organelles has proven to be immensely successful for drug therapy. Newer technologies that accommodate multiple signals (e.g., protein switch and virus-like delivery systems) mimic nature and allow for a more sophisticated approach to drug delivery. Harnessing different methods of targeting multiple organelles in a cell will lead to better drug delivery and improvements in disease therapy. PMID:21113240

  7. Visualizing chemical structure-subcellular localization relationships using fluorescent small molecules as probes of cellular transport

    PubMed Central

    2013-01-01

    Background To study the chemical determinants of small molecule transport inside cells, it is crucial to visualize relationships between the chemical structure of small molecules and their associated subcellular distribution patterns. For this purpose, we experimented with cells incubated with a synthetic combinatorial library of fluorescent, membrane-permeant small molecule chemical agents. With an automated high content screening instrument, the intracellular distribution patterns of these chemical agents were microscopically captured in image data sets, and analyzed off-line with machine vision and cheminformatics algorithms. Nevertheless, it remained challenging to interpret correlations linking the structure and properties of chemical agents to their subcellular localization patterns in large numbers of cells, captured across large number of images. Results To address this challenge, we constructed a Multidimensional Online Virtual Image Display (MOVID) visualization platform using off-the-shelf hardware and software components. For analysis, the image data set acquired from cells incubated with a combinatorial library of fluorescent molecular probes was sorted based on quantitative relationships between the chemical structures, physicochemical properties or predicted subcellular distribution patterns. MOVID enabled visual inspection of the sorted, multidimensional image arrays: Using a multipanel desktop liquid crystal display (LCD) and an avatar as a graphical user interface, the resolution of the images was automatically adjusted to the avatar’s distance, allowing the viewer to rapidly navigate through high resolution image arrays, zooming in and out of the images to inspect and annotate individual cells exhibiting interesting staining patterns. In this manner, MOVID facilitated visualization and interpretation of quantitative structure-localization relationship studies. MOVID also facilitated direct, intuitive exploration of the relationship between the

  8. Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

    PubMed

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2016-01-01

    Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (p<0.05). The extent of calcium deposits in the cortical alveoli of mature oocytes was substantially lower than in earlier stages. Basic information about calcium distribution during zebrafish oogenesis may contribute to better understanding of its role in oogenesis. PMID:26402915

  9. Subcellular Localization of Hexokinases I and II Directs the Metabolic Fate of Glucose

    PubMed Central

    John, Scott; Weiss, James N.; Ribalet, Bernard

    2011-01-01

    Background The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use). Methodology/Principal Findings To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P. Conclusions/Significance These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an

  10. Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

    PubMed Central

    Tiessen, Axel; Nerlich, Annika; Faix, Benjamin; Hümmer, Christine; Fox, Simon; Trafford, Kay; Weber, Hans; Weschke, Winfriede; Geigenberger, Peter

    2012-01-01

    Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios

  11. Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

    PubMed

    Tiessen, Axel; Nerlich, Annika; Faix, Benjamin; Hümmer, Christine; Fox, Simon; Trafford, Kay; Weber, Hans; Weschke, Winfriede; Geigenberger, Peter

    2012-03-01

    Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios

  12. Monoterpene biosynthesis potential of plant subcellular compartments.

    PubMed

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

    2016-01-01

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

  13. Tau regulates the subcellular localization of calmodulin

    SciTech Connect

    Barreda, Elena Gomez de

    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.

  14. Subcellular proteomics of Trypanosoma cruzi reservosomes

    PubMed Central

    Sant’Anna, Celso; Nakayasu, Ernesto S.; Pereira, Miria G.; Lourenço, Daniela; de Souza, Wanderley; Almeida, Igor C.; Cunha-e-Silva, Narcisa L.

    2009-01-01

    Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles. PMID:19288526

  15. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    PubMed

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

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

  16. A Formal Ontology of Subcellular Neuroanatomy

    PubMed Central

    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

  17. Can subcellular organization be explained only by physical principles?

    PubMed Central

    Witzany, Guenther; Baluška, František

    2015-01-01

    In a recent forum article, Dan Needleman and Jan Brugues argue that, despite the astonishing advances in cell biology, a fundamental understanding of even the most well-studied subcellular biological processes is lacking.1 This lack of understanding is evidenced by our inability to make precise predictions of subcellular and cellular behaviors. They suggest that to achieve such an understanding, we need to apply a combination of quantitative experiments with new theoretical concepts and determine the physical principles of subcellular biological organization.1 We discuss these issues and suggest that, besides biophysics, we need strong theoretical inputs from biocommunication theory in order to understand all the core agents of the cellular life and subcellular organization. PMID:26478776

  18. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    DOEpatents

    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.

  19. Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

    PubMed

    Okada, Sabrina Sayori; de Oliveira, Edson Mendes; de Araújo, Tomaz Henrique; Rodrigues, Maria Rita; Albuquerque, Renata Chaves; Mortara, Renato Arruda; Taniwaki, Noemi Nosomi; Nakaya, Helder Imoto; Campa, Ana; Moreno, Ana Carolina Ramos

    2016-02-01

    Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes. PMID:26632272

  20. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

    PubMed

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

    2012-12-12

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

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

    PubMed

    Saravanan, Vijayakumar; Lakshmi, P T V

    2013-12-01

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

  2. Proteome-wide subcellular topologies of E. coli polypeptides database (STEPdb).

    PubMed

    Orfanoudaki, Georgia; Economou, Anastassios

    2014-12-01

    Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance

  3. Subcellular location and molecular mobility of human cytosolic sulfotransferase 1C1 in living human embryonic kidney 293 cells.

    PubMed

    Sheng, Jonathan J; Acquaah-Mensah, George K

    2011-08-01

    Cytosolic sulfotransferases were first isolated from the hepatic cytosol, and they have been localized in the cytoplasm of formaldehyde-fixed human cell samples. The current work was carried out to determine the subcellular localization and molecular mobility of cytosolic sulfotransferases in living human embryonic kidney (HEK) 293 cells. In this work, the subcellular location of human cytosolic sulfotransferase 1C1 (SULT1C1) was studied in cultured HEK293 cells using confocal laser-scanning microscopy. A green fluorescent protein (GFP)-tagged SULT1C1 protein was localized in the cytoplasm of living HEK293 cells. This is consistent with results from previous studies on several other cytosolic sulfotransferase isoforms. Fluorescence recovery after photobleaching microscopy was performed to assess the molecular mobility of the expressed GFP-SULT1C1 molecules. The results suggested that the expressed recombinant GFP-SULT1C1 molecules in living HEK293 cells may include both mobile and immobile populations. To obtain additional insights into the subcellular location of SULT1C1, two machine learning algorithms, Sequential Minimal Optimization and Multilayer Perceptron, were used to compute the probability distribution for the localization of SULT1C1 in nine selected cellular compartments. The resulting probability distribution suggested that the most likely subcellular location of SULT1C1 is the cytosol. PMID:21546557

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

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

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

    PubMed

    Kamunde, Collins; MacPhail, Ruth

    2011-10-01

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

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

    SciTech Connect

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

    2013-09-20

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

  7. Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

    PubMed Central

    2013-01-01

    Background Most proteins have evolved in specific cellular compartments that limit their functions and potential interactions. On the other hand, motifs define amino acid arrangements conserved between protein family members and represent powerful tools for assigning function to protein sequences. The ideal motif would identify all members of a protein family but in practice many motifs identify both family members and unrelated proteins, referred to as True Positive (TP) and False Positive (FP) sequences, respectively. Results To address the relationship between protein motifs, protein function and cellular localization, we systematically assigned subcellular localization data to motif sequences from the comprehensive PROSITE sequence motif database. Using this data we analyzed relationships between localization and function. We find that TPs and FPs have a strong tendency to localize in different compartments. When multiple localizations are considered, TPs are usually distributed between related cellular compartments. We also identified cases where FPs are concentrated in particular subcellular regions, indicating possible functional or evolutionary relationships with TP sequences of the same motif. Conclusions Our findings suggest that the systematic examination of subcellular localization has the potential to uncover evolutionary and functional relationships between motif-containing sequences. We believe that this type of analysis complements existing motif annotations and could aid in their interpretation. Our results shed light on the evolution of cellular organelles and potentially establish the basis for new subcellular localization and function prediction algorithms. PMID:23865897

  8. Subcellular Localized Chemical Imaging of Benthic Algal Nutritional Content via HgCdTe Array FT-IR

    SciTech Connect

    Wetzel, D.; Murdock, J; Dodds, W

    2008-01-01

    Algae respond rapidly and uniquely to changes in nutrient availability by adjusting pigment, storage product, and organelle content and quality. Cellular and subcellular variability of the relative abundance of macromolecular pools (e.g. protein, lipid, carbohydrate, and phosphodiesters) within the benthic (bottom dwelling) alga Cladophora glomerata (a common nuisance species in fresh and saline waters) was revealed by FT-IR microspectroscopic imaging. Nutrient heterogeneity was compared at the filament, cellular, and subcellular level, and localized nutrient uptake kinetics were studied by detecting the gradual incorporation of isotopically labeled nitrogen (N) (as K15NO3) from surrounding water into cellular proteins. Nutritional content differed substantially among filament cells, with differences driven by protein and lipid abundance. Whole cell imaging showed high subcellular macromolecular variability in all cells, including adjacent cells on a filament that developed clonally. N uptake was also very heterogeneous, both within and among cells, and did not appear to coincide with subcellular protein distribution. Despite high intercellular variability, some patterns emerged. Cells acquired more 15N the further they were away from the filament attachment point, and 15N incorporation was more closely correlated with phosphodiester content than protein, lipid, or carbohydrate content. Benthic algae are subject to substantial environmental heterogeneity induced by microscale hydrodynamic factors and spatial variability in nutrient availability. Species specific responses to nutrient heterogeneity are central to understanding this key component of aquatic ecosystems. FT-IR microspectroscopy, modified for benthic algae, allows determination of algal physiological responses at scales not available using current techniques.

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

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  10. Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs

    PubMed Central

    2010-01-01

    Background Process formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs. Results The subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild

  11. Subcellular optogenetics – controlling signaling and single-cell behavior

    PubMed Central

    Karunarathne, W. K. Ajith; O'Neill, Patrick R.; Gautam, Narasimhan

    2015-01-01

    ABSTRACT Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide. PMID:25433038

  12. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

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

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

    PubMed

    Bettendorff, L; Wins, P; Lesourd, M

    1994-05-26

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

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

    PubMed Central

    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. PMID:25941807

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

    PubMed

    Dubois, Maïtée; Hare, Landis

    2009-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  17. Ion microscopy: a new approach for subcellular localization of labelled molecules

    SciTech Connect

    Hindie, E.; Hallegot, P.; Chabala, J.M.; Thorne, N.A.; Coulomb, B.; Levi-Setti, R.; Galle, P.

    1988-12-01

    Secondary ion mass spectroscopy (SIMS) was used to obtain images representing the intracellular distribution of molecules labelled with carbon 14. Deoxyadenosine labelled with carbon 14 was added to a cultured human fibroblast cell medium, and the intracellular distribution of this molecule was studied using three different SIMS instruments: the CAMECA IMS 3F and SMI 300 ion microscopes and the UC-HRL scanning ion microprobe. Carbon 14 distribution images obtained by this method show that deoxyadenosine U-C14 is present in the cytoplasm as well as the nucleus, with a higher concentration in the nucleoli. Our study clearly demonstrates that ion microscopy is well suited for carbon 14 detection and localization at the subcellular level, permitting a wide variety of microanalytical tracer experiments.

  18. SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

    EPA Science Inventory

    Abstract

    Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

  19. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

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

    2016-01-01

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

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

    PubMed

    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. PMID:27528661

  1. Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms

    PubMed Central

    Tabuchi, Mitsuaki; Tanaka, Naotaka; Nishida-Kitayama, Junko; Ohno, Hiroshi; Kishi, Fumio

    2002-01-01

    Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3′ exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3′ untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type–specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y555XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type–specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types. PMID:12475959

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

    PubMed

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

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture. PMID:27059239

  3. Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

    PubMed Central

    Mulvey, Claire M.; Tudzarova, Slavica; Crawford, Mark; Williams, Gareth H.; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations. PMID:23320540

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

    NASA Astrophysics Data System (ADS)

    Yuan, Y.; Chen, S.; Gleber, S. C.; Lai, B.; Brister, K.; Flachenecker, C.; Wanzer, B.; Paunesku, T.; Vogt, S.; Woloschak, G. E.

    2013-10-01

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

  5. Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments.

    PubMed

    Maity, Biswanath; Sheff, David; Fisher, Rory A

    2013-01-01

    The purpose of this protocol is to describe various methodologies used to detect the distribution and localization of specific proteins within individual cells or tissues using immunostaining, defined as the use of specific antibodies to detect a single target protein. Detection of antigens in cultured cells is referred to as immunocytochemistry, whereas their detection in tissues is generally referred to as immunohistochemistry. Both methods involve exposure of fixed cells or tissues to primary antibodies directed against one or more proteins of interest. Bound antibodies are then detected using commercially available secondary antibodies directed against the invariant portion of the primary antibody. Two primary methodologies exist to visualize antigen-antibody complexes: immunofluorescence using fluorophore-conjugated antibodies or chemiluminescence using antibodies coupled to horse-radish peroxidase. This protocol details the steps involved and appropriate use of both methodologies. Immunostaining is used in cell biology to study differential protein expression, localization and distribution at the tissue, cellular, and subcellular level. PMID:23317899

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

    NASA Astrophysics Data System (ADS)

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

    1995-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    SciTech Connect

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

    2009-10-25

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

  10. Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders

    PubMed Central

    Kreis, Patricia; Leondaritis, George; Lieberam, Ivo; Eickholt, Britta J.

    2014-01-01

    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(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), 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. PMID:24744697

  11. Different subcellular localization of muscarinic and serotonin (S2) receptors in human, dog, and rat brain.

    PubMed

    Luabeya, M K; Maloteaux, J M; De Roe, C; Trouet, A; Laduron, P M

    1986-02-01

    Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain. PMID:2934515

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

    PubMed

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

    2016-06-01

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

  13. Subcellular Localization of the Sigma-1 Receptor in Retinal Neurons — an Electron Microscopy Study

    PubMed Central

    Mavlyutov, Timur A.; Epstein, Miles; Guo, Lian-Wang

    2015-01-01

    The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations. We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina. Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well. A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons. PMID:26033680

  14. Subcellular localization of the sigma-1 receptor in retinal neurons - an electron microscopy study.

    PubMed

    Mavlyutov, Timur A; Epstein, Miles; Guo, Lian-Wang

    2015-01-01

    The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations. We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina. Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well. A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons. PMID:26033680

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

    NASA Astrophysics Data System (ADS)

    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%.

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

    PubMed

    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. PMID:26183934

  17. Subcellular patterning: axonal domains with specialized structure and function

    PubMed Central

    Normand, Elizabeth A.; Rasband, Matthew N.

    2015-01-01

    Myelinated axons are patterned into discrete and often repeating domains responsible for the efficient and rapid transmission of electrical signals. These domains include nodes of Ranvier and axon initial segments. Disruption of axonal patterning leads to nervous system dysfunction. In this review we introduce the concept of subcellular patterning as applied to axons and discuss how these patterning events depend on both intrinsic, cytoskeletal mechanisms, and extrinsic, myelinating-glia dependent mechanisms. PMID:25710532

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

    PubMed Central

    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

  19. High Sequence Variability, Diverse Subcellular Localizations, and Ecological Implications of Alkaline Phosphatase in Dinoflagellates and Other Eukaryotic Phytoplankton

    PubMed Central

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort

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

    SciTech Connect

    Wolff, Horst; Hadian, Kamyar; Ziegler, Manja; Weierich, Claudia; Kramer-Hammerle, Susanne; Kleinschmidt, Andrea; Erfle, Volker; Brack-Werner, Ruth . E-mail: brack@gsf.de

    2006-02-15

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

  1. Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP-1

    PubMed Central

    Gay, Frédérique; Calvo, Dominica; Lo, Miao-Chia; Ceron, Julian; Maduro, Morris; Lin, Rueyling; Shi, Yang

    2003-01-01

    Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors. PMID:12651889

  2. In vivo imaging of specific drug-target binding at subcellular resolution

    NASA Astrophysics Data System (ADS)

    Dubach, J. M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L. A.; Weissleder, R.

    2014-05-01

    The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.

  3. Prediction of protein structural classes and subcellular locations.

    PubMed

    Chou, K C

    2000-09-01

    The structural class and subcellular location are the two important features of proteins that are closely related to their biological functions. With the rapid increase in new protein sequences entering into data banks, it is highly desirable to develop a fast and accurate method for predicting the attributes of these features for them. This can expedite the functionality determination of new proteins and the process of prioritizing genes and proteins identified by genomics efforts as potential molecular targets for drug design. Various prediction methods have been developed during the last two decades. This review is devoted to presenting a systematic introduction and comparison of the existing methods in respect to the prediction algorithm and classification scheme. The attention is focused on the state-of-the-art, which is featured by the covarient-discriminant algorithm developed very recently, as well as some new classification schemes for protein structural classes and subcellular locations. Particularly, addressed are also the physical chemistry foundation of the existing prediction methods, and the essence why the covariant-discriminant algorithm is so powerful. PMID:12369916

  4. Subcellular targeting and trafficking of nitric oxide synthases

    PubMed Central

    Oess, Stefanie; Icking, Ann; Fulton, David; Govers, Roland; Müller-Esterl, Werner

    2006-01-01

    Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action. PMID:16722822

  5. Alternative splicing affects the subcellular localization of Drosha

    PubMed Central

    Link, Steffen; Grund, Stefanie E.; Diederichs, Sven

    2016-01-01

    The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity. PMID:27185895

  6. Discovery of putative pancreatic cancer biomarkers using subcellular proteomics.

    PubMed

    McKinney, Kimberly Q; Lee, Yong-Yook; Choi, Hyun-Su; Groseclose, Gale; Iannitti, David A; Martinie, John B; Russo, Mark W; Lundgren, Deborah H; Han, David K; Bonkovsky, Herbert L; Hwang, Sun-Il

    2011-01-01

    Pancreatic cancer (PC) is a highly aggressive disease that frequently remains undetected until it has progressed to an advanced, systemic stage. Successful treatment of PC is hindered by the lack of early detection. The application of proteomic analysis to PC combined with subcellular fractionation has introduced new possibilities in the field of biomarker discovery. We utilized matched pairs of pancreas tumor and non-tumor pancreas from patients undergoing tumor resection. The tissues were treated to obtain cellular protein fractions corresponding to cytosol, membrane, nucleus and cytoskeleton. The fractions were then separated by molecular weight and digested with trypsin, followed by liquid chromatography and tandem mass spectrometry. The spectra obtained were searched using Sequest engine and combined into a single analysis file to obtain a semi-quantitative number, spectral count, using Scaffold software. We identified 2393 unique proteins in non-tumor and cancer pancreas. Utilizing PLGEM statistical analysis we determined 104 proteins were significantly changed in cancer. From these, we further validated four secreted proteins that are up-regulated in cancer and have potential for development as minimally-invasive diagnostic markers. We conclude that subcellular fractionation followed by gel electrophoresis and tandem mass spectrometry is a powerful strategy for identification of differentially expressed proteins in pancreatic cancer. PMID:20807598

  7. Subcellular tissue proteomics of hepatocellular carcinoma for molecular signature discovery.

    PubMed

    Lee, Yong-Yook; McKinney, Kimberly Q; Ghosh, Sriparna; Iannitti, David A; Martinie, John B; Caballes, F Ryan; Russo, Mark W; Ahrens, William A; Lundgren, Deborah H; Han, David K; Bonkovsky, Herbert L; Hwang, Sun-Il

    2011-11-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of mortality from solid organ malignancy worldwide. Because of the complexity of proteins within liver cells and tissues, the discovery of therapeutic targets of HCC has been difficult. To investigate strategies for decreasing the complexity of tissue samples for detecting meaningful protein mediators of HCC, we employed subcellular fractionation combined with 1D-gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. Moreover, we utilized a statistical method, namely, the Power Law Global Error Model (PLGEM), to distinguish differentially expressed proteins in a duplicate proteomic data set. Mass spectrometric analysis identified 3045 proteins in nontumor and HCC from cytosolic, membrane, nuclear, and cytoskeletal fractions. The final lists of highly differentiated proteins from the targeted fractions were searched for potentially translocated proteins in HCC from soluble compartments to the nuclear or cytoskeletal compartments. This analysis refined our targets of interest to include 21 potential targets of HCC from these fractions. Furthermore, we validated the potential molecular targets of HCC, MATR3, LETM1, ILF2, and IQGAP2 by Western blotting, immunohistochemisty, and immunofluorescent microscopy. Here we demonstrate an efficient strategy of subcellular tissue proteomics toward molecular target discovery of one of the most complicated human disease, HCC. PMID:21913717

  8. Alternative splicing affects the subcellular localization of Drosha.

    PubMed

    Link, Steffen; Grund, Stefanie E; Diederichs, Sven

    2016-06-20

    The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity. PMID:27185895

  9. Nanodiamond landmarks for subcellular multimodal optical and electron imaging.

    PubMed

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

    2013-01-01

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

  10. Protein Subcellular Relocalization Increases the Retention of Eukaryotic Duplicate Genes

    PubMed Central

    Byun, S. Ashley; Singh, Sarabdeep

    2013-01-01

    Gene duplication is widely accepted as a key evolutionary process, leading to new genes and novel protein functions. By providing the raw genetic material necessary for functional expansion, the mechanisms that involve the retention and functional diversification of duplicate genes are one of the central topics in evolutionary and comparative genomics. One proposed source of retention and functional diversification is protein subcellular relocalization (PSR). PSR postulates that changes in the subcellular location of eukaryotic duplicate proteins can positively modify function and therefore be beneficial to the organism. As such, PSR would promote retention of those relocalized duplicates and result in significantly lower death rates compared with death rates of nonrelocalized duplicate pairs. We surveyed both relocalized and nonrelocalized duplicate proteins from the available genomes and proteomes of 59 eukaryotic species and compared their relative death rates over a Ks range between 0 and 1. Using the Cox proportional hazard model, we observed that the death rates of relocalized duplicate pairs were significantly lower than the death rates of the duplicates without relocalization in most eukaryotic species examined in this study. These observations suggest that PSR significantly increases retention of duplicate genes and that it plays an important, but currently underappreciated, role in the evolution of eukaryotic genomes. PMID:24265504