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Sample records for identify intracellular accumulation

  1. Pulsed magneto-motive ultrasound imaging to detect intracellular accumulation of magnetic nanoparticles

    International Nuclear Information System (INIS)

    Mehrmohammadi, Mohammad; Qu Min; Sokolov, Konstantin V; Emelianov, Stanislav Y; Ma, Li L; Johnston, Keith P; Romanovicz, Dwight K

    2011-01-01

    As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular accumulation of nanoparticles-an important part of cell-nanoparticle interaction-has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique-pulsed magneto-motive ultrasound (pMMUS)-to identify intracellular accumulation of endocytosed magnetic nanoparticles. In pMMUS imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to the signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular accumulation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular accumulation non-invasively and in real-time.

  2. Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk.

    Science.gov (United States)

    Zhang, De-Liang; Wu, Jian; Shah, Binal N; Greutélaers, Katja C; Ghosh, Manik C; Ollivierre, Hayden; Su, Xin-Zhuan; Thuma, Philip E; Bedu-Addo, George; Mockenhaupt, Frank P; Gordeuk, Victor R; Rouault, Tracey A

    2018-03-30

    Malaria parasites invade red blood cells (RBCs), consume copious amounts of hemoglobin, and severely disrupt iron regulation in humans. Anemia often accompanies malaria disease; however, iron supplementation therapy inexplicably exacerbates malarial infections. Here we found that the iron exporter ferroportin (FPN) was highly abundant in RBCs, and iron supplementation suppressed its activity. Conditional deletion of the Fpn gene in erythroid cells resulted in accumulation of excess intracellular iron, cellular damage, hemolysis, and increased fatality in malaria-infected mice. In humans, a prevalent FPN mutation, Q248H (glutamine to histidine at position 248), prevented hepcidin-induced degradation of FPN and protected against severe malaria disease. FPN Q248H appears to have been positively selected in African populations in response to the impact of malaria disease. Thus, FPN protects RBCs against oxidative stress and malaria infection. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  3. Heavy metals toxicity after acute exposure of cultured renal cells. Intracellular accumulation and repartition

    International Nuclear Information System (INIS)

    Khodja, Hicham; Carriere, Marie; Avoscan, Laure; Gouget, Barbara

    2005-01-01

    Lead (Pb), cadmium (Cd) and uranium (U) present no known biological function but are toxic in various concentration ranges. Pb and Cd lead generally to nephrotoxicity consisting in proximal renal tubular dysfunction and accumulation while U has been reported to induce chemical kidney toxicity, functional and histological damages being as well mainly observed in proximal tubule cells. This work address the question of Cd, Pb, and U cytotoxicity, intracellular accumulation and repartition after acute intoxication of renal proximal tubule epithelial cells. After cells exposure to different concentrations of metals for various times, morphological changes were observed and intracellular concentrations and distributions of toxic metals were specified by PIXE coupled to RBS. Cell viability, measured by biochemical tests, was used as toxicity indicator. A direct correlation between cytotoxicity and intracellular accumulation in renal epithelial cells have been established. Finally, intracellular Pb and U localizations were detected while Cd was found to be uniformly distributed in renal cells. (author)

  4. Intracellular accumulation of potent amiloride analogues by human neutrophils

    International Nuclear Information System (INIS)

    Simchowitz, L.; Woltersdorf, O.W. Jr.; Cragoe, E.J. Jr.

    1987-01-01

    The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [ 14 C]amiloride and the 14 C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids

  5. Analysis method of intracellular iron accumulated in phytoplankton

    Directory of Open Access Journals (Sweden)

    Jorge Falcão Paredes

    1977-01-01

    Full Text Available A colorimetric method for the assessment of iron accumulated in the cells of phytoplankton utilizing potassium ferrocyanide was developed. Two experiences with Phaeodactylum tricornutum and Chlorella sp. were carried out: the first one, on uptake of the iron relatively to the age of the culture: the second one, on the iron uptake by the cells, relatively to different concentrations of the chelator EDTA.Foi desenvolvido um método para análise do ferro acumulado em células de fitoplâncton, usando ferrocianeto de potássio como agente complexante. Com esse objetivo montaram-se duas experiências: uma em que se mostra o acúmulo de ferro em relação à idade das células e uma segunda experiência onde a assimilação é plotada em função da concentração de EDTA no meio de cultura.

  6. Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells

    NARCIS (Netherlands)

    Salerno, Milena; Yahia, Dalila; Dzamitika, Simplice; de Vries, Elisabeth G. E.; Pereira-Maia, Elene; Garnier-Suillerot, Arlette

    Resistance to cisplatin [cis-diamminedichloroplatinum(II), CDDP] chemotherapy is a major problem in the clinic. Understanding the molecular basis of the intracellular accumulation of CDDP and other platinum-based anticancer drugs is of importance in delineating the mechanism of resistance to these

  7. Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production

    DEFF Research Database (Denmark)

    Cao, Yi; Roursgaard, Martin; Danielsen, Pernille Høgh

    2014-01-01

    , the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production....... and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular...... GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects...

  8. Molecular mechanism of intracellular lipid accumulation: Suppressive effect of PycnogenolR in liver cells

    Directory of Open Access Journals (Sweden)

    Shoichiro Ikuyama

    2013-09-01

    Full Text Available ABSTRACTCells are physiologically ready to accumulate lipids such as triacylglycerides in the cytoplasm.Five classes of perilipin (PLIN family proteins are known to be involved in the process of intracellular lipid accumulation. PLIN2 is expressed ubiquitously including adipocytes, hepatocytes and macrophages. Over-expression of PLIN2 is demonstrated in the lesions of fatty liver diseases and atherosclerosis. Suppression of PLIN2 expression prevents from developing these pathological conditions in animal models, suggesting that PLIN2 could be a therapeutic target molecule for excessive intracellular lipid accumulation which leads to various metabolic derangements. The PLIN2 gene promoter has two important cis-acting elements in close proximity:AP-1 element which mediates inflammatory signals and PPRE which mediates free fatty acid effect. In NMuLi mouse liver cells, FFA such as oleic acid requires both functional AP-1 and PPRE simultaneously to stimulate the promoter activity, indicating the presence of intimate interaction of inflammatory and metabolic signals on this gene. PycnogenolR, French maritime pine bark extracts, suppressed the oleic acid-induced PLIN2 expression and lipid accumulation in NMuLi cells. We found that PycnogenolR did not suppress the PLIN2 promoter activity or AP-1 binding to DNA. Instead, PycnogenolRfacilitates the PLIN2 mRNA degradation, leading to suppression of lipid accumulation. This effect seems to be independent of antioxidant effect of PycnogenolR.We raise the idea that PLIN2 is a putative target molecule for prevention of pathological condition induced by excessive lipid accumulation, and this class of natural compounds could be putative therapeutic modalities.Key words: PycnogenolR, lipid droplet, perilipin, fatty liver disease

  9. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  10. Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation.

    Science.gov (United States)

    Liu, Di; Zhang, Hong; Gu, Wenjuan; Zhang, Mengren

    2014-06-01

    Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFH-DA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucose-treated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation.

  11. Intracellular vorinostat accumulation and its relationship to histone deacetylase activity in soft tissue sarcoma patients.

    Science.gov (United States)

    Burhenne, Jürgen; Liu, Lu; Heilig, Christoph E; Meid, Andreas D; Leisen, Margarete; Schmitt, Thomas; Kasper, Bernd; Haefeli, Walter E; Mikus, Gerd; Egerer, Gerlinde

    2017-08-01

    In the regulation of chromatin-structure and histone function, histone deacetylases (HDACs) are key enzymes and thus modulators of epigenetic regulation and gene expression. Accesses of the HDAC inhibitor vorinostat to intracellular compartments are essential to exert epigenetic effects. In ten sarcoma patients receiving oral Zolinza (400 mg qd) vorinostat concentrations in plasma and peripheral blood mononuclear cells (PBMCs) were quantified using validated LC/MS/MS assays to determine intracellular and extracellular pharmacokinetic data. Cellular HDAC activity was evaluated using a fluorogenic assay. Concentration-response relationships were established between intracellular and extracellular vorinostat concentrations and HDAC inhibition in PBMCs. Pharmacokinetics of vorinostat and its two main inactive metabolites were determined over 8 h in plasma and PBMCs. Steady state AUCs (±SD) and T 1/2 (±SD) were calculated to 4.61 ± 0.87 h µM and 1.73 ± 0.69 h (plasma) and 15.2 ± 9.03 h µM and 5.30 ± 4.27 h (PBMCs). Intracellular accumulation of vorinostat was determined together with prolonged vorinostat elimination in PBMCs. Cellular HDAC inhibition increased parallel with vorinostat concentrations in plasma and PBMCs. For effective inhibition of cellular HDACs (IC 50 ) vorinostat concentrations of 0.05 µM in plasma and 0.17 µM in PBMCs were necessary. HDAC inhibition closely followed intracellular vorinostat concentrations and was short-lasting, which may contribute to the limited efficacy seen with vorinostat in solid tumors so far.

  12. Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds

    International Nuclear Information System (INIS)

    Nam, Dong-Ha; Anan, Yasumi; Ikemoto, Tokutaka; Tanabe, Shinsuke

    2005-01-01

    This study was aimed at determining multielemental concentration and its intracellular distribution in selected tissues of cormorant and waterfowl species. Non-essential elements such as Hg, Tl, Cd, Pb and V in tissues were generally consistent with those in ingested items, indicating the significance of food sources of non-essential metal accumulation in great cormorants and mallards. Great cormorants and four waterfowl species examined reflected natural background levels of toxic metals such as Cd, Hg and Pb as well as some essential elements, indicating no specific metal exposure from local sources. Most of Cu, Zn, Se, Rb, Ag, Cd, Cs, and Hg contents were present in the hepatocytosolic fraction, whereas a large percentage of V and Mo were present in insoluble fraction in great cormorant, mallard, and spot-billed duck. The major role of these subcelluar fractions in elemental regulation accounts for the high percentage contribution of each cellular fraction to the total metal contents. Cadmium and Cu are chiefly sequestered through binding to metallothioneins (MTs) of hepatocytosolic fraction in these three avian species. Both MTs and high-molecular-weight substance (HMWS) for Zn and low-molecular-weight substance (LMWS) for Rb were also involved in their sequestration in cytosolic fractions. Relatively different species-specific cytosolic substances were responsible for varying degrees of Ag, Mn, and Co accumulation. It is worth noting that these intracellular metal levels in birds are closely regulated by metal-associated cellular constituents. Therefore, risk assessment studies of metal accumulation in such wild birds should take intracellular metal distribution and specific cellular constituents into account

  13. Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kerdsomboon, Kittikhun; Tatip, Supinda; Kosasih, Sattawat; Auesukaree, Choowong

    2016-05-01

    Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. INTRACELLULAR COPPER ACCUMULATION ENHANCES THE GROWTH OF KINEOCOCCUS RADIOTOLERANS DURING CHRONIC IRRADIATION

    International Nuclear Information System (INIS)

    Bagwell, C; Charles Milliken, C

    2007-01-01

    The actinobacteria Kineococcus radiotolerans is highly resistant to ionizing radiation, desiccation, and oxidative stress; though the underlying biochemical mechanisms are unknown. The purpose of this study was to explore a possible linkage between the uptake of transition metals and extreme resistance to ionizing radiation and oxidative stress. The effects of 6 different divalent cationic metals on growth were examined in the absence of ionizing radiation. None of the metals tested were stimulatory, though cobalt was inhibitory to growth. In contrast, copper supplementation dramatically increased cell growth during chronic irradiation. K. radiotolerans exhibited specific uptake and intracellular accumulation of copper compared to only a weak response to both iron and manganese supplementation. Copper accumulation sensitized cells to hydrogen peroxide. Acute irradiation induced DNA damage was similar between the copper-loaded culture as the age-synchronized no copper control culture, though low molecular weight DNA was more persistent during post-irradiation recovery in the Cu-loaded culture. Still, the estimated times for genome restoration differed by only 1 hr between treatments. While we cannot discount the possibility that copper fulfills an unexpectedly important biochemical role in a radioactive environment; K. radiotolerans has a high capacity for intracellular copper sequestration, and presumably efficiently coordinated oxidative stress defenses and detoxification systems, which confers cross-protection from the damaging affects ionizing radiation

  15. Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Alexei Vazquez

    Full Text Available The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

  16. Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy.

    Science.gov (United States)

    Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

    2013-03-01

    Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellulare pulmonary disease, the patient was clinically well at a 1-month follow-up.

  17. Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy

    OpenAIRE

    Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

    2013-01-01

    Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellu...

  18. Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

    Science.gov (United States)

    Meza, Beatriz; de-Bashan, Luz E; Hernandez, Juan-Pablo; Bashan, Yoav

    2015-06-01

    Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

    Science.gov (United States)

    Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

    2010-02-01

    Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

  20. Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation

    Directory of Open Access Journals (Sweden)

    Emmanuelle Goubert

    2017-05-01

    Full Text Available The solute carrier family 25 (SLC25 drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1 have been identified in early epileptic encephalopathy (EEE and migrating partial seizures in infancy (MPSI but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate (NAD(PH formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in

  1. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    Science.gov (United States)

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  3. Cross talk between increased intracellular zinc (Zn2+) and accumulation of reactive oxygen species in chemical ischemia.

    Science.gov (United States)

    Slepchenko, Kira G; Lu, Qiping; Li, Yang V

    2017-10-01

    Both zinc (Zn 2+ ) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn 2+ and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn 2+ rises that were divided into four phases in the course of 30 min of OGD. The first Zn 2+ rise was a transient, which was followed by a latent phase during which Zn 2+ levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn 2+ rise, which reached a sustained plateau called Zn 2+ overload. Zn 2+ rises were not observed when Zn 2+ was removed by TPEN (a Zn 2+ chelator) or thapsigargin (depleting Zn 2+ from intracellular stores) treatment, indicating that Zn 2+ was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn 2+ rise, indicating that the mitochondrial Zn 2+ accumulation contributes to Zn 2+ overload. We also detected two OGD-induced ROS rises. Two Zn 2+ rises preceded two ROS rises. Removal of Zn 2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn 2+ contributes to mitochondrial ROS generation. There was a Zn 2+ -induced increase in the functional component of NADPH oxidase, p47 phox , thus suggesting that NADPH oxidase may mediate Zn 2+ -induced ROS accumulation. We suggest a new mechanism of cross talk between Zn 2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn 2+ and ROS accumulations during the course of ischemic stress. Copyright © 2017 the American Physiological Society.

  4. Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2015-02-20

    Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights:

  5. Biochemical diagnosis of mucopolysaccharidoses by estimation of intracellular 35S-sulfate accumulation

    International Nuclear Information System (INIS)

    Petruschka, L.; Machill, G.; Wehnert, M.

    1985-01-01

    The investigation of 35 S-sulfate accumulation and of 35 SO 4 -labelled glycosaminoglucane molecules (chase-experiments) in cultured cells was used in post- and prenatal diagnosis of mucopolysaccharidosis (MPS). Cell lines which accumulate 35 S-sulfate can be differentiated by means of cross correction or measurement of enzyme activity. 34 patients with the MPS type I, II, III A, III B and VI, respectively, were diagnosed in this way. Four pregnancies at risk for MPS were prenatally monitored by the sulfate accumulation in cultured amniotic fluid cells. One fetus suffering from MPS II was found. (author)

  6. Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation.

    Science.gov (United States)

    Wang, Dingkun; Tian, Min; Qi, Yuan; Chen, Guang; Xu, Lijun; Zou, Xin; Wang, Kaifu; Dong, Hui; Lu, Fuer

    2015-02-23

    Jinlida granule (JLDG), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating diabetes mellitus. However, the mechanism underlying this effect remains unclear. To determine the main components in JLDG and to explore the effect of JLDG on autophagy and lipid accumulation in NIT-1 pancreatic β cells exposed to politic acid (PA) through AMP activated protein kinase (AMPK) signaling pathway. JLDG was prepared and the main components contained in the granules were identified by ultra performance liquid chromatography (UPLC) fingerprint. Intracellular lipid accumulation in NIT-1 cells was induced by culturing with medium containing PA. Intracellular lipid droplets were observed by Oil Red O staining and triglyceride (TG) content was measured by colorimetric assay. The formation of autophagosomes was observed under transmission electron microscope. The expression of AMPK and phospho-AMPK (pAMPK) proteins as well as its downstream fatty acid metabolism-related proteins (fatty acid synthase, FAS; acetyl-coA carboxylase, ACC; carnitine acyltransferase 1, CPT-1) and autophagy-related genes (mammal target of rapamycin, mTOR; tuberous sclerosis complex 1, TSC1; microtubule-associated protein 1 light chain 3, LC3-II) were determined by Western blot. The expression of sterol regulating element binding protein 1c (SREBP-1c) mRNA was examined by real time PCR (RT-PCR). Our data showed that JLDG could significantly reduce PA-induced intracellular lipid accumulation in NIT-1 pancreatic β cells. This effect was associated with increased protein expression of pAMPK and AMPK in NIT-1 cells. Treatment with JLDG also decreased the expression of AMPK downstream lipogenic genes (SREBP-1c mRNA, FAS and ACC proteins) whereas increased the expression of fatty acid oxidation gene (CPT-1 protein). Additionally, JLDG-treated cells displayed a markedly increase in the number of autophagosomes which was accompanied by the down-regulation of m

  7. An atmospheric-pressure cold plasma leads to apoptosis in Saccharomyces cerevisiae by accumulating intracellular reactive oxygen species and calcium

    International Nuclear Information System (INIS)

    Ma, R N; Zhang, Q; Tian, Y; Su, B; Zhang, J; Fang, J; Feng, H Q; Liang, Y D

    2013-01-01

    A non-thermal plasma is known to induce apoptosis of various cells but the mechanism is not yet clear. A eukaryotic model organism Saccharomyces cerevisiaewas used to investigate the cellular and biochemical regulations of cell apoptosis and cell cycle after an atmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca 2+ ) was first involved in monitoring the process of plasma-induced apoptosis in this study. We analysed the cell apoptosis and cell cycle by flow cytometry and observed the changes in intracellular reactive oxygen species (ROS) and Ca 2+ concentration, cell mitochondrial membrane potential (Δψ m ) as well as nuclear DNA morphology via fluorescence staining assay. All experimental results indicated that plasma-generated ROS leads to the accumulation of intracellular ROS and Ca 2+ that ultimately contribute to apoptosis associated with cell cycle arrest at G1 phase through depolarization of Δψ m and fragmenting nuclear DNA. This work provides a novel insight into the physical and biological mechanism of apoptosis induced by a plasma which could benefit for promoting the development of plasmas applied to cancer therapy. (paper)

  8. Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Huai-Ren Chang

    2018-04-01

    Full Text Available Background/Aims: Intermittent hypoxia (IH has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA group. When we treated cardiomyocytes with Na+-H+ exchange (NHE inhibitors (Amiloride and HOE642 or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline, intracellular calcium chelator or Na+-Ca2+ exchange (NCX inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

  9. Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses.

    Directory of Open Access Journals (Sweden)

    Nicole Schöbel

    Full Text Available Intracellular Cl(- concentrations ([Cl(-](i of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG and olfactory sensory neurons (OSNs, Cl(- is accumulated by the Na(+-K(+-2Cl(- cotransporter 1 (NKCC1, resulting in a [Cl(-](i above electrochemical equilibrium and a depolarizing Cl(- efflux upon Cl(- channel opening. Here, we investigate the [Cl(-](i and function of Cl(- in primary sensory neurons of trigeminal ganglia (TG of wild type (WT and NKCC1(-/- mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl(-](i of WT TG neurons indicated active NKCC1-dependent Cl(- accumulation. Gamma-aminobutyric acid (GABA(A receptor activation induced a reduction of [Cl(-](i as well as Ca(2+ transients in a corresponding fraction of TG neurons. Ca(2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca(2+ channels (VGCCs. Ca(2+ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1 were diminished in NKCC1(-/- TG neurons, but elevated under conditions of a lowered [Cl(-](o suggesting a Cl(--dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS, we found expression of different Ca(2+-activated Cl(- channels (CaCCs in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca(2+ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1(-/- mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca(2+-activated Cl(--dependent signal amplification mechanism in TG neurons that requires intracellular Cl(- accumulation by NKCC1 and the activation of CaCCs.

  10. Triiodothyronine enhances accumulation of intracellular lipids in adipocytes through thyroid hormone receptor α via direct and indirect mechanisms.

    Science.gov (United States)

    Gambo, Yurina; Matsumura, Miki; Fujimori, Ko

    2016-08-15

    Triiodothyronine (T3) enhanced the expression of adipogenic and lipogenic genes with elevation of the intracellular lipids through thyroid hormone receptor (TR) α in mouse 3T3-L1 cells. However, the transcription of the SREBP-1c and HSL genes was decreased by T3. Such T3-mediated alterations were negated by TRα siRNA. Chromatin immunoprecipitation assay showed that the binding of TRα to the TR-responsive element (TRE) of the FAS promoter was elevated by T3. In contrast, the ability of TRα to bind to the TRE of the SREBP-1c promoter was decreased by T3. In addition, the binding of SREBP-1c to the SRE of the HSL promoter was lowered by T3. These results indicate that T3 increased the accumulation of intracellular lipids by enhancing the expression of the FAS gene through direct binding of TRα to the FAS promoter and simultaneously lowered the amount of lipolysis via reduced binding of T3-decreased SREBP-1c to the HSL promoter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  12. Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

    Science.gov (United States)

    Ali, F; Khan, A Q; Khan, R; Sultana, S

    2016-02-01

    Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar. © The Author(s) 2015.

  13. Role of ATP-binding cassette and solute carrier transporters in erlotinib CNS penetration and intracellular accumulation.

    Science.gov (United States)

    Elmeliegy, Mohamed A; Carcaboso, Angel M; Tagen, Michael; Bai, Feng; Stewart, Clinton F

    2011-01-01

    To study the role of drug transporters in central nervous system (CNS) penetration and cellular accumulation of erlotinib and its metabolite, OSI-420. After oral erlotinib administration to wild-type and ATP-binding cassette (ABC) transporter-knockout mice (Mdr1a/b(-/-), Abcg2(-/-), Mdr1a/b(-/-)Abcg2(-/-), and Abcc4(-/-)), plasma was collected and brain extracellular fluid (ECF) was sampled using intracerebral microdialysis. A pharmacokinetic model was fit to erlotinib and OSI-420 concentration-time data, and brain penetration (P(Brain)) was estimated by the ratio of ECF-to-unbound plasma area under concentration-time curves. Intracellular accumulation of erlotinib was assessed in cells overexpressing human ABC transporters or SLC22A solute carriers. P(Brain) in wild-type mice was 0.27 ± 0.11 and 0.07 ± 0.02 (mean ± SD) for erlotinib and OSI-420, respectively. Erlotinib and OSI-420 P(Brain) in Abcg2(-/-) and Mdr1a/b(-/-)Abcg2(-/-) mice were significantly higher than in wild-type mice. Mdr1a/b(-/-) mice showed similar brain ECF penetration as wild-type mice (0.49 ± 0.37 and 0.04 ± 0.02 for erlotinib and OSI-420, respectively). In vitro, erlotinib and OSI-420 accumulation was significantly lower in cells overexpressing breast cancer resistance protein (BCRP) than in control cells. Only OSI-420, not erlotinib, showed lower accumulation in cells overexpressing P-glycoprotein (P-gp) than in control cells. The P-gp/BCRP inhibitor elacridar increased erlotinib and OSI-420 accumulation in BCRP-overexpressing cells. Erlotinib uptake was higher in OAT3- and OCT2-transfected cells than in empty vector control cells. Abcg2 is the main efflux transporter preventing erlotinib and OSI-420 penetration in mouse brain. Erlotinib and OSI-420 are substrates for SLC22A family members OAT3 and OCT2. Our findings provide a mechanistic basis for erlotinib CNS penetration, cellular uptake, and efflux mechanisms. ©2010 AACR.

  14. Intracellular recording, sensory field mapping, and culturing identified neurons in the leech, Hirudo medicinalis.

    Science.gov (United States)

    Titlow, Josh; Majeed, Zana R; Nicholls, John G; Cooper, Robin L

    2013-11-04

    The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

  15. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  16. A new type of intracellular retention signal identified in a pestivirus structural glycoprotein.

    Science.gov (United States)

    Burrack, Sandra; Aberle, Daniel; Bürck, Jochen; Ulrich, Anne S; Meyers, Gregor

    2012-08-01

    Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.

  17. CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

    Directory of Open Access Journals (Sweden)

    Hong-Wai Tham

    2014-12-01

    Full Text Available Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV. To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H screenings against DENV2 envelope (E protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1 was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  18. Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens.

    Science.gov (United States)

    Kim, K S; Baek, C H; Lee, J K; Yang, J M; Farrand, S K

    2001-06-01

    pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses

  19. Identifying change in spatial accumulation of soil salinity in an inland river watershed, China.

    Science.gov (United States)

    Wang, Yugang; Deng, Caiyun; Liu, Yan; Niu, Ziru; Li, Yan

    2018-04-15

    Soil salinity accumulation is strong in arid areas and it has become a serious environmental problem. Knowledge of the process and spatial changes of accumulated salinity in soil can provide an insight into the spatial patterns of soil salinity accumulation. This is especially useful for estimating the spatial transport of soil salinity at the watershed scale. This study aimed to identify spatial patterns of salt accumulation in the top 20cm soils in a typical inland watershed, the Sangong River watershed in arid northwest China, using geostatistics, spatial analysis technology and the Lorenz curve. The results showed that: (1) soil salt content had great spatial variability (coefficient variation >1.0) in both in 1982 and 2015, and about 56% of the studied area experienced transition the degree of soil salt content from one class to another during 1982-2015. (2) Lorenz curves describing the proportions of soil salinity accumulation (SSA) identified that the boundary between soil salinity migration and accumulation regions was 24.3m lower in 2015 than in 1982, suggesting a spatio-temporal inequality in loading of the soil salinity transport region, indicating significant migration of soil salinity from the upstream to the downstream watershed. (3) Regardless of migration or accumulation region, the mean value of SSA per unit area was 0.17kg/m 2 higher in 2015 than 1982 (pwatershed during the studied period in the arid northwest of China. This study demonstrates the spatial patterns of soil salinity accumulation, which is particularly useful for estimating the spatial transport of soil salinity at the watershed scale. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Russell, J.B. (Cornell Univ., Ithaca, NY (USA))

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y{sub ATP} (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up ({sup 14}C)acetate and ({sup 14}C)benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.

  1. Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

    International Nuclear Information System (INIS)

    Russell, J.B.

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y ATP (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [ 14 C]acetate and [ 14 C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation

  2. A simple algorithm for identifying periods of snow accumulation on a radiometer

    Science.gov (United States)

    Lapo, Karl E.; Hinkelman, Laura M.; Landry, Christopher C.; Massmann, Adam K.; Lundquist, Jessica D.

    2015-09-01

    Downwelling solar, Qsi, and longwave, Qli, irradiances at the earth's surface are the primary energy inputs for many hydrologic processes, and uncertainties in measurements of these two terms confound evaluations of estimated irradiances and negatively impact hydrologic modeling. Observations of Qsi and Qli in cold environments are subject to conditions that create additional uncertainties not encountered in other climates, specifically the accumulation of snow on uplooking radiometers. To address this issue, we present an automated method for estimating these periods of snow accumulation. Our method is based on forest interception of snow and uses common meteorological observations. In this algorithm, snow accumulation must exceed a threshold to obscure the sensor and is only removed through scouring by wind or melting. The algorithm is evaluated at two sites representing different mountain climates: (1) Snoqualmie Pass, Washington (maritime) and (2) the Senator Beck Basin Study Area, Colorado (continental). The algorithm agrees well with time-lapse camera observations at the Washington site and with multiple measurements at the Colorado site, with 70-80% of observed snow accumulation events correctly identified. We suggest using the method for quality controlling irradiance observations in snow-dominated climates where regular, daily maintenance is not possible.

  3. Intracellular accumulation of amyloid-beta - a predictor for synaptic dysfunction and neuron loss in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Thomas A Bayer

    2010-03-01

    Full Text Available Despite of long-standing evidence that beta-amyloid (Aβ peptides have detrimental effects on synaptic function, the relationship between Aβ, synaptic and neuron loss is largely unclear. During the last years there is growing evidence that early intraneuronal accumulation of Aβ peptides is one of the key events leading to synaptic and neuronal dysfunction. Many studies have been carried out using transgenic mouse models of Alzheimer’s disease (AD which have been proven to be valuable model system in modern AD research. The present review discusses the impact of intraneuronal Aβ accumulation on synaptic impairment and neuron loss and provides an overview of currently available AD mouse models showing these pathological alterations.

  4. The expression of cholesterol metabolism genes in monocytes from HIV-infected subjects suggests intracellular cholesterol accumulation.

    Science.gov (United States)

    Feeney, Eoin R; McAuley, Nuala; O'Halloran, Jane A; Rock, Clare; Low, Justin; Satchell, Claudette S; Lambert, John S; Sheehan, Gerald J; Mallon, Patrick W G

    2013-02-15

    Human immunodeficiency virus (HIV) infection is associated with increased cardiovascular risk and reduced high-density lipoprotein cholesterol (HDL-c). In vitro, HIV impairs monocyte-macrophage cholesterol efflux, a major determinant of circulating HDL-c, by increasing ABCA1 degradation, with compensatory upregulation of ABCA1 messenger RNA (mRNA). We examined expression of genes involved in cholesterol uptake, metabolism, and efflux in monocytes from 22 HIV-positive subjects on antiretroviral therapy (ART-Treated), 30 untreated HIV-positive subjects (ART-Naive), and 22 HIV-negative controls (HIV-Neg). HDL-c was lower and expression of ABCA1 mRNA was higher in ART-Naive subjects than in both ART-Treated and HIV-Neg subjects (both P ART-Treated and ART-Naive subjects than in HIV-Neg controls. In vivo, increased monocyte ABCA1 expression in untreated HIV-infected patients and normalization of ABCA1 expression with virological suppression by ART supports direct HIV-induced impairment of cholesterol efflux previously demonstrated in vitro. However, decreased expression of cholesterol sensing, uptake, and synthesis genes in both untreated and treated HIV infection suggests that both HIV and ART affect monocyte cholesterol metabolism in a pattern consistent with accumulation of intramonocyte cholesterol.

  5. TransDetect Identifies a New Regulatory Module Controlling Phosphate Accumulation.

    Science.gov (United States)

    Pal, Sikander; Kisko, Mushtak; Dubos, Christian; Lacombe, Benoit; Berthomieu, Pierre; Krouk, Gabriel; Rouached, Hatem

    2017-10-01

    Identifying transcription factor (TFs) cooperation controlling target gene expression is still an arduous challenge. The accuracy of current methods at genome scale significantly drops with the increase in number of genes, which limits their applicability to more complex genomes, like animals and plants. Here, we developed an algorithm, TransDetect, able to predict TF combinations controlling the expression level of a given gene. TransDetect was used to identify novel TF modules regulating the expression of Arabidopsis ( Arabidopsis thaliana ) phosphate transporter PHO1;H3 comprising MYB15, MYB84, bHLH35, and ICE1. These TFs were confirmed to interact between themselves and with the PHO1;H3 promoter. Phenotypic and genetic analyses of TF mutants enable the organization of these four TFs and PHO1;H3 in a new gene regulatory network controlling phosphate accumulation in zinc-dependent manner. This demonstrates the potential of TransDetect to extract directionality in nondynamic transcriptomes and to provide a blueprint to identify gene regulatory network involved in a given biological process. © 2017 American Society of Plant Biologists. All Rights Reserved.

  6. Overexpression of rice glutaredoxin OsGrx_C7 and OsGrx_C2.1 reduces intracellular arsenic accumulation and increases tolerance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Pankaj Kumar Verma

    2016-06-01

    Full Text Available Glutaredoxins (Grxs are a family of small multifunctional proteins involved in various cellular functions, including redox regulation and protection under oxidative stress. Despite the high number of Grx genes in plant genomes (48 Grxs in rice, the biological functions and physiological roles of most of them remain unknown. Here, the functional characterization of the two arsenic-responsive rice Grx family proteins, OsGrx_C7 and OsGrx_C2.1 are reported. Over-expression of OsGrx_C7 and OsGrx_C2.1 in transgenic Arabidopsis thaliana conferred arsenic (As tolerance as reflected by germination, root growth assay, and whole plant growth. Also, the transgenic expression of OsGrxs displayed significantly reduced As accumulation in A. thaliana seeds and shoot tissues compared to WT plants during both AsIII and AsV stress. Thus, OsGrx_C7 and OsGrx_C2.1 seem to be an important determinant of As-stress response in plants. OsGrx_C7 and OsGrx_C2.1 transgenic showed to maintain intracellular GSH pool and involved in lowering AsIII accumulation either by extrusion or reducing uptake by altering the transcript of A. thaliana AtNIPs. Overall, OsGrx_C7 and OsGrx_C2.1 may represent a Grx family protein involved in As stress response and may allow a better understanding of the As induced stress pathways and the design of strategies for the improvement of stress tolerance as well as decreased As content in crops.

  7. Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

    International Nuclear Information System (INIS)

    Loew, Karin; Wagner, Sylvia; Briesen, Hagen von; Knobloch, Thomas; Wiehe, Arno; Engel, Andrea; Langer, Klaus

    2011-01-01

    The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

  8. Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Loew, Karin; Wagner, Sylvia; Briesen, Hagen von [Fraunhofer-Institute for Biomedical Engineering, D-66386 Strasse Ingbert (Germany); Knobloch, Thomas [Institute of Pharmaceutical Technology, Biocenter of Goethe-University, D-60438 Frankfurt (Germany); Wiehe, Arno [Biolitec AG, D-07745 Jena (Germany); Engel, Andrea; Langer, Klaus, E-mail: hagen.briesen@ibmt.fraunhofer.de [Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, D-48149 Muenster (Germany)

    2011-06-17

    The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

  9. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    Science.gov (United States)

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Colloidal stability, surface characterisation and intracellular accumulation of Rhodium(II) citrate coated superparamagnetic iron oxide nanoparticles in breast tumour: a promising platform for cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Silva Nunes, Eloiza da [Universidade Federal de Goias, Campus Samambaia, Instituto de Quimica (Brazil); Lemos Brettas Carneiro, Marcella; Guirelli Simoes de Oliveira, Ricardo; Nair Bao, Sonia [Universidade de Brasilia (UnB), Instituto de Ciencias Biologicas (Brazil); Ribeiro de Souza, Aparecido, E-mail: ardsouza@quimica.ufg.br [Universidade Federal de Goias, Campus Samambaia, Instituto de Quimica (Brazil)

    2013-06-15

    The colloidal stability of a rhodium(II) citrate, Rh{sub 2}(H{sub 2}cit){sub 4}, coating on the surface of maghemite ({gamma}-Fe{sub 2}O{sub 3}) nanoparticles was studied and compared in different dispersion media. The adsorption of Rh{sub 2}(H{sub 2}cit){sub 4} at the water-maghemite interface was evaluated as a function of pH and complex concentration. A slight pH-dependent adsorption of the complex was observed with a maximum at pH 3. The colloidal stability of the functionalised nanoparticles with different amounts of Rh{sub 2}(H{sub 2}cit){sub 4} as a function of pH was evaluated using dynamic light scattering measurements. The particles have a mean magnetic core size of 5.6 nm and the hydrodynamic diameters are approximately 60 nm, which remained unchanged in the pH range in which the samples were a stable sol. The tolerance to different dispersion media, which were deionised water, saline, phosphate-buffered saline (PBS), foetal bovine serum (FBS) and NaCl solutions with different concentrations, was investigated. At moderate ionic strength, the colloidal stability of the dispersions was similar in saline and in PBS compared to the stability of dispersions diluted in water. Moreover, the intracellular accumulation of nanoparticles in 4T1 breast tumour was examined by ultrastructural analysis performed by transmission electron microscopy. The rhodium(II) citrate-coated nanoparticles were found mostly in the cytoplasm and nucleus. Thus, we suggest that these SPIO nanoparticles functionalized with Rh{sub 2}(H{sub 2}Cit){sub 4} can be potential tools for anticancer therapy.

  11. Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources

    Science.gov (United States)

    Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław

    2017-01-01

    The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua, Debaryomyces hansenii, Kluyveromyces marxianus, Kazachstania unispora, and Zygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L−1. Kazachstania unispora was able to accumulate the high amount of palmitoleic acid. PMID:29098157

  12. Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources

    Directory of Open Access Journals (Sweden)

    Iwona Gientka

    2017-01-01

    Full Text Available The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua, Debaryomyces hansenii, Kluyveromyces marxianus, Kazachstania unispora, and Zygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L−1. Kazachstania unispora was able to accumulate the high amount of palmitoleic acid.

  13. Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources.

    Science.gov (United States)

    Gientka, Iwona; Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław

    2017-01-01

    The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua , Debaryomyces hansenii , Kluyveromyces marxianus , Kazachstania unispora , and Zygotorulaspora florentina . We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L -1 . Kazachstania unispora was able to accumulate the high amount of palmitoleic acid.

  14. The lipid accumulation product as a useful index for identifying abnormal glucose regulation in young Korean women.

    Science.gov (United States)

    Oh, J-Y; Sung, Y-A; Lee, H J

    2013-04-01

    The lipid accumulation product, a combination of waist circumference and triglycerides concentration, has been suggested as a better marker for abnormal glucose regulation than BMI. We aimed to compare the lipid accumulation product and BMI as useful markers for abnormal glucose regulation in young Korean women. The lipid accumulation product was calculated using the formula [waist circumference (cm) - 58] × triglycerides (mmol/l). Glucose tolerance status was determined using a 75-g oral glucose tolerance test in 2810 Korean women aged 18-39 years from the general population. The prevalence of abnormal glucose regulation was 6.8% (isolated impaired fasting glucose 1.8%, isolated impaired glucose tolerance 4.0%; impaired fasting glucose + impaired glucose tolerance 0.4% and diabetes mellitus 0.6%). According to the quintile distributions of the lipid accumulation product and BMI, women with a lipid accumulation product quintile greater than their BMI quintile exhibited significantly greater areas under the curve and higher levels of 2-h post-load glucose, insulin, homeostasis model analysis of insulin resistance and lipid profiles than did women with a BMI quintile greater than their lipid accumulation product quintile. Multiple logistic regression revealed that the lipid accumulation product exhibited a higher odds ratio for abnormal glucose regulation than did BMI after adjusting for age, systolic blood pressure, HDL cholesterol, previous history of gestational diabetes and family history of diabetes (odds ratios 3.5 and 2.6 of the highest vs. the lowest quintiles of lipid accumulation product and BMI, respectively). The lipid accumulation product could be useful for identifying the young Korean women with abnormal glucose regulation. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

  15. Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

    Science.gov (United States)

    Ihara, Kohei; Sato, Kazuki; Hori, Hatsuhiro; Makino, Yumiko; Shigenobu, Shuji; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2017-04-01

    The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent K D , 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  17. The use of diagnostic imaging for identifying abnormal gas accumulations in cetaceans and pinnipeds.

    Directory of Open Access Journals (Sweden)

    Sophie eDennison

    2012-06-01

    Full Text Available Recent dogma suggested that marine mammals are not at risk of decompression sickness (DCS due to a number of evolutionary adaptations. Several proposed adaptations exist. Lung compression and alveolar collapse that terminate gas exchange before a depth is reached where supersaturation is significant and bradycardia with peripheral vasoconstriction affecting the distribution, and dynamics of blood and tissue nitrogen levels. Published accounts of gas and fat emboli and dysbaric osteonecrosis in marine mammals and theoretical modeling have challenged this view-point, suggesting that decompression-like symptoms may occur under certain circumstances, contrary to common belief. Diagnostic imaging modalities are invaluable tools for the non-invasive examination of animals for evidence of gas and have been used to demonstrate the presence of incidental decompression-related renal gas accumulations in some stranded cetaceans. Diagnostic imaging has also contributed to the recognition of clinically significant gas accumulations in live and dead cetaceans and pinnipeds. Understanding the appropriate application and limitations of the available imaging modalities is important for accurate interpretation of results. The presence of gas may be incidental and must be interpreted cautiously alongside all other available data including clinical examination, clinical laboratory testing, gas analysis, necropsy examination and histology results.

  18. Investigation of intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-pseudomonas syringae interactions using a fast neutron-generated mutant allele of EDS5 identified by genetic mapping and whole-genome sequencing.

    Directory of Open Access Journals (Sweden)

    Jessie L Carviel

    Full Text Available A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5. RPS2-AvrRpt2-initiated effector-triggered immunity (ETI was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst, little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space.

  19. High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

    Directory of Open Access Journals (Sweden)

    Olga Mestre

    Full Text Available Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS. Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

  20. Abnormal intracellular accumulation and extracellular Aβ deposition in idiopathic and Dup15q11.2-q13 autism spectrum disorders.

    Directory of Open Access Journals (Sweden)

    Jerzy Wegiel

    Full Text Available It has been shown that amyloid ß (Aβ, a product of proteolytic cleavage of the amyloid β precursor protein (APP, accumulates in neuronal cytoplasm in non-affected individuals in a cell type-specific amount.In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15 and autism spectrum disorder (ASD. In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15 autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test. In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001. The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ(17-40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ(1-40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15 compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links

  1. Reduced intracellular drug accumulation in drug-resistant leukemia cells is not solely due to MDR-mediated efflux but also to decreased uptake

    Directory of Open Access Journals (Sweden)

    Angela Oliveira Pisco

    2014-10-01

    Full Text Available Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes was also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not solely achieved by enhanced efflux capacity but also by supressed intake of the drug offering an alternative target to overcome drug resistance or potentiate chemotherapy.

  2. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

    Science.gov (United States)

    Smith, Mark L; Mason, Hugh S; Shuler, Michael L

    2002-12-30

    The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed. Copyright 2002 Wiley Periodicals, Inc.

  3. Lipophilic ester and amide derivatives of rosmarinic acid protect cells against H2O2-induced DNA damage and apoptosis: The potential role of intracellular accumulation and labile iron chelation

    Directory of Open Access Journals (Sweden)

    Paraskevi S. Gerogianni

    2018-05-01

    Full Text Available Phenolic acids represent abundant components contained in human diet. However, the negative charge in their carboxylic group limits their capacity to diffuse through biological membranes, thus hindering their access to cell interior. In order to promote the diffusion of rosmarinic acid through biological membranes, we synthesized several lipophilic ester- and amide-derivatives of this compound and evaluated their capacity to prevent H2O2-induced DNA damage and apoptosis in cultured human cells. Esterification of the carboxylic moiety with lipophilic groups strongly enhanced the capacity of rosmarinic acid to protect cells. On the other hand, the amide-derivatives were somewhat less effective but exerted less cytotoxicity at high concentrations. Cell uptake experiments, using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS, illustrated different levels of intracellular accumulation among the ester- and amide-derivatives, with the first being more effectively accumulated, probably due to their extensive hydrolysis inside the cells. In conclusion, these results highlight the hitherto unrecognized fundamental importance of derivatization of diet-derived phenolic acids to unveil their biological potential.

  4. Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

    Science.gov (United States)

    Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

    2018-06-11

    Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    Science.gov (United States)

    Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715

  6. Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

    Directory of Open Access Journals (Sweden)

    Kumagai Yoshinori

    2010-12-01

    Full Text Available Abstract Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium, several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

  7. Characterization of Leptin Intracellular Trafficking

    Directory of Open Access Journals (Sweden)

    E Walum

    2009-12-01

    Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

  8. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

    Science.gov (United States)

    Xie, Junjun; Lei, Bo; Niu, Mengliang; Huang, Yuan; Kong, Qiusheng; Bie, Zhilong

    2015-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the

  9. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

    Directory of Open Access Journals (Sweden)

    Junjun Xie

    Full Text Available MicroRNAs (miRNAs, a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch. and N15 (Cucurbita. moschata Duch., with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small

  10. Visceral Adiposity Index and Lipid Accumulation Product Index: Two Alternate Body Indices to Identify Chronic Kidney Disease among the Rural Population in Northeast China

    Directory of Open Access Journals (Sweden)

    Dongxue Dai

    2016-12-01

    Full Text Available We aimed to compare the relative strength of the association between anthropometric obesity indices and chronic kidney disease (CKD. Another objective was to examine whether the visceral adiposity index (VAI and lipid accumulation product index (LAPI can identify CKD in the rural population of China. There were 5168 males and 6024 females involved in this cross-sectional study, and 237 participants (2.12% suffered from CKD. Obesity indices included body mass index (BMI, waist circumference (WC, waist-to-height ratio (WHtR, VAI and LAPI. VAI and LAPI were calculated with triglyceride (TG, high-density lipoprotein (HDL, BMI and WC. VAI = [WC/39.68 + (1.88 × BMI] × (TG /1.03 × (1.31/ HDL for males; VAI = [WC/36.58 + (1.89 × BMI] × (TG/0.81 × (1.52/HDL for females. LAPI = (WC-65 × TG for males, LAPI = (WC-58 × TG for females. CKD was defined as an estimated glomerular filtration rate (eGFR of less than 60 mL/min per 1.73 m2. The prevalence of CKD increased across quartiles for WHtR, VAI and LAPI. A multivariate logistic regression analysis of the presence of CKD for the highest quartile vs. the lowest quartile of each anthropometric measure showed that the VAI was the best predictor of CKD in females (OR: 4.21, 95% CI: 2.09–8.47, p < 0.001. VAI showed the highest AUC for CKD (AUC: 0.68, 95% CI: 0.65–0.72 and LAPI came second (AUC: 0.66, 95% CI: 0.61–0.70 in females compared with BMI (both p-values < 0.001. However, compared with the traditional index of the BMI, the anthropometric measures VAI, LAPI, WC, and WHtR had no statistically significant capacity to predict CKD in males. Our results showed that both VAI and LAPI were significantly associated with CKD in the rural population of northeast China. Furthermore, VAI and LAPI were superior to BMI, WC and WHtR for predicting CKD only in females.

  11. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

  12. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Science.gov (United States)

    Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

    2011-01-01

    Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  13. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  14. Intracellular sodium hydrogen exchange inhibition and clinical myocardial protection.

    Science.gov (United States)

    Mentzer, Robert M; Lasley, Robert D; Jessel, Andreas; Karmazyn, Morris

    2003-02-01

    Although the mechanisms underlying ischemia/reperfusion injury remain elusive, evidence supports the etiologic role of intracellular calcium overload and oxidative stress induced by reactive oxygen species. Activation of the sodium hydrogen exchanger (NHE) is associated with intracellular calcium accumulation. Inhibition of the NHE-1 isoform may attenuate the consequences of this injury. Although there is strong preclinical and early clinical evidence that NHE inhibitors may be cardioprotective, definitive proof of this concept in humans awaits the results of ongoing clinical trials.

  15. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  16. Single-cell intracellular nano-pH probes†

    OpenAIRE

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

  17. A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryziaslatipes) eggs and eleutheroembryos as lipid rich models

    International Nuclear Information System (INIS)

    Mondon, J.A.; Howitt, J.; Tosiano, M.; Kwok, K.W.H.; Hinton, D.E.

    2011-01-01

    Highlights: → Hepatic lipidosis in fish liver is often misdiagnosed or overlooked. → Specific histological fat stains and cryostat sections are not commonly used. → Standard paraffin processing removes lipid leaving vacuoles of unknown origin. → Osmium post-fixed paraffin-embedment is a cost effective alternative. → Medaka trials show suitability for lipid visualization in tissues from egg to adult. - Abstract: Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

  18. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Directory of Open Access Journals (Sweden)

    Yu-Hsuan Tu

    Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  19. Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

    Science.gov (United States)

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-03-01

    Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Apoplastic and intracellular plant sugars regulate developmental transitions in witches’ broom disease of cacao

    Science.gov (United States)

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-01-01

    Witches’ broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant–fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. PMID:25540440

  1. Describing the Diapause-Preparatory Proteome of the Beetle Colaphellus bowringi and Identifying Candidates Affecting Lipid Accumulation Using Isobaric Tags for Mass Spectrometry-Based Proteome Quantification (iTRAQ

    Directory of Open Access Journals (Sweden)

    Daniel A. Hahn

    2017-04-01

    Full Text Available Prior to entering diapause, insects must prepare themselves physiologically to withstand the stresses of arresting their development for a lengthy period. While studies describing the biochemical and cellular milieu of the maintenance phase of diapause are accumulating, few studies have taken an “omics” approach to describing molecular events during the diapause preparatory phase. We used isobaric tags and mass spectrometry (iTRAQ to quantitatively compare the expression profiles of proteins identified during the onset of diapause preparation phase in the heads of adult female cabbage beetles, Colaphellus bowringi. A total of 3,175 proteins were identified, 297 of which were differentially expressed between diapause-destined and non-diapause-destined female adults and could therefore be involved in diapause preparation in this species. Comparison of identified proteins with protein function databases shows that many of these differentially expressed proteins enhanced in diapause destined beetles are involved in energy production and conversion, carbohydrate metabolism and transport, and lipid metabolism. Further hand annotation of differentially abundant peptides nominates several associated with stress hardiness, including HSPs and antioxidants, as well as neural development. In contrast, non-diapause destined beetles show substantial increases in cuticle proteins, suggesting additional post-emergence growth. Using RNA interference to silence a fatty acid-binding protein (FABP that was highly abundant in the head of diapause-destined females prevented the accumulation of lipids in the fat body, a common product of diapause preparation in this species and others. Surprisingly, RNAi against the FABP also affected the transcript abundance of several heat shock proteins. These results suggest that the identified differentially expressed proteins that play vital roles in lipid metabolism may also contribute somehow to enhanced hardiness to

  2. Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Fabian Arenas

    2017-11-01

    Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

  3. A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

    Science.gov (United States)

    2000-03-31

    have a major impact on neuronal intracellular signaling. Most of the ER in neurons and glia appears to accumulate calcium by energy driven ion pumps...secretion of exocrine, endocrine, and neurocrine products, regulation of glycogenolysis and gluconeogenesis , intracellular transport, secretion of fluids...the RyRs [140]. Furthermore, the intracellular expression of these receptor-channels in neuronal ER is also reciprocal with RyRs located primarily in

  4. Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

    Science.gov (United States)

    Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

    2017-06-26

    Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

  5. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

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

  6. Lipid Accumulation in Peripheral Blood Dendritic Cells and Anticancer Immunity in Patients with Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ryo Arai

    2018-01-01

    Full Text Available We studied the subsets of peripheral blood dendritic cells (DCs and lipid accumulation in DCs to investigate the involvement of DCs in the decreased anticancer immunity of advanced lung cancer patients. We analyzed the population of DC subsets in peripheral blood using flow cytometry. We then determined lipid accumulation in the DCs using BODIPY 650/665, a fluorophore with an affinity for lipids. Compared with healthy controls, the number of DCs in the peripheral blood of treatment-naive cancer patients was significantly reduced. In patients with stage III + IV disease, the numbers of myeloid DCs (mDCs and plasmacytoid DCs were also significantly reduced. Lipid accumulation in DCs evaluated based on the fluorescence intensity of BODIPY 650/665 was significantly higher in stage III + IV lung cancer patients than in the controls. In the subset analysis, the fluorescence was highest for mDCs. The intracellularly accumulated lipids were identified as triglycerides. A decreased mixed leukocyte reaction was observed in the mDCs from lung cancer patients compared with those from controls. Taken together, the results show that lung cancer patients have a notably decreased number of peripheral blood DCs and their function as antigen-presenting cells is decreased due to their high intracellular lipid accumulation. Thereby, anticancer immunity is suppressed.

  7. Secretome of obligate intracellular Rickettsia

    Science.gov (United States)

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  8. Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

    Science.gov (United States)

    Miyamoto, Yuji; Mukai, Tetsu; Matsuoka, Masanori; Kai, Masanori; Maeda, Yumi; Makino, Masahiko

    2016-08-01

    Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

  9. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually

  10. Microbial accumulation of uranium, radium, and cesium

    International Nuclear Information System (INIS)

    Strandberg, G.W.; Shumate, S.E. II; Parrott, J.R. Jr.; North, S.E.

    1981-05-01

    Diverse microbial species varied considerably in their ability to accumulate uranium, cesium, and radium. Mechanistic differences in uranium uptake by Saccharomyces cerevisiae and Pseudomonas aeruginosa were indicated. S. serevisiae exhibited a slow (hours) surface accumulation of uranium which was subject to environmental factors, while P. aeruginosa accumulated uranium rapidly (minutes) as dense intracellular deposits and did not appear to be affected by environmental parameters. Metabolism was not required for uranium uptake by either organism. Cesium and radium were concentrated to a considerably lesser extent than uranium by the several species tested

  11. Intracellular localization of Arabidopsis sulfurtransferases.

    Science.gov (United States)

    Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

    2004-06-01

    Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

  12. Drosophila VAMP7 regulates Wingless intracellular trafficking.

    Science.gov (United States)

    Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

    2017-01-01

    Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

  13. Intracellular sphingosine releases calcium from lysosomes.

    Science.gov (United States)

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-11-27

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

  14. Influence of intracellular Na + , K + and Cl - on the salt tolerance in ...

    African Journals Online (AJOL)

    In the process of selection for salt tolerance, it is important to understand the physiological basis of ion management executed by the cells through the exclusion, accumulation or maintenance of ratios of specific ions. Intracellular accumulation of Na+, K+ and Cl- ions in the cells in vitro was studied as a factor in salt ...

  15. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  16. A bacteriophage endolysin that eliminates intracellular streptococci

    Science.gov (United States)

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  17. External validation of the fatty liver index and lipid accumulation product indices, using 1H-magnetic resonance spectroscopy, to identify hepatic steatosis in healthy controls and obese, insulin-resistant individuals.

    Science.gov (United States)

    Cuthbertson, Daniel J; Weickert, Martin O; Lythgoe, Daniel; Sprung, Victoria S; Dobson, Rebecca; Shoajee-Moradie, Fariba; Umpleby, Margot; Pfeiffer, Andreas F H; Thomas, E Louise; Bell, Jimmy D; Jones, Helen; Kemp, Graham J

    2014-11-01

    Simple clinical algorithms including the fatty liver index (FLI) and lipid accumulation product (LAP) have been developed as surrogate markers for non-alcoholic fatty liver disease (NAFLD), constructed using (semi-quantitative) ultrasonography. This study aimed to validate FLI and LAP as measures of hepatic steatosis, as determined quantitatively by proton magnetic resonance spectroscopy (1H-MRS). Data were collected from 168 patients with NAFLD and 168 controls who had undergone clinical, biochemical and anthropometric assessment. Values of FLI and LAP were determined and assessed both as predictors of the presence of hepatic steatosis (liver fat>5.5%) and of actual liver fat content, as measured by 1H-MRS. The discriminative ability of FLI and LAP was estimated using the area under the receiver operator characteristic curve (AUROC). As FLI can also be interpreted as a predictive probability of hepatic steatosis, we assessed how well calibrated it was in our cohort. Linear regression with prediction intervals was used to assess the ability of FLI and LAP to predict liver fat content. Further validation was provided in 54 patients with type 2 diabetes mellitus. FLI, LAP and alanine transferase discriminated between patients with and without steatosis with an AUROC of 0.79 (IQR=0.74, 0.84), 0.78 (IQR=0.72, 0.83) and 0.83 (IQR=0.79, 0.88) respectively although could not quantitatively predict liver fat. Additionally, the algorithms accurately matched the observed percentages of patients with hepatic steatosis in our cohort. FLI and LAP may be used to identify patients with hepatic steatosis clinically or for research purposes but could not predict liver fat content. © 2014 European Society of Endocrinology.

  18. Selenium accumulation by plants

    Science.gov (United States)

    White, Philip J.

    2016-01-01

    Background Selenium (Se) is an essential mineral element for animals and humans, which they acquire largely from plants. The Se concentration in edible plants is determined by the Se phytoavailability in soils. Selenium is not an essential element for plants, but excessive Se can be toxic. Thus, soil Se phytoavailability determines the ecology of plants. Most plants cannot grow on seleniferous soils. Most plants that grow on seleniferous soils accumulate 100 mg Se kg–1 dry matter. These plants are considered to be Se accumulators. Some species can even accumulate Se concentrations of 1000–15 000 mg Se kg–1 dry matter and are called Se hyperaccumulators. Scope This article provides an overview of Se uptake, translocation and metabolism in plants and highlights the possible genetic basis of differences in these between and within plant species. The review focuses initially on adaptations allowing plants to tolerate large Se concentrations in their tissues and the evolutionary origin of species that hyperaccumulate Se. It then describes the variation in tissue Se concentrations between and within angiosperm species and identifies genes encoding enzymes limiting the rates of incorporation of Se into organic compounds and chromosomal loci that might enable the development of crops with greater Se concentrations in their edible portions. Finally, it discusses transgenic approaches enabling plants to tolerate greater Se concentrations in the rhizosphere and in their tissues. Conclusions The trait of Se hyperaccumulation has evolved several times in separate angiosperm clades. The ability to tolerate large tissue Se concentrations is primarily related to the ability to divert Se away from the accumulation of selenocysteine and selenomethionine, which might be incorporated into non-functional proteins, through the synthesis of less toxic Se metabilites. There is potential to breed or select crops with greater Se concentrations in their edible tissues, which

  19. Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates

    DEFF Research Database (Denmark)

    Kamp, Anja; Stief, Peter; Bristow, Laura A.

    2016-01-01

    % was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before...... store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom......-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly...

  20. Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity

    NARCIS (Netherlands)

    Goosen, C.; Yuan, X.L.; Munster, J.M. van; Ram, A.F.J.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2007-01-01

    A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger.

  1. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    Wagner, G.J.

    1979-01-01

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

  2. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  3. Selenium accumulation by plants.

    Science.gov (United States)

    White, Philip J

    2016-02-01

    Selenium (Se) is an essential mineral element for animals and humans, which they acquire largely from plants. The Se concentration in edible plants is determined by the Se phytoavailability in soils. Selenium is not an essential element for plants, but excessive Se can be toxic. Thus, soil Se phytoavailability determines the ecology of plants. Most plants cannot grow on seleniferous soils. Most plants that grow on seleniferous soils accumulate plant species have evolved tolerance to Se, and commonly accumulate tissue Se concentrations >100 mg Se kg(-1) dry matter. These plants are considered to be Se accumulators. Some species can even accumulate Se concentrations of 1000-15 000 mg Se kg(-1 )dry matter and are called Se hyperaccumulators. This article provides an overview of Se uptake, translocation and metabolism in plants and highlights the possible genetic basis of differences in these between and within plant species. The review focuses initially on adaptations allowing plants to tolerate large Se concentrations in their tissues and the evolutionary origin of species that hyperaccumulate Se. It then describes the variation in tissue Se concentrations between and within angiosperm species and identifies genes encoding enzymes limiting the rates of incorporation of Se into organic compounds and chromosomal loci that might enable the development of crops with greater Se concentrations in their edible portions. Finally, it discusses transgenic approaches enabling plants to tolerate greater Se concentrations in the rhizosphere and in their tissues. The trait of Se hyperaccumulation has evolved several times in separate angiosperm clades. The ability to tolerate large tissue Se concentrations is primarily related to the ability to divert Se away from the accumulation of selenocysteine and selenomethionine, which might be incorporated into non-functional proteins, through the synthesis of less toxic Se metabilites. There is potential to breed or select crops

  4. ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

    Science.gov (United States)

    Wu, Yu; Pons, Valérie; Goudet, Amélie; Panigai, Laetitia; Fischer, Annette; Herweg, Jo-Ana; Kali, Sabrina; Davey, Robert A; Laporte, Jérôme; Bouclier, Céline; Yousfi, Rahima; Aubenque, Céline; Merer, Goulven; Gobbo, Emilie; Lopez, Roman; Gillet, Cynthia; Cojean, Sandrine; Popoff, Michel R; Clayette, Pascal; Le Grand, Roger; Boulogne, Claire; Tordo, Noël; Lemichez, Emmanuel; Loiseau, Philippe M; Rudel, Thomas; Sauvaire, Didier; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien

    2017-11-14

    Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

  5. Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

    Science.gov (United States)

    Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

    2015-06-01

    Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Electron Microscopy of Intracellular Protozoa

    Science.gov (United States)

    1988-12-20

    Classification) " ELECTRON MICROSCOPY OF INTRACELLULAR PROTOZOA 12. PERSONAL AUTHOR(S) Aikawa, Masamichi 13a. TYPE OF REPORT I13b. TIME COVERED 114...authors suggest that anti-CS protein antibody is important in reducing the prevalence of malaria with increasing age among persons in such areas and... Hygine 33, 220-226. 0Giudice, G.D., Engers, H.D., Tougne, C., Biro, S.S., Weiss, N., Verdini, A.S., Pessi, A., Degremont, A.A., Freyvogel, T.A., Lambert

  7. Pathogenic mechanisms of intracellular bacteria.

    Science.gov (United States)

    Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

    2017-06-01

    We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

  8. Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van; Kesbeke, Fanja

    1983-01-01

    Chemoattractants induce a transient accumulation of cGMP levels in Dictyostelium. Intracellular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extracellular cGMP levels were detected simultaneously after stimulation of D. lacteum cells

  9. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  10. Enhanced intracellular delivery and antibacterial efficacy of enrofloxacin-loaded docosanoic acid solid lipid nanoparticles against intracellular Salmonella.

    Science.gov (United States)

    Xie, Shuyu; Yang, Fei; Tao, Yanfei; Chen, Dongmei; Qu, Wei; Huang, Lingli; Liu, Zhenli; Pan, Yuanhu; Yuan, Zonghui

    2017-01-23

    Enrofloxacin-loaded docosanoic acid solid lipid nanoparticles (SLNs) with different physicochemical properties were developed to enhance activity against intracellular Salmonella. Their cellular uptake, intracellular elimination and antibacterial activity were studied in RAW 264.7 cells. During the experimental period, SLN-encapsulated enrofloxacin accumulated in the cells approximately 27.06-37.71 times more efficiently than free drugs at the same extracellular concentration. After incubation for 0.5 h, the intracellular enrofloxacin was enhanced from 0.336 to 1.147 μg/mg of protein as the sizes of nanoparticles were increased from 150 to 605 nm, and from 0.960 to 1.147 μg/mg of protein when the charge was improved from -8.1 to -24.9 mv. The cellular uptake was more significantly influenced by the size than it was by the charge, and was not affected by whether the charge was positive or negative. The elimination of optimal SLN-encapsulated enrofloxacin from the cells was significantly slower than that of free enrofloxacin after removing extracellular drug. The inhibition effect against intracellular Salmonella CVCC541 of 0.24 and 0.06 μg/mL encapsulated enrofloxacin was stronger than 0.6 μg/mL free drug after all of the incubation periods and at 48 h, respectively. Docosanoic acid SLNs are thus considered as a promising carrier for intracellular bacterial treatment.

  11. Intracellular Ca2+ Regulation in Calcium Sensitive Phenotype of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    HERMANSYAH

    2010-03-01

    Full Text Available Intracellular cytosolic Ca2+ concentration accumulation plays an essential information in Saccharomyces cerevisiae i.e. to explain cellular mechanism of Ca2+ sensitive phenotype. Disruption both S. cerevisiae PPase PTP2 and MSG5 genes showed an inhibited growth in the presence of Ca2+. On the other hand, by using Luminocounter with apoaequorin system, a method based upon luminescent photoprotein aequorin, intracellular Ca2+ concentration was accumulated as a consequence of calcium sensitive phenotype of S. cerevisiae. This fact indicated that PPase ptp2Δ and msg5Δ were involved in intracellular Ca2+ transport in addition their already known pathways i.e Mitogen Activated Protein Kinase cell wall integrity pathway, high osmolarity glycerol (HOG pathway, and pheromone response FUS3 pathway.

  12. Advanced accumulator for PWR

    International Nuclear Information System (INIS)

    Ichimura, Taiki; Chikahata, Hideyuki

    1997-01-01

    Advanced accumulators have been incorporated into the APWR design in order to simplify the safety system configuration and to improve reliability. The advanced accumulators refill the reactor vessel with a large discharge flow rate in a large LOCA, then switch to a small flow rate to continue safety injection for core reflooding. The functions of the conventional accumulator and the low head safety injection pump are integrated into this advanced accumulator. Injection performance tests simulating LOCA conditions and visualization tests for new designs have been carried out. This paper describes the APWR ECCS configuration, the advanced accumulator design and some of the injection performance and visualization test results. It was verified that the flow resistance of the advanced accumulator is independent of the model scale. The similarity law and performance data of the advanced accumulator for applying APWR was established. (author)

  13. MR imaging of intracellular and extracellular deoxyhemoglobin

    International Nuclear Information System (INIS)

    Janick, P.A.; Grossman, R.I.; Asakura, T.

    1989-01-01

    MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

  14. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  15. Dynamics of intracellular information decoding

    International Nuclear Information System (INIS)

    Kobayashi, Tetsuya J; Kamimura, Atsushi

    2011-01-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity

  16. Dynamics of intracellular information decoding.

    Science.gov (United States)

    Kobayashi, Tetsuya J; Kamimura, Atsushi

    2011-10-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

  17. HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

    NARCIS (Netherlands)

    STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

    1993-01-01

    It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

  18. Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates

    Directory of Open Access Journals (Sweden)

    Anja Kamp

    2016-11-01

    Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

  19. Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

    Science.gov (United States)

    Grimm, Marcus O. W.; Grösgen, Sven; Rothhaar, Tatjana L.; Burg, Verena K.; Hundsdörfer, Benjamin; Haupenthal, Viola J.; Friess, Petra; Müller, Ulrike; Fassbender, Klaus; Riemenschneider, Matthias; Grimm, Heike S.; Hartmann, Tobias

    2011-01-01

    Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology. PMID:21660213

  20. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

    NARCIS (Netherlands)

    Pannekoek, Yvonne; Morelli, Giovanna; Kusecek, Barica; Morré, Servaas A.; Ossewaarde, Jacobus M.; Langerak, Ankie A.; van der Ende, Arie

    2008-01-01

    BACKGROUND: The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome.

  1. Intracellular calcium homeostasis and signaling.

    Science.gov (United States)

    Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

    2013-01-01

    Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

  2. Microbial accumulation of uranium

    International Nuclear Information System (INIS)

    Zhang Wei; Dong Faqin; Dai Qunwei

    2005-01-01

    The mechanism of microbial accumulation of uranium and the effects of some factors (including pH, initial uranium concentration, pretreatment of bacteria, and so on) on microbial accumulation of uranium are discussed briefly. The research direction and application prospect are presented. (authors)

  3. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shangrong; Wang, Yifan; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

    2014-06-13

    Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

  4. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

    International Nuclear Information System (INIS)

    Zhang, Shangrong; Wang, Yifan; Li, Shu Jie

    2014-01-01

    Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor

  5. Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery

    DEFF Research Database (Denmark)

    Martinez, Virginia; Herencias, Cristina; Jurkevitch, Edouard

    2016-01-01

    This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered......% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures....

  6. Single-cell intracellular nano-pH probes†

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  7. Single-cell intracellular nano-pH probes.

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  8. Intracellular recording from a spider vibration receptor.

    Science.gov (United States)

    Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G

    2006-05-01

    The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

  9. Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

    Science.gov (United States)

    Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

    2018-01-01

    Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

  10. Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

    Directory of Open Access Journals (Sweden)

    Nor ‘Aini, A. R.

    2006-01-01

    Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

  11. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    International Nuclear Information System (INIS)

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-01-01

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  12. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jun [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Ren, Pingping; Zhang, Lin [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Wang, Xing Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Chen, Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Shen, Ying H., E-mail: hyshen@bcm.edu [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States)

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  13. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  14. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    thods of reduction of metal ions using plants or microorganisms are often ... have several advantages over bacteria, they are often pre- ferred. ... in static condition for a period of 7 days. ... work was focused on the production of intracellular gold.

  15. Mycobacterium intracellulare Infection Mimicking Progression of Scleroderma

    DEFF Research Database (Denmark)

    Krabbe, Simon; Engelhart, Merete; Thybo, Sören

    2017-01-01

    This case report describes a patient with scleroderma who developed Mycobacterium intracellulare infection, which for more than a year mimicked worsening of her connective tissue disorder. The patient was diagnosed with scleroderma based on puffy fingers that developed into sclerodactyly, abnormal......, unfortunately with significant scarring. Immunodeficiency testing was unremarkable. In summary, an infection with Mycobacterium intracellulare was mistaken for an unusually severe progression of scleroderma....

  16. Intracellular Localization of Arabidopsis Sulfurtransferases1

    Science.gov (United States)

    Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

    2004-01-01

    Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

  17. Ortholog-based screening and identification of genes related to intracellular survival.

    Science.gov (United States)

    Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

    2018-04-20

    Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

  18. Characteristics of cesium accumulation in the filamentous soil bacterium Streptomyces sp. K202

    International Nuclear Information System (INIS)

    Kuwahara, Chikako; Fukumoto, Atsushi; Nishina, Masami; Sugiyama, Hideo; Anzai, Yojiro; Kato, Fumio

    2011-01-01

    A filamentous soil bacterium, strain K202, was isolated from soil where an edible mushroom (Boletopsis leucomelas) was growing and identified as belonging to the genus Streptomyces on the basis of its morphological characteristics and the presence of LL-2, 6-diaminopimelic acid. We studied the existence states of Cs and its migration from extracellular to intracellular fluid in the mycelia of Streptomyces sp. K202. The results indicated that Cs accumulated in the cells through at least 2 steps: in the first step, Cs + was immediately and non-specifically adsorbed on the negatively charged cell surface, and in the second step, this adsorbed Cs + was taken up into the cytoplasm, and a part of the Cs entering the cytoplasm was taken up by an energy-dependent transport system(s). Further, we confirmed that a part of the Cs + was taken up into the mycelia competitively with K + , because K + uptake into the intact mycelia of the strain was significantly inhibited by the presence of Cs + in the culture media. This suggested that part of the Cs is transported by the potassium transport system. Moreover, 133 Cs-NMR spectra and SEM-EDX spectra of the mycelia that accumulated Cs showed the presence of at least 2 intracellular Cs states: Cs + trapped by intercellular materials such as polyphosphate and Cs + present in a cytoplasmic pool. - Research highlights: → Cs was taken up into the cells of Streptomyces sp. K202 via 2 steps. → The existence states of Cs accumulated in strain K202 were at least 2 types. → The localized Cs in the cells would be trapped by granules such as polyphosphate. → The localized Cs in the cells might involve in Cs detoxification of strain K202.

  19. Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

    Science.gov (United States)

    Bahr, Ben A; Wisniewski, Meagan L; Butler, David

    2012-04-01

    Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance

  20. Horizontal Transmission of Intracellular Insect Symbionts via Plants

    Directory of Open Access Journals (Sweden)

    Ewa Chrostek

    2017-11-01

    Full Text Available Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  1. Complexation of intracellular cyanide by hydroxocobalamin using a human cellular model.

    Science.gov (United States)

    Astier, A; Baud, F J

    1996-01-01

    1. The rational for administering hydroxocobalamin (OHCbl) as an antidote to cyanide poisoning is based on the high affinity of CN ion for cobalt compounds. However, only few data are available on the influence of OHCbl on the intracellular cyanide pool. 2. In human fibroblasts incubated for 10 min with 500 microM of [14C] cyanide, the accumulation ratio was 25 at 37 degrees C (10.45 +/- 1.51 mM) and 11.9 at 4 degrees C. 3. Using the monoblastic U-937 cell line, a rapid uptake of radioactive cyanide was observed with a maximum accumulation ratio of 1.97 at 5 min. 4. A linear relationship between cyanide uptake by U-937 cells and cyanide concentration in incubation medium (10-500 microM; 5 min) was found suggesting a first order process (k = 0.25 min-1). 5. After incubation of fibroblasts with 500 microM of OHCbl, a 75% decrease of intracellular cyanide was observed, with concomittant formation of intracellular cyanocobalamin CNCbl (intracellular/extracellular ratio: 158). 6. These findings suggest that OHCbl is able to penetrate into heavily cyanide loaded cells and to complex cyanide to the non-toxic CNCbl form.

  2. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Science.gov (United States)

    Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

    2012-01-01

    Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  3. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Directory of Open Access Journals (Sweden)

    Yves Briers

    Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  4. (13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

    Science.gov (United States)

    Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2015-12-01

    The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Uptake and intracellular accumulation of diamond nanoparticles – a metabolic and cytotoxic study

    Directory of Open Access Journals (Sweden)

    Antonín Brož

    2017-08-01

    Full Text Available Diamond nanoparticles, known as nanodiamonds (NDs, possess several medically significant properties. Having a tailorable and easily accessible surface gives them great potential for use in sensing and imaging applications and as a component of cell growth scaffolds. In this work we investigate in vitro interactions of human osteoblast-like SAOS-2 cells with four different groups of NDs, namely high-pressure high-temperature (HPHT NDs (diameter 18–210 nm, oxygen-terminated, photoluminescent HPHT NDs (diameter 40 nm, oxygen-terminated, detonation NDs (diameter 5 nm, H-terminated, and the same detonation NDs further oxidized by annealing at 450 °C. The influence of the NDs on cell viability and cell count was measured by the mitochondrial metabolic activity test and by counting cells with stained nuclei. The interaction of NDs with cells was monitored by phase contrast live-cell imaging in real time. For both types of oxygen-terminated HPHT NDs, the cell viability and the cell number remained almost the same for concentrations up to 100 µg/mL within the whole range of ND diameters tested. The uptake of hydrogen-terminated detonation NDs caused the viability and the cell number to decrease by 80–85%. The oxidation of the NDs hindered the decrease, but on day 7, a further decrease was observed. While the O-terminated NDs showed mechanical obstruction of cells by agglomerates preventing cell adhesion, migration and division, the H-terminated detonation NDs exhibited rapid penetration into the cells from the beginning of the cultivation period, and also rapid cell congestion and a rapid reduction in viability. These findings are discussed with reference to relevant properties of NDs such as surface chemical bonds, zeta potential and nanoparticle types.

  6. Uptake and intracellular accumulation of diamond nanoparticles – a metabolic and cytotoxic study

    Czech Academy of Sciences Publication Activity Database

    Brož, Antonín; Bačáková, Lucie; Štenclová, Pavla; Kromka, Alexander; Potocký, Štěpán

    2017-01-01

    Roč. 8, Aug 10 (2017), s. 1649-1657 ISSN 2190-4286 R&D Projects: GA ČR(CZ) GA17-19968S; GA MŠk LD15003; GA MZd(CZ) NV15-32497A Institutional support: RVO:67985823 ; RVO:68378271 Keywords : cell viability * FTIR * live-cell imaging * MTS * nanodiamond * SAOS-2 cells Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Biomaterials (as related to medical implants, devices, sensors) Impact factor: 3.127, year: 2016

  7. Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

    Czech Academy of Sciences Publication Activity Database

    Zelenka, Jaroslav; Muchová, L.; Zelenková, M.; Váňová, K.; Vreman, H.J.; Wong, R.J.; Vítek, L.

    2012-01-01

    Roč. 94, č. 8 (2012), s. 1821-1827 ISSN 0300-9084 Grant - others:GA MZd(CZ) NT11327 Institutional research plan: CEZ:AV0Z50110509 Keywords : bilirubin * heme oxygenase * hyperbilirubinemia * lipopolysacccharide * oxidative stress Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 3.142, year: 2012

  8. Cadmium accumulation by the marine red alga Porphyra umbilicalis

    Energy Technology Data Exchange (ETDEWEB)

    McLean, M.W.; Williamson, F.B.

    1977-01-01

    The characteristics of cadmium accumulation by the marine red alga Porphyra umbilicalis L. in culture are reported. The time course of uptake under various light conditions shows that cadmium is concentrated as the result of an on-going anabolic process and not as a consequence of a pH gradient as provided by photosynthesis. The effect of cycloheximide is in agreement with de novo protein synthesis being a prerequisite for cadmium accumulation. Autoradiography suggests a specific intracellular location for bound cadmium--apparently the nucleus.

  9. Plastids and Carotenoid Accumulation.

    Science.gov (United States)

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants.

  10. Mechanisms of intrahepatic triglyceride accumulation

    Science.gov (United States)

    Ress, Claudia; Kaser, Susanne

    2016-01-01

    Hepatic steatosis defined as lipid accumulation in hepatocytes is very frequently found in adults and obese adolescents in the Western World. Etiologically, obesity and associated insulin resistance or excess alcohol intake are the most frequent causes of hepatic steatosis. However, steatosis also often occurs with chronic hepatitis C virus (HCV) infection and is also found in rare but potentially life-threatening liver diseases of pregnancy. Clinical significance and outcome of hepatic triglyceride accumulation are highly dependent on etiology and histological pattern of steatosis. This review summarizes current concepts of pathophysiology of common causes of hepatic steatosis, including non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, chronic HCV infections, drug-induced forms of hepatic steatosis, and acute fatty liver of pregnancy. Regarding the pathophysiology of NAFLD, this work focuses on the close correlation between insulin resistance and hepatic triglyceride accumulation, highlighting the potential harmful effects of systemic insulin resistance on hepatic metabolism of fatty acids on the one side and the role of lipid intermediates on insulin signalling on the other side. Current studies on lipid droplet morphogenesis have identified novel candidate proteins and enzymes in NAFLD. PMID:26819531

  11. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    Science.gov (United States)

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

  12. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling.

    Science.gov (United States)

    Noctor, Graham; Foyer, Christine H

    2016-07-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. © 2016 American Society of Plant Biologists. All Rights Reserved.

  13. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

    Science.gov (United States)

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-07-17

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

  14. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence.

    Directory of Open Access Journals (Sweden)

    Robert A Colvin

    Full Text Available Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX and two regions of the hippocampus: dentate gyrus (DG and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2-3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.

  16. LDL Receptors as Gateways for Intracellular Porphyrin Uptake

    International Nuclear Information System (INIS)

    Novick, S.; Laster, B.; Quastel, M.

    2004-01-01

    Boronated compounds are currently being studied for possible use in Boron Neutron Capture Therapy (BNCT). We found that one of these agents, BOPP (tetrakis-carborane-carboxylate, esters of 2,4-bis (a,b- dihydroxyethyl) deuteroporphyrin IX), could also be labeled with indium (In-BOPP) and, therefore, could also be used potentially to transport high Z atoms into tumor cell DNA for AET (Auger Electron Therapy). In order to assess the uptake of these agents into cells, the role of the LDL receptor in the intracellular accumulation of BOPP and In-BOPP was investigated. Pre-incubation of V-79 Chinese hamster cells in medium containing delipidized fetal bovine serum (FBS) markedly increased the subsequent uptake of intracellular boron transported by both BOPP and In-BOPP when compared with cells that had been pre-incubated with medium containing 10% normal FBS (lipidized). The increased uptake was characterized by elevated levels of receptor, and greater affinity was shown for both BOPP and In-BOPP, although less marked with the latter. Positive cooperativity was demonstrated by sigmoid saturation curves, Scatchard analysis and Hill plots. Increasing the amount of LDL in the incubation medium had a relatively small effect on the total accumulation of either indium or boron atoms inside the cell. Furthermore, chemical acetylation of LDL did not decrease the intracellular uptake of either boron or indium transported by BOPP or In-BOPP. It is thus concluded that BOPP and In-BOPP preferentially enter the cells directly by way of the LDL receptor and that only a small fraction of these molecules are transported into the cells indirectly using serum LDLs as their carriers. These data suggest a novel way of bringing greater amounts of boron and indium (and perhaps other agents) into tissues. Porphyrins can be used to transport different agents into tumor cells because they are tumor affinic molecules. Tumors express a higher number of LDL receptors than do most normal tissues

  17. The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

    Science.gov (United States)

    Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin

    2015-03-24

    Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside

  18. Accumulation by Conservation

    NARCIS (Netherlands)

    Büscher, Bram; Fletcher, Robert

    2015-01-01

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

  19. Accumulation by Conservation

    NARCIS (Netherlands)

    Büscher, Bram; Fletcher, Robert

    2014-01-01

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

  20. Creation / accumulation city

    NARCIS (Netherlands)

    Doevendans, C.H.; Schram, A.L.

    2005-01-01

    A distinction between basic archetypes of urban form was made by Bruno Fortier: the accumulation city as opposed to the creation city. These archetypes derive from archaeology - being based on the Roman and the Egyptian city - but are interpreted as morphological paradigms, as a set of assumptions

  1. Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

    Science.gov (United States)

    Eid, Islam; Elsebaei, Mohamed M; Mohammad, Haroon; Hagras, Mohamed; Peters, Christine E; Hegazy, Youssef A; Cooper, Bruce; Pogliano, Joe; Pogliano, Kit; Abulkhair, Hamada S; Seleem, Mohamed N; Mayhoub, Abdelrahman S

    2017-10-20

    The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing

    DEFF Research Database (Denmark)

    Thomas Danielsen, E.; E. Møller, Morten; Yamanaka, Naoki

    2016-01-01

    Steroid hormones control important developmental processes and are linked to many diseases. To systematically identify genes and pathways required for steroid production, we performed a Drosophila genome-wide in vivo RNAi screen and identified 1,906 genes with potential roles in steroidogenesis...... and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. We identify a conserved fatty acid elongase that underlies a mechanism that adjusts cholesterol trafficking and steroidogenesis with nutrition...... and developmental programs. In addition, we demonstrate the existence of an autophagosomal cholesterol mobilization mechanism and show that activation of this system rescues Niemann-Pick type C1 deficiency that causes a disorder characterized by cholesterol accumulation. These cholesterol-trafficking mechanisms...

  3. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    Science.gov (United States)

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  4. Cationic polymers for intracellular delivery of proteins

    NARCIS (Netherlands)

    Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Samal, Sangram; Dubruel, Peter

    2015-01-01

    Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering

  5. Molecular detection and characterization of sustainable intracellular ...

    African Journals Online (AJOL)

    3Centre for Biopolymer and Bio-Molecular Research, Athlone College of Technology, Republic of Ireland. ... cells was associated with the elongation of micro-villar extension that ... Keywords: Intracellular contaminants, cell cultures, bacteria culture, pre-clinical studies. ... production work involving culture technology.

  6. Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

    Science.gov (United States)

    Hafner, Anne E.; Rieger, Heiko

    2018-03-01

    The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

  7. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent. Production of nanoparticles was confirmed by the colour ...

  8. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

    Science.gov (United States)

    Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

    2018-03-20

    Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

  9. Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

    International Nuclear Information System (INIS)

    Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

    1987-01-01

    We studied total cell-associated A14-[ 125 I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[ 125 I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[ 125 I]insulin, [ 125 I]iodide, or [ 125 I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients

  10. Accumulation of satellites

    International Nuclear Information System (INIS)

    Safronov, V.S.; Ruskol, E.L.

    1977-01-01

    Formation and evolution of circumplanetary satellite swarms are investigated. Characteristic times of various processes are estimated. The characteristic time for the accumulation of the bodies in the swarm was several orders of magnitude shorter than that of the planet, i.e. than the time of the replenishment of the material by the swarm (10 8 yr). The model of the accumulation of the swarm is constructed taking into account the increase of its mass due to trapping of heliocentrically moving particles and its decrease due to outfall of the inner part of the swarm onto the growing planet. The accumulation of circumplanetary bodies is also considered. The main features of the evolution of the swarm essentially depend on the size distribution of bodies in the swarm and in the zone of the planet and also on the degree of the concentration of the swarm mass toward the planet. If the sum of the exponents of the inverse power laws of these distributions is less than 7, the model of the transparent swarm developed in this paper should be preferred. When this sum is greater than 7, the model of opaque swarm suggested by A. Harris and W.M. Kaula is better. There is predominant trapping of small particles into the swarm due to their more frequent collisions. Optical thickness of the protoplanetary cloud in radial direction is estimated. It is shown that at the final stage of the planetary accumulation, the cloud was semitransparent in the region of terrestrial planets and volatile substances evaporated at collisions could be swept out from the outer parts of the satellite swarm by the solar wind

  11. Antiproton Accumulator (AA)

    CERN Multimedia

    Photographic Service

    1980-01-01

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

  12. Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

    Science.gov (United States)

    Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Valius, Mindaugas; Rotomskis, Ricardas

    2013-01-01

    Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison

  13. A new mercury-accumulating Mucor hiemalis strain EH8 from cold sulfidic spring water biofilms.

    Science.gov (United States)

    Hoque, Enamul; Fritscher, Johannes

    2016-10-01

    Here, we report about a unique aquatic fungus Mucor hiemalisEH8 that can remove toxic ionic mercury from water by intracellular accumulation and reduction into elemental mercury (Hg 0 ). EH8 was isolated from a microbial biofilm grown in sulfidic-reducing spring water sourced at a Marching's site located downhill from hop cultivation areas with a history of mercury use. A thorough biodiversity survey and mercury-removal function analyses were undertaken in an area of about 200 km 2 in Bavaria (Germany) to find the key biofilm and microbe for mercury removal. After a systematic search using metal removal assays we identified Marching spring's biofilm out of 18 different sulfidic springs' biofilms as the only one that was capable of removing ionic Hg from water. EH8 was selected, due to its molecular biological identification as the key microorganism of this biofilm with the capability of mercury removal, and cultivated as a pure culture on solid and in liquid media to produce germinating sporangiospores. They removed 99% of mercury from water within 10-48 h after initial exposure to Hg(II). Scanning electron microscopy demonstrated occurrence of intracellular mercury in germinating sporangiospores exposed to mercury. Not only associated with intracellular components, but mercury was also found to be released and deposited as metallic-shiny nanospheres. Electron-dispersive x-ray analysis of such a nanosphere confirmed presence of mercury by the HgM α peak at 2.195 keV. Thus, a first aquatic eukaryotic microbe has been found that is able to grow even at low temperature under sulfur-reducing conditions with promising performance in mercury removal to safeguard our environment from mercury pollution. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  14. Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

    Directory of Open Access Journals (Sweden)

    Jiang LQ

    2017-08-01

    Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

  15. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    Science.gov (United States)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  16. Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

  17. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  18. Dynamics of gradient formation by intracellular shuttling

    Energy Technology Data Exchange (ETDEWEB)

    Berezhkovskii, Alexander M. [Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 (United States); Shvartsman, Stanislav Y. [Department of Chemical and Biological Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544 (United States)

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  19. Leishmania hijacking of the macrophage intracellular compartments.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Loiseau, Philippe M

    2016-02-01

    Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

  20. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-05-01

    The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

  1. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-01-01

    The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

  2. Intracellular serpins, firewalls and tissue necrosis.

    Science.gov (United States)

    Marciniak, Stefan J; Lomas, David A

    2008-02-01

    Luke and colleagues have recently attributed a new role to a member of the serpin superfamily of serine proteinase inhibitors. They have used Caenorhabditis elegans to show that an intracellular serpin is crucial for maintaining lysosomal integrity. We examine the role of this firewall in preventing necrosis and attempt to integrate this with current theories of stress-induced protein degradation. We discuss how mutant serpins cause disease either through polymerization or now, perhaps, by unleashing necrosis.

  3. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  4. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  5. Impact of intracellular metallothionein on metal biouptake and partitioning dynamics at bacterial interfaces.

    Science.gov (United States)

    Présent, Romain M; Rotureau, Elise; Billard, Patrick; Pagnout, Christophe; Sohm, Bénédicte; Flayac, Justine; Gley, Renaud; Pinheiro, José P; Duval, Jérôme F L

    2017-11-08

    Genetically engineered microorganisms are alternatives to physicochemical methods for remediation of metal-contaminated aquifers due to their remarkable bioaccumulation capacities. The design of such biosystems would benefit from the elaboration of a sound quantitative connection between performance in terms of metal removal from aqueous solution and dynamics of the multiscale processes leading to metal biouptake. In this work, this elaboration is reported for Escherichia coli cells modified to overexpress intracellular metallothionein (MTc), a strong proteinaceous metal chelator. Depletion kinetics of Cd(ii) from bulk solution following biouptake and intracellular accumulation is addressed as a function of cell volume fraction using electroanalytical probes and ligand exchange-based analyses. It is shown that metal biouptake in the absence and presence of MTc is successfully interpreted on the basis of a formalism recently developed for metal partitioning dynamics at biointerfaces with integration of intracellular metal speciation. The analysis demonstrates how fast sequestration of metals by intracellular MTc bypasses metal excretion (efflux) and enhances the rate of metal depletion to an extent such that complete removal is achieved at sufficiently large cell volume fractions. The magnitude of the stability constant of nanoparticulate metal-MTc complexes, as derived from refined analysis of macroscopic bulk metal depletion data, is further confirmed by independent electrochemical measurement of metal binding by purified MTc extracts.

  6. Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

    Directory of Open Access Journals (Sweden)

    Bush Ashley I

    2009-01-01

    Full Text Available Abstract Background The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic and V18A (NIC. Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism.

  7. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

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

  8. Solids Accumulation Scouting Studies

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-26

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

  9. The Original Linguistic Accumulation

    DEFF Research Database (Denmark)

    Bjerre, Henrik Jøker

    2008-01-01

    This paper represents an attempt at identifying a lack (of a lack) in analytic philosophy. It claims that one of the central features common to a variety of analytic philosophies is the absence of an investigation of what Jacques Lacan has identified as the lack of being (manque à être). This lac...

  10. [Hyperosmolarity: Intracellular effects and implication in dry eye disease].

    Science.gov (United States)

    Warcoin, E; Clouzeau, C; Brignole-Baudouin, F; Baudouin, C

    2016-09-01

    Dry eye disease is a multifactorial disease affecting the lacrimal functional unit and which has a significant impact on the quality of life of patients. This pathology works as a vicious circle at the ocular surface in which hyperosmolarity of the tear film plays a key role. This review intends to describe the different reported intracellular effects induced by hyperosmolarity in cells: alteration of cytoskeleton, cell cycle slowdown, adaptation mechanisms triggered as restoration of cell volume and accumulation of compatible osmolytes, the crucial role of the osmoprotectant factor Nuclear Factor of the Activated T cells-5 (NFAT5), apoptosis, as well as oxidative stress and inflammatory responses caused by this particular condition. Reported effects of hyperosmolarity in the experimental studies specific of dry eye disease concerning ocular surface cells will be described in parallel. Indeed, these data allow to understand a part of the pathophysiology of the disease, and specially the links between tear hyperosmolarity and inflammation of the ocular surface, the second key of the pathology phenomenon. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

    International Nuclear Information System (INIS)

    Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C.

    2007-01-01

    Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane

  12. Intracellular fate of Ureaplasma parvum entrapped by host cellular autophagy.

    Science.gov (United States)

    Nishiumi, Fumiko; Ogawa, Michinaga; Nakura, Yukiko; Hamada, Yusuke; Nakayama, Masahiro; Mitobe, Jiro; Hiraide, Atsushi; Sakai, Norio; Takeuchi, Makoto; Yoshimori, Tamotsu; Yanagihara, Itaru

    2017-06-01

    Genital mycoplasmas, including Ureaplasma spp., are among the smallest human pathogenic bacteria and are associated with preterm birth. Electron microscopic observation of U. parvum showed that these prokaryotes have a regular, spherical shape with a mean diameter of 146 nm. U. parvum was internalized into HeLa cells by clathrin-mediated endocytosis and survived for at least 14 days around the perinuclear region. Intracellular U. parvum reached endosomes in HeLa cells labeled with EEA1, Rab7, and LAMP-1 within 1 to 3 hr. After 3 hr of infection, U. parvum induced the cytosolic accumulation of galectin-3 and was subsequently entrapped by the autophagy marker LC3. However, when using atg7 -/- MEF cells, autophagy was inadequate for the complete elimination of U. parvum in HeLa cells. U. parvum also colocalized with the recycling endosome marker Rab11. Furthermore, the exosomes purified from infected HeLa cell culture medium included U. parvum. In these purified exosomes ureaplasma lipoprotein multiple banded antigen, host cellular annexin A2, CD9, and CD63 were detected. This research has successfully shown that Ureaplasma spp. utilize the host cellular membrane compartments possibly to evade the host immune system. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  13. TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2016-01-01

    Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.

  14. Azithromycin effectiveness against intracellular infections of Francisella

    Directory of Open Access Journals (Sweden)

    Mann Barbara J

    2010-04-01

    Full Text Available Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F. tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS (Type B was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA were found to be less sensitive in vitro to Az compared to the wild

  15. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    International Nuclear Information System (INIS)

    Akter, Jesmin; Takatori, Atsushi; Islam, Md. Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

    2014-01-01

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas

  16. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Akter, Jesmin [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Islam, Md. Sazzadul [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakazawa, Atsuko [Department of Pathology, National Center for Child Health and Development, Tokyo (Japan); Ozaki, Toshinori, E-mail: tozaki@chiba-cc.jp [Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nagase, Hiroki [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakagawara, Akira [Saga Medical Centre, 840-8571 (Japan)

    2014-10-10

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.

  17. Disseminated Mycobacterium avium--intracellulare complex infection in a miniature schnauzer.

    Science.gov (United States)

    Miller, M A; Greene, C E; Brix, A E

    1995-01-01

    A two-year-old, spayed female, miniature schnauzer was evaluated for respiratory distress associated with a compressive cervical mass. Generalized mycobacterial infection was diagnosed from aspirates of several enlarged lymph nodes. Tissue specimens further identified Mycobacterium avium--intracellulare using polymerase chain reaction followed by nucleic acid hybridization. Treatment with enrofloxacin, clofazamine, rifampin, and interferon did not result in long-term success.

  18. Evaluation of cell factory performance through determination of intracellular metabolites using LC-MS/MS

    DEFF Research Database (Denmark)

    Magdenoska, Olivera; Martinussen, Jan; Nielsen, Kristian Fog

    2012-01-01

    . By quantification of intracellular metabolite changes over time, under given genetic and environmental perturbations, key regulatory nodes may be identified in the cellular metabolic network. In target metabolome analysis, the quantification is applied to only one or a small group of metabolites and is very useful...

  19. The role of intracellular thyroid hormone metabolism in innate immune cells

    NARCIS (Netherlands)

    van der Spek, A.H.

    2018-01-01

    Innate immune cells have recently been identified as important thyroid hormone target cells. This thesis studies the role of intracellular thyroid hormone metabolism in the function of neutrophils and macrophages, two essential cell types of the innate immune system. Neutrophils, monocytes and

  20. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

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

  1. Selenium accumulation in the cockle Anadara trapezia

    International Nuclear Information System (INIS)

    Jolley, Dianne F.; Maher, William A.; Kyd, Jennelle

    2004-01-01

    An extensive study on Se accumulation in a population of Anadara trapezia from a marine lake is reported. The effects of organism mass, gender, reproductive cycle, and season on Se accumulation and tissue distribution were investigated. Analyses showed that gender and reproductive cycle had no significant effect on Se accumulation. A. trapezia showed a strong positive correlation between Se burden and tissue mass. Constant Se concentrations were observed within individual populations but varied spatially with sediment Se concentrations. Se concentrations in tissues decreased from gills > gonad/intestine > mantle > muscle > foot, which remained constant over 12 months, however, significantly lower concentrations were observed in the summer compared to winter. A. trapezia is a good biomonitor for Se, as gender and size do not effect concentration, however, season of collection must be reported if changes in Se bioavailability are to be identified in short term studies, or during intersite comparisons. - Capsule: The marine bivalve Anadara trapezia is a good bioindicator for marine selenium contamination

  2. Batteries and accumulators in France

    International Nuclear Information System (INIS)

    2012-12-01

    The present report gives an overview of the batteries and accumulators market in France in 2011 based on the data reported through ADEME's Register of Batteries and accumulators. In 2001, the French Environmental Agency, known as ADEME, implemented a follow-up of the batteries and accumulators market, creating the Observatory of batteries and accumulators (B and A). In 2010, ADEME created the National Register of producers of Batteries and Accumulators in the context of the implementation of the order issued on November 18, 2009. This is one of the four enforcement orders for the decree 2009-1139 issued on September 22, 2009, concerning batteries and accumulators put on the market and the disposal of waste batteries and accumulators, and which transposes the EU-Directive 2006/66/CE into French law. This Register follows the former Observatory for batteries and accumulators. This Register aims to record the producers on French territory and to collect the B and A producers and recycling companies' annual reporting: the regulation indeed requires that all B and A producers and recycling companies report annually on the Register the quantities of batteries and accumulators they put on the market, collect and treat. Based on this data analysis, ADEME issues an annual report allowing both the follow-up of the batteries and accumulators market in France and communication regarding the achievement of the collection and recovery objectives set by EU regulation. This booklet presents the situation in France in 2011

  3. Model Linking Plasma and Intracellular Tenofovir/Emtricitabine with Deoxynucleoside Triphosphates.

    Directory of Open Access Journals (Sweden)

    Xinhui Chen

    Full Text Available The coformulation of the nucleos(tide analogs (NA tenofovir (TFV disoproxil fumarate (TDF and emtricitabine (FTC is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP and FTC-triphosphate (FTC-TP. Such complexities manifest in nonlinear intracellular pharmacokinetics (PK. In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD study among forty subjects receiving daily TDF/FTC (300 mg/200 mg from the first-dose to pharmacological intracellular steady-state (30 days. TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM. Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP and deoxycytidine triphosphate (dCTP production were 1020 fmol/106 cells (130% and 44.4 pmol/106 cells (82.5%, resulting in (90% prediction interval 11% (0.45%, 53% and 14% (2.6%, 35% reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP. Simulation

  4. Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

    Directory of Open Access Journals (Sweden)

    M. Malatesta

    2012-04-01

    Full Text Available Chitosan-based nanoparticles (NPs deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

  5. Quantification and isotopic analysis of intracellular sulfur metabolites in the dissimilatory sulfate reduction pathway

    Science.gov (United States)

    Sim, Min Sub; Paris, Guillaume; Adkins, Jess F.; Orphan, Victoria J.; Sessions, Alex L.

    2017-06-01

    Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in 34S and producing sulfide that is depleted in 34S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfur isotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellular mechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopic ratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis. Intracellular sulfate and APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulates in the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in 34S. The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfate and sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the net fractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This 'reservoir effect' diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations. Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the overall (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for the well-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while at the same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in this relationship. Our approach, together with ongoing investigations of the kinetic isotope

  6. Intracellular bacteria: the origin of dinoflagellate toxicity.

    Science.gov (United States)

    Silva, E S

    1990-01-01

    Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

  7. Fluorescent nanoparticles for intracellular sensing: a review.

    Science.gov (United States)

    Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

    2012-11-02

    Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Mechanisms of cellular invasion by intracellular parasites.

    Science.gov (United States)

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  9. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  10. Detection of a variable intracellular acid-labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system.

    Science.gov (United States)

    Isensee, Kirsten; Erez, Jonathan; Stoll, Heather M

    2014-02-01

    Accumulation of an intracellular pool of carbon (C(i) pool) is one strategy by which marine algae overcome the low abundance of dissolved CO2 (CO2 (aq) ) in modern seawater. To identify the environmental conditions under which algae accumulate an acid-labile C(i) pool, we applied a (14) C pulse-chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid-labile C(i) pools. C(i) pools are measureable in cells cultured in media with 2-30 µmol l(-1) CO2 (aq), corresponding to a medium pH of 8.6-7.9. The absolute C(i) pool was greater for the larger celled diatoms. For both algal classes, the C(i) pool became a negligible contributor to photosynthesis once CO2 (aq) exceeded 30 µmol l(-1) . Combining the (14) C pulse-chase method and (14) C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO2 (aq) . We showed that the C(i) pool decreases with higher CO2 :HCO3 (-) uptake rates. © 2013 Scandinavian Plant Physiology Society.

  11. Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

    Science.gov (United States)

    Hirokawa, N.; Takemura, R.

    Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

  12. Measuring intracellular redox conditions using GFP-based sensors

    DEFF Research Database (Denmark)

    Björnberg, Olof; Ostergaard, Henrik; Winther, Jakob R

    2006-01-01

    Recent years have seen the development of methods for analyzing the redox conditions in specific compartments in living cells. These methods are based on genetically encoded sensors comprising variants of Green Fluorescent Protein in which vicinal cysteine residues have been introduced at solvent......-exposed positions. Several mutant forms have been identified in which formation of a disulfide bond between these cysteine residues results in changes of their fluorescence properties. The redox sensors have been characterized biochemically and found to behave differently, both spectroscopically and in terms...... of redox properties. As genetically encoded sensors they can be expressed in living cells and used for analysis of intracellular redox conditions; however, which parameters are measured depends on how the sensors interact with various cellular redox components. Results of both biochemical and cell...

  13. An active matter analysis of intracellular Active Transport

    Science.gov (United States)

    Wang, Bo; Chen, Kejia; Bae, Sung Chul; Granick, Steve

    2012-02-01

    Tens of thousands of fluorescence-based trajectories at nm resolution have been analyzed, regarding active transport along microtubules in living cells. The following picture emerges. Directed motion to pre-determined locations is certainly an attractive idea, but cannot be pre-programmed as to do so would sacrifice adaptability. The polarity of microtubules is inadequate to identify these directions in cells, and no other mechanism is currently known. We conclude that molecular motors carry cargo through disordered intracellular microtubule networks in a statistical way, with loud cellular ``noise'' both in directionality and speed. Programmed random walks describe how local 1D active transport traverses crowded cellular space efficiently, rapidly, minimizing the energy waste that would result from redundant activity. The mechanism of statistical regulation is not yet understood, however.

  14. Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

    Directory of Open Access Journals (Sweden)

    Audrey Bernut

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

  15. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    Science.gov (United States)

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

    Science.gov (United States)

    Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

    2017-01-01

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

  17. Characterization of distinct Arctic aerosol accumulation modes and their sources

    DEFF Research Database (Denmark)

    Lange, R.; Dall'Osto, M.; Skov, H.

    2018-01-01

    -August). By association to chemical composition, cloud condensation nuclei properties, and meteorological variables, three typical accumulation mode aerosol clusters were identified: Haze (32% of the time), Bimodal (14%) and Aged (6%). In brief: (1) Haze accumulation mode aerosol shows a single mode at 150 nm, peaking...

  18. Colchicine induced intraneuronal free zinc accumulation and dentate granule cell degeneration.

    Science.gov (United States)

    Choi, Bo Young; Lee, Bo Eun; Kim, Jin Hee; Kim, Hyun Jung; Sohn, Min; Song, Hong Ki; Chung, Tae Nyoung; Suh, Sang Won

    2014-08-01

    Colchicine has been discovered to inhibit many inflammatory processes such as gout, familial Mediterranean fever, pericarditis and Behcet disease. Other than these beneficial anti-inflammatory effects, colchicine blocks microtubule-assisted axonal transport, which results in the selective loss of dentate granule cells of the hippocampus. The mechanism of the colchicine-induced dentate granule cell death and depletion of mossy fiber terminals still remains unclear. In the present study, we hypothesized that colchicine-induced dentate granule cell death may be caused by accumulation of labile intracellular zinc. 10 μg kg(-1) of colchicine was injected into the adult rat hippocampus and then brain sections were evaluated at 1 day or 1 week later. Neuronal cell death was evaluated by H&E staining or Fluoro-Jade B. Zinc accumulation and vesicular zinc were detected by N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide (TSQ) staining. To test whether an extracellular zinc chelator can prevent this process, CaEDTA was injected into the hippocampus over a 5 min period with colchicine. To test whether other microtubule toxins also produce similar effects as colchicine, vincristine was injected into the hippocampus. The present study found that colchicine injection induced intracellular zinc accumulation in the dentate granule cells and depleted vesicular zinc from mossy fiber terminals. Injection of a zinc chelator, CaEDTA, did not block the zinc accumulation and neuronal death. Vincristine also produced intracellular zinc accumulation and neuronal death. These results suggest that colchicine-induced dentate granule cell death is caused by blocking axonal zinc flow and accumulation of intracellular labile zinc.

  19. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich

    2018-01-01

    Abstract Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. PMID:28913971

  20. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F L; Leonhardt, Heinrich; Hackenberger, Christian P R

    2018-02-23

    Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  3. Cardiac lipid accumulation associated with diastolic dysfunction in obese mice

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Bollano, Entela; Lindegaard, Marie L S

    2003-01-01

    Obesity may confer cardiac dysfunction due to lipid accumulation in cardiomyocytes. To test this idea, we examined whether obese ob/ob mice display heart lipid accumulation and cardiac dysfunction. Ob/ob mouse hearts had increased expression of genes mediating extracellular generation, transport....../ob mice and 2.5 +/- 0.1 in ob/+ mice (P = 0.0001). In contrast, the indexes of systolic function and heart brain natriuretic peptide mRNA expression were only marginally affected and unaffected, respectively, in ob/ob compared with ob/+ mice. The results suggest that ob/ob mouse hearts have increased...... across the myocyte cell membrane, intracellular transport, mitochondrial uptake, and beta-oxidation of fatty acids compared with ob/+ mice. Accordingly, ob/ob mouse hearts contained more triglyceride (6.8 +/- 0.4 vs. 2.3 +/- 0.4 microg/mg; P hearts. Histological examinations...

  4. [Limbic encephalitis with antibodies against intracellular antigens].

    Science.gov (United States)

    Morita, Akihiko; Kamei, Satoshi

    2010-04-01

    Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

  5. Implications of metal accumulation mechanisms to phytoremediation.

    Science.gov (United States)

    Memon, Abdul R; Schröder, Peter

    2009-03-01

    example, glutathione (GSH), a precursor of phytochelatin synthesis, plays a key role not only in metal detoxification but also in protecting plant cells from other environmental stresses including intrinsic oxidative stress reactions. In the last decade, tremendous developments in molecular biology and success of genomics have highly encouraged studies in molecular genetics, mainly transcriptomics, to identify functional genes implied in metal tolerance in plants, largely belonging to the metal homeostasis network. Analyzing the genetics of metal accumulation in these accumulator plants has been greatly enhanced through the wealth of tools and the resources developed for the study of the model plant Arabidopsis thaliana such as transcript profiling platforms, protein and metabolite profiling, tools depending on RNA interference (RNAi), and collections of insertion line mutants. To understand the genetics of metal accumulation and adaptation, the vast arsenal of resources developed in A. thaliana could be extended to one of its closest relatives that display the highest level of adaptation to high metal environments such as A. halleri and T. caerulescens. This review paper deals with the mechanisms of heavy metal accumulation and tolerance in plants. Detailed information has been provided for metal transporters, metal chelation, and oxidative stress in metal-tolerant plants. Advances in phytoremediation technologies and the importance of metal accumulator plants and strategies for exploring these immense and valuable genetic and biological resources for phytoremediation are discussed. A number of species within the Brassicaceae family have been identified as metal accumulators. To understand fully the genetics of metal accumulation, the vast genetic resources developed in A. thaliana must be extended to other metal accumulator species that display traits absent in this model species. A. thaliana microarray chips could be used to identify differentially expressed genes in

  6. Novel robust biomarkers for human bladder cancer based on activation of intracellular signaling pathways

    OpenAIRE

    Lezhnina, Ksenia; Kovalchuk, Olga; Zhavoronkov, Alexander A.; Korzinkin, Mikhail B.; Zabolotneva, Anastasia A.; Shegay, Peter V.; Sokov, Dmitry G.; Gaifullin, Nurshat M.; Rusakov, Igor G.; Aliper, Alexander M.; Roumiantsev, Sergey A.; Alekseev, Boris Y.; Borisov, Nikolay M.; Buzdin, Anton A.

    2014-01-01

    We recently proposed a new bioinformatic algorithm called OncoFinder for quantifying the activation of intracellular signaling pathways. It was proved advantageous for minimizing errors of high-throughput gene expression analyses and showed strong potential for identifying new biomarkers. Here, for the first time, we applied OncoFinder for normal and cancerous tissues of the human bladder to identify biomarkers of bladder cancer. Using Illumina HT12v4 microarrays, we profiled gene expression ...

  7. Bladder-type hydropneumatic accumulators

    International Nuclear Information System (INIS)

    Anigas, F.

    1985-01-01

    Hydropneumatic pressure accumulators allow liquids to be stored under pressure, their operating principle being based on the inherent compressibility of elements in a liquid and gaseous state. A wide range of fluids can be covered by means of the appropriate choice of the material for the body and bladder. Their main applications are: energy accumulation, safety reserve, suspension. (author)

  8. Nuclear translocation of IGF1R by intracellular amphiregulin contributes to the resistance of lung tumour cells to EGFR-TKI.

    Science.gov (United States)

    Guerard, Marie; Robin, Thomas; Perron, Pascal; Hatat, Anne-Sophie; David-Boudet, Laurence; Vanwonterghem, Laetitia; Busser, Benoit; Coll, Jean-Luc; Lantuejoul, Sylvie; Eymin, Beatrice; Hurbin, Amandine; Gazzeri, Sylvie

    2018-04-28

    Many Receptor Tyrosine Kinases translocate from the cell surface to the nucleus in normal and pathological conditions, including cancer. Here we report the nuclear expression of insulin-like growth factor-1 receptor (IGF1R) in primary human lung tumours. Using lung cancer cell lines and lung tumour xenografts, we demonstrate that the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib induces the nuclear accumulation of IGF1R in mucinous lung adenocarcinoma by a mechanism involving the intracellular re-localization of the growth factor amphiregulin. Amphiregulin allows the binding of IGF1R to importin-β1 and promotes its nuclear transport. The nuclear accumulation of IGF1R by amphiregulin induces cell cycle arrest through p21 WAF1/CIP1 upregulation, and prevents the induction of apoptosis in response to gefitinib. These results identify amphiregulin as the first nuclear localization signal-containing protein that interacts with IGF1R and allows its nuclear translocation. Furthermore they indicate that nuclear expression of IGF1R contributes to EGFR-TKI resistance in lung cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Credit Market Development and Human Capital Accumulation

    OpenAIRE

    Ho, Wai-Hong

    2008-01-01

    In a two period overlapping generations economy with asymmetric information, we investigate the interaction between credit market development and human capital accumulation. As is typical, young borrowers supply their endowed unit of labor time to earn wage income which is used as internal funds. In contrast to conventional setups, young lenders distribute theirs between acquiring education and working for earnings. Through identifying the risk types of borrowers by a costly screening tech...

  10. Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

    Directory of Open Access Journals (Sweden)

    Muriaux Delphine

    2007-08-01

    Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

  11. Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ming-Ju Chou

    2018-02-01

    Full Text Available Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

  12. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    Science.gov (United States)

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  13. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    Science.gov (United States)

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  14. MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

    International Nuclear Information System (INIS)

    Liu Xiaolong; Yuan Zhenglong; Chung, Maureen

    2008-01-01

    MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5

  15. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    International Nuclear Information System (INIS)

    Verdin, Anthony; Lounes-Hadj Sahraoui, Anissa; Newsam, Ray; Robinson, Gary; Durand, Roger

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities

  16. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Verdin, Anthony [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France); Lounes-Hadj Sahraoui, Anissa [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)]. E-mail: lounes@univ-littoral.fr; Newsam, Ray [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Robinson, Gary [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Durand, Roger [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities.

  17. Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Allen, J.C.; Navran, S.S.; Seidel, C.L.; Dennison, D.K.; Amann, J.M.; Jemelka, S.K.

    1989-01-01

    Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3 H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86 Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [ 3 H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation

  18. The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

    Science.gov (United States)

    Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-06-27

    Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Strain accumulation in quasicrystalline solids

    International Nuclear Information System (INIS)

    Nori, F.; Ronchetti, M.; Elser, V.

    1988-01-01

    We study the relaxation of 2D quasicrystalline elastic networks when their constituent bonds are perturbed homogeneously. Whereas ideal, quasiperiodic networks are stable against such perturbations, we find significant accumulations of strain in a class of disordered networks generated by a growth process. The grown networks are characterized by root mean square phason fluctuations which grow linearly with system size. The strain accumulation we observe in these networks also grows linearly with system size. Finally, we find a dependence of strain accumulation on cooling rate

  20. Gypsum accumulation on carbonate stone

    Science.gov (United States)

    McGee, E.S.; Mossotti, V.G.

    1992-01-01

    The accumulation of gypsum on carbonate stone has been investigated through exposure of fresh samples of limestone and marble at monitored sites, through examination of alteration crusts from old buildings and through laboratory experiments. Several factors contribute to gypsum accumulation on carbonate stone. Marble or limestone that is sheltered from direct washing by rain in an urban environment with elevated pollution levels is likely to accumulate a gypsum crust. Crust development may be enhanced if the stone is porous or has an irregular surface area. Gypsum crusts are a surficial alteration feature; gypsum crystals form at the pore opening-air interface, where evaporation is greatest.

  1. Cholic acid is accumulated spontaneously, driven by membrane Delta pH, in many lactobacilli

    NARCIS (Netherlands)

    Kurdi, P; van Veen, HW; Tanaka, H; Mierau, [No Value; Konings, WN; Tannock, GW; Tomita, F; Yokota, A

    2000-01-01

    Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids

  2. Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

    Science.gov (United States)

    Ruta, Lavinia Liliana; Lin, Ya-Fen; Kissen, Ralph; Nicolau, Ioana; Neagoe, Aurora Daniela; Ghenea, Simona; Bones, Atle M; Farcasanu, Ileana Cornelia

    2017-01-01

    In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

  3. Comparison of the biological effects of {sup 18}F at different intracellular levels

    Energy Technology Data Exchange (ETDEWEB)

    Kashino, Genro, E-mail: kashino@oita-u.ac.jp [Advanced Molecular Imaging Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan); Hayashi, Kazutaka; Douhara, Kazumasa [Advanced Molecular Imaging Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan); Kobashigawa, Shinko; Mori, Hiromu [Department of Radiology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu City, Oita 879-5593 (Japan)

    2014-11-07

    Highlights: • We estimated the inductions of DNA DSB in cell treated with {sup 18}F-FDG. • We found that inductions of DNA DSB are dependent on accumulation of {sup 18}F in cell. • Accumulation of {sup 18}F in cell may be indispensable for risk estimation of PET. - Abstract: We herein examined the biological effects of cells treated with {sup 18}F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of {sup 18}F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of {sup 18}F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with {sup 18}F-FDG than in those treated with {sup 18}F-HF. The clonogenic survival of cells was significantly lower with {sup 18}F-FDG than with {sup 18}F-HF. We concluded that the efficient accumulation of {sup 18}F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.

  4. LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION

    Science.gov (United States)

    Stein, Olga; Stein, Yechezkiel

    1967-01-01

    In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-3H or 9, 10-palmitic acid-3H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk. PMID:6033535

  5. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  6. Intracellular events regulating cross-presentation

    Directory of Open Access Journals (Sweden)

    Peter eCresswell

    2012-06-01

    Full Text Available Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation (ERAD machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on MHC class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation.

  7. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  8. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  9. Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

    Science.gov (United States)

    Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

    2018-06-04

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

  10. Choice Rules and Accumulator Networks

    Science.gov (United States)

    2015-01-01

    This article presents a preference accumulation model that can be used to implement a number of different multi-attribute heuristic choice rules, including the lexicographic rule, the majority of confirming dimensions (tallying) rule and the equal weights rule. The proposed model differs from existing accumulators in terms of attribute representation: Leakage and competition, typically applied only to preference accumulation, are also assumed to be involved in processing attribute values. This allows the model to perform a range of sophisticated attribute-wise comparisons, including comparisons that compute relative rank. The ability of a preference accumulation model composed of leaky competitive networks to mimic symbolic models of heuristic choice suggests that these 2 approaches are not incompatible, and that a unitary cognitive model of preferential choice, based on insights from both these approaches, may be feasible. PMID:28670592

  11. Disruption of the Arabidopsis CGI-58 homologue produces Chanarin–Dorfman-like lipid droplet accumulation in plants

    OpenAIRE

    James, Christopher N.; Horn, Patrick J.; Case, Charlene R.; Gidda, Satinder K.; Zhang, Daiyuan; Mullen, Robert T.; Dyer, John M.; Anderson, Richard G. W.; Chapman, Kent D.

    2010-01-01

    CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common...

  12. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens.

    Science.gov (United States)

    Voigt, Anja; Schöfl, Gerhard; Saluz, Hans Peter

    2012-01-01

    Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis. A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins. This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.

  13. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens.

    Directory of Open Access Journals (Sweden)

    Anja Voigt

    Full Text Available Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.

  14. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

    Science.gov (United States)

    Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

    2017-10-23

    Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  15. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

    Directory of Open Access Journals (Sweden)

    Farhana R. Pinu

    2017-10-01

    Full Text Available Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  16. Modelling the extra and intracellular uptake and discharge of heavy metals in Fontinalis antipyretica transplanted along a heavy metal and pH contamination gradient

    International Nuclear Information System (INIS)

    Fernandez, J.A.; Vazquez, M.D.; Lopez, J.; Carballeira, A.

    2006-01-01

    Samples of the aquatic bryophyte Fontinalis antipyretica Hedw. were transplanted to different sites with the aim of characterizing the kinetics of the uptake and discharge of heavy metals in the extra and intracellular compartments. The accumulation of metals in extracellular compartments, characterized by an initial rapid accumulation, then a gradual slowing down over time, fitted perfectly to a Michaelis-Menten model. The discharge of metals from the same compartment followed an inverse linear model or an inverse Michaelis-Menten model, depending on the metal. In intracellular sites both uptake and discharge occurred more slowly and progressively, following a linear model. We also observed that the acidity of the environment greatly affected metal accumulation in extracellular sites, even when the metals were present at relatively high concentrations, whereas the uptake of metals within cells was much less affected by pH. - The kinetics of uptake and discharge of heavy metals, in different cellular locations, were studied in transplanted aquatic mosses

  17. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    Science.gov (United States)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  18. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

    Science.gov (United States)

    Knapp, W; Strobl, H; Majdic, O

    1994-12-15

    New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

  19. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Science.gov (United States)

    Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

    2013-01-01

    Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  20. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Directory of Open Access Journals (Sweden)

    Jinli Wang

    Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  1. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  2. A method for functional trans-complementation of intracellular Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Shaun Steele

    Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

  3. Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments

    DEFF Research Database (Denmark)

    Kolko, Miriam; Wang, Jinmei; Zhan, Chen

    2007-01-01

    PURPOSE: To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS). METHODS: PCR amplification and Western blot analysis were used to identify m......)-VIA activity was found to be specifically increased 12 hours after ARPE-19 cells were fed with POS. Finally, RPE phagocytosis was inhibited by the iPLA(2)-VIA inhibitor bromoenol lactone. CONCLUSIONS: Various intracellular PLA(2) subtypes are present in the human retina. iPLA(2)-VIA may play...

  4. HALOGENATED AROMATIC HYDROCARBON-MEDIATED PORPHYRIN ACCUMULATION AND INDUCTION OF CYTOCHROME P4501A IN CHICKEN EMBRYO HEPATOCYTES. (R823889)

    Science.gov (United States)

    Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'...

  5. Induced Plant Accumulation of Lithium

    Directory of Open Access Journals (Sweden)

    Laurence Kavanagh

    2018-02-01

    Full Text Available Lithium’s (Li value has grown exponentially since the development of Li-ion batteries. It is usually accessed in one of two ways: hard rock mineral mining or extraction from mineral-rich brines. Both methods are expensive and require a rich source of Li. This paper examines the potential of agro-mining as an environmentally friendly, economically viable process for extracting Li from low grade ore. Agro-mining exploits an ability found in few plant species, to accumulate substantial amounts of metals in the above ground parts of the plant. Phyto-mined metals are then retrieved from the incinerated plants. Although the actual amount of metal collected from a crop may be low, the process has been shown to be profitable. We have investigated the suitability of several plant species including: Brassica napus and Helianthus annuus, as Li-accumulators under controlled conditions. Large plant trials were carried out with/without chelating agents to encourage Li accumulation. The question we sought to answer was, can any of the plant species investigated accumulate Li at levels high enough to justify using them to agro-mine Li. Results show maximum accumulated levels of >4000 mg/kg Li in some species. Our data suggests that agro-mining of Li is a potentially viable process.

  6. New perspective in the assessment of total intracellular magnesium

    Directory of Open Access Journals (Sweden)

    Azzurra Sargenti

    2014-01-01

    Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

  7. Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

    Science.gov (United States)

    da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

    2017-08-09

    Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

  8. The accumulation of metals in lichens

    Directory of Open Access Journals (Sweden)

    Smaranda Mâșu

    2017-05-01

    Full Text Available The capacity to accumulate metal in the lichen communities has been used to identify the level of air pollution due road traffic. Several analyses have been conducted in the study, on tissue from lichens collected from six areas: the park of a town, various sites on the town’s freeway and on a county road segment with moderate traffic. The analyzed lichens were from the Parmelia spp. species which grow naturally on trees found in borderline lanes of motorways. Based on the degree of heavy metal accumulation such as Cd, Cr, Cu, Fe Mn, Pb, Ni and Zn in the Parmelia spp. lichens, a correlation has been made, with the road traffic. According to the metal bioaccumulation degree in lichens tissue, it has been established that the sources such as traffic from the outskirts of cities, from the perimeter of gas stations and of county roads continuously spread products which contain these elements, into the atmosphere. The fast information regarding the quality of the air in the environment allows the use of lichens as organism which can indicate environmental conditions and their modification by accumulating substances.

  9. Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

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    Satdip Kaur

    Full Text Available CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.

  10. Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

    Science.gov (United States)

    Ludwar, Bjoern Ch; Evans, Colin G; Cropper, Elizabeth C

    2012-07-15

    synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

  11. Identification of key amino acid residues modulating intracellular and in vitro microcin E492 amyloid formation

    Directory of Open Access Journals (Sweden)

    Paulina eAguilera

    2016-01-01

    Full Text Available Microcin E492 (MccE492 is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well characterized, however it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in E. coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophillic probes, 2-4´-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59, which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  12. Effect of minerals on accumulation of Cs by fungus Saccaromyces cerevisiae.

    Science.gov (United States)

    Ohnuki, Toshihiko; Sakamoto, Fuminori; Yamasaki, Shinya; Kozai, Naofumi; Shiotsu, Hiroyuki; Utsunomiya, Satoshi; Watanabe, Naoko; Kozaki, Tamotsu

    2015-06-01

    The accumulation of Cs by unicellular fungus of Saccharomyces cerevisiae in the presence of minerals has been studied to elucidate the role of microorganisms in the migration of radioactive Cs in the environment. Two different types of experiments were employed: experiments using stable Cs to examine the effect of a carbon source on the accumulation of Cs, and accumulation experiments of radioactive Cs from agar medium containing (137)Cs and zeolite, vermiculite, phlogopite, smectite, mica, or illite as mineral supplements. In the former type of experiments, the Cs-accumulated cells were analyzed by scanning electron microscopy equipped with energy dispersive X-ray analysis (SEM-EDS). In the latter type, the radioactivity in the yeast cells was measured by an autoradiography technique. When a carbon source was present, higher amounts of Cs accumulated in the cells than in the resting condition without a carbon source. Analyses with SEM-EDS showed that no mineral formed on the cell surface. These results indicate that the yeast cells accumulate Cs by adsorption on the cell surface and intracellular accumulation. In the presence of minerals in the agar medium, the radioactivity in the yeast cells was in the order of mica > smectite, illite > vermiculite, phlogopite, zeolite. This order is inversely correlated to the ratio of the concentration of radioactive Cs between the minerals and the medium solution. These results strongly suggest that the yeast accumulates radioactive Cs competitively with minerals. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Digitonin abolishes free 2-deoxy-D-glucose accumulation in isolated rat adipocytes

    International Nuclear Information System (INIS)

    Thompson, K.; Kleinzeller, A.

    1986-01-01

    The hypothesis that accumulation against sizable chemical gradients of free (non-phosphorylated) 2-deoxy-D-glucose (2dGlc) in isolated rat adipocytes results from an intracellular compartmentation of free hexose was investigated. Cells exposed to 20 μg/ml digitonin for 10' demonstrated an increased plasma membrane permeability indexed by increased L-glucose entry rates and cellular (presumably cytosolic) protein and K + loss. Functional integrity of intracellular organelles was indicated by the ability of the cells to support ATP-driven 45 Ca 2+ -uptake. Equilibrium 3-O-methylglucose (3-O-MG, a non-accumulated hexose) levels were unaffected. These data suggest a specific permeabilizing action of digitonin at the plasma membrane having no effect on intracellular organelles or passively distributed solutes. Upon addition of digitonin, free 2dGlc fell from 66.5 +/- 8.9 to 7.4 +/- 2.3 pmol/10 5 cells, a value not significantly different from 3-O-MG levels. The gradient of 2dGlc-phosphate was also abolished, as was the increased steady-state free 2dGlc levels induced by insulin. The data argue against a compartmentation model as either the mechanism of adipocyte sugar accumulation or the basis of the steady-state free 2dGlc increase seen with insulin and suggest that an intact plasma membrane is essential to the process

  14. Intracellular trafficking of new anticancer therapeutics: antibody–drug conjugates

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody–drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs. PMID:28814834

  15. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-01-01

    Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

  16. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    Science.gov (United States)

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  17. Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

    DEFF Research Database (Denmark)

    Ernstsen, Christina Lundgaard; Login, Frédéric H.; Jensen, Helene Halkjær

    2017-01-01

    Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacteria...

  18. Conformational determinants of the intracellular localization of midkine

    International Nuclear Information System (INIS)

    Dai Lichen; Xu, Diyong; Yao Xing; Lu Yongliang; Xu Zhengping

    2005-01-01

    Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus, however, the data are not consistent and the signals responsible for the localization are unknown. Here we reported that human MK exclusively localized to the nucleus and nucleolus in HepG2 cells by using GFP as a tracking molecule. In order to identify the motifs required for the nuclear localization and nucleolar accumulation, point- and deletion-mutations were introduced and the corresponding subcellular localizations were analyzed. Data revealed that (i) K79R81, K86K87, and the C-terminal tail of MK constitute the nuclear localization determinant of MK, and (ii) the C-terminal tail is the key element controlling MK nucleolar accumulation though the N-terminal tail, K79R81, and K86K87 also contribute to this process. Taken together, our results provide the first documentation about the determinants required for MK nuclear and nucleolar localization

  19. Uptake and intracellular activity of AM-1155 in phagocytic cells.

    Science.gov (United States)

    Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

    1996-01-01

    The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

  20. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

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    Xianglu Li

    2009-01-01

    Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

  1. Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets

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    Fuhrer Erna

    2011-05-01

    Full Text Available Abstract Background Adipocyte volume (fat accumulation and cell number (adipogenesis is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation. Methods C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation. Results The ω-3 PUFAs docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA, the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E-lycopene and epigallocatechin gallate (EGCG showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β. Conclusion This in

  2. Intracellular trafficking pathways of Cx43 gap junction channels.

    Science.gov (United States)

    Epifantseva, Irina; Shaw, Robin M

    2018-01-01

    Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

    Science.gov (United States)

    Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

    2017-01-01

    Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

  4. Intracellular and membrane-damaging activities of methyl gallate isolated from Terminalia chebula against multidrug-resistant Shigella spp.

    Science.gov (United States)

    Acharyya, Saurabh; Sarkar, Prodipta; Saha, Dhira R; Patra, Amarendra; Ramamurthy, T; Bag, Prasanta K

    2015-08-01

    Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24  h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20  h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.

  5. Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

    Directory of Open Access Journals (Sweden)

    Lavinia Liliana Ruta

    Full Text Available In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I, Zn(II or Cd(II. The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3 were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyperaccumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II, Zn(II or Cd(II, but also non-canonical metal ions, such as Co(II, Mn(II or Ni(II, myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

  6. Comparative Salt Stress Study on Intracellular Ion Concentration in Marine and Salt-adapted Freshwater Strains of Microalgae

    Directory of Open Access Journals (Sweden)

    Ahmad Farhad TALEBI

    2013-08-01

    Full Text Available Salinity imposes significant stresses in various living organisms including microalgae. High extracellular concentration of Na+ directly influences ionic balance inside the cell and subsequently the cellular activities. In the present study, the effect of such stress on growth and intracellular ions concentration (IIC of Dunaliella salina and Chlorella Spp. was investigated. IIC was analyzed using Ion chromatography technique. D. salina showed the highest degree of resistance to increase in salinity as little changes occurred both in IIC and in growth parameters. D. salina could maintain the balance of K+ inside the cell and eject the excess Na+ even at NaCl concentrations above 1M. Moreover, D. salina accumulated β-carotene in order to protect its photosynthetic apparatus. Among Chlorella species, C. vulgaris showed signs of adaptation to high content of salinity, though it is a fresh water species by nature. Moreover, the response shown by C. vulgaris to rise in salinity was even stronger than that of C. salina, which is presumably a salt-water resistant species. In fact, C. vulgaris could maintain intracellular K+ better than C. salina in response to increasing salinity, and as a result, it could survive at NaCl concentrations as high as 0.75 M. Marine strains such as D. salina well cope with the fluctuations in salinity through the existing adaptation mechanisms i.e. maintaining the K+/N+ balance inside the cell, K+ accumulation and Na+ ejection, accumulation of photosynthetic pigments like β-carotene.

  7. An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

    Science.gov (United States)

    Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

    2008-05-20

    Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

  8. An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

    Directory of Open Access Journals (Sweden)

    Welsh Gavin I

    2008-05-01

    Full Text Available Abstract Background Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Results Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. Conclusion The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

  9. Mi Casa es Su Casa: how an intracellular symbiont manipulates host biology.

    Science.gov (United States)

    Bhattacharya, Tamanash; Newton, Irene L G

    2017-10-27

    Wolbachia pipientis, the most common intracellular infection on the planet, infects 40% of insects as well as nematodes, isopods and arachnids. Wolbachia are obligately intracellular and challenging to study; there are no genetic tools for manipulating Wolbachia nor can they be cultured outside of host cells. Despite these roadblocks, the research community has defined a set of Wolbachia loci involved in host interaction: Wolbachia effectors. Through the use of Drosophila genetics, surrogate systems and biochemistry, the field has begun to define the toolkit Wolbachia use for host manipulation. Below we review recent findings identifying these Wolbachia effectors and point to potential, as yet uncharacterized, links between known phenotypes induced by Wolbachia infection and predicted effectors. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. Calcium, membranes and accumulation of alkaloids in plants

    International Nuclear Information System (INIS)

    Lovkova, M.Ya.; Buzuk, G.N.; Grinkevich, N.I.

    1983-01-01

    Ca 2+ effect upon metabolism of aporphines and protopines has been studied in Glaucium flavun, which alkaloids are of an essential interest for the medicine practice. It has been shown that calcium produces the inhibiting effect both on catabolitic splitting and metabolism of glaucine and protopine. It has been anticipated that calcuium introduced into an expert plant stabilizes membranes of intracellular structures and prevents 14 C alkaloid entering from an environment to metabolically active cell compartments, which contain ferments realizing transformations of the above compounds. The level of membrane permeability is probably the main mechanism, through which a control of metabolism processes occurs, and hence, a control of alkaloid accumulation processes under in vivo conditions

  11. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner.

    Directory of Open Access Journals (Sweden)

    Josef Köck

    Full Text Available Persistence of hepatitis B virus (HBV infection requires covalently closed circular (cccDNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

  12. Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

    Science.gov (United States)

    Vandenboom, Rene

    2014-01-01

    Ca2+ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca2+-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca2+ transient (ICT) and the baseline Ca2+ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca2+ accumulated during SP such that baseline Ca2+ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca2+ coincide with greater +df/dt but not necessarily with higher twitch force. PMID:25422504

  13. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    Science.gov (United States)

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. © FASEB.

  14. Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding

    DEFF Research Database (Denmark)

    Loland, Claus Juul; Grånäs, Charlotta; Javitch, Jonathan A

    2004-01-01

    Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-26...

  15. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

    Directory of Open Access Journals (Sweden)

    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  16. Type IV pili in Francisella – A virulence trait in an intracellular pathogen

    Directory of Open Access Journals (Sweden)

    Emelie eNäslund Salomonsson

    2011-02-01

    Full Text Available Francisella tularensis is a highly virulent intracellular human pathogen that is capable of rapid proliferation in the infected host. Mutants affected in intracellular survival and growth are highly attenuated which highlights the importance of the intracellular phase of the infection. Genomic analysis has revealed that Francisella encodes all genes required for expression of functional type IV pili (Tfp, and in this focused review we summarise recent findings regarding this system in the pathogenesis of tularemia. Tfp are dynamic adhesive structures that have been identified as major virulence determinants in several human pathogens, but it is not obvious what role these structures could have in an intracellular pathogen like Francisella. In the human pathogenic strains, genes required for secretion and assembly of Tfp and one pilin, PilA, have shown to be required for full virulence. Importantly, specific genetic differences have been identified between the different Francisella subspecies where in the most pathogenic type A variants all genes are intact while several Tfp genes are pseudogenes in the less pathogenic type B strains. This suggests that there has been a selection for expression of Tfp with different properties in the different subspecies. There is also a possibility that the genetic differences reflect adaption to different environmental niches of the subspecies and plays a role in transmission of tularemia. This is also in line with recent findings where Tfp pilins are found to be glycosylated which could reflect a role for Tfp in the environment to promote survival and transmission. We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but with the genomic information and genetic tools available we are in a good position to address these issues in the future.

  17. Negotiation of intracellular membrane barriers by TAT-modified gold nanoparticles.

    Science.gov (United States)

    Krpetić, Zeljka; Saleemi, Samia; Prior, Ian A; Sée, Violaine; Qureshi, Rumana; Brust, Mathias

    2011-06-28

    This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.

  18. Effects of potentially acidic air pollutants on the intracellular distribution and transport of plant growth regulators in mesophyll cells of leaves. Consequences on stress- and developmental physiology

    Energy Technology Data Exchange (ETDEWEB)

    Kremer, H.; Pfanz, H.; Hartung, W.

    1987-07-11

    The influence of SO/sub 2/ on the intracellular distribution of abscisic acid (ABA) and indole-acetic acid (IAA) in mesophyll cells of Picea abies, Tsuga americana and Hordeum vulgare was investigated. The compartmentation of ABA and IAA depends on intracellular pH-gradients. The hydrophilic anions ABA and IAA are accumulated in the alkaline cell compartments cytosol and chloroplasts, which act as anion traps for weak acids. Uptake of sulfur dioxide into leaves leads to an acidification of alkaline cell compartments, thus decreasing intracellular pH-gradients. Consequently this results in an increased release of plant growth regulators from the cell interior into the apoplast. Therefore the target cells of plant hormones i.e. meristems and stomates are exposed to altered hormone concentrations. Obviously this influences the regulation of cellular metabolism plant development and growth.

  19. Increased intracellular proteolysis reduces disease severity in an ER stress-associated dwarfism.

    Science.gov (United States)

    Mullan, Lorna A; Mularczyk, Ewa J; Kung, Louise H; Forouhan, Mitra; Wragg, Jordan Ma; Goodacre, Royston; Bateman, John F; Swanton, Eileithyia; Briggs, Michael D; Boot-Handford, Raymond P

    2017-10-02

    The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress-associated dwarfism MCDS.

  20. Extra and intracellular synthesis of nickel oxide nanoparticles mediated by dead fungal biomass.

    Directory of Open Access Journals (Sweden)

    Marcia Regina Salvadori

    Full Text Available The use of dead biomass of the fungus Hypocrea lixii as a biological system is a new, effective and environmentally friendly bioprocess for the production and uptake of nickel oxide nanoparticles (NPs, which has become a promising field in nanobiotechnology. Dead biomass of the fungus was successfully used to convert nickel ions into nickel oxide NPs in aqueous solution. These NPs accumulated intracellularly and extracellularly on the cell wall surface through biosorption. The average size, morphology and location of the NPs were characterized by transmission electron microscopy, high-resolution transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. The NPs were mainly spherical and extra and intracellular NPs had an average size of 3.8 nm and 1.25 nm, respectively. X-ray photoelectron spectroscopy analysis confirmed the formation of nickel oxide NPs. Infrared spectroscopy detected the presence of functional amide groups, which are probable involved in particle binding to the biomass. The production of the NPs by dead biomass was analyzed by determining physicochemical parameters and equilibrium concentrations. The present study opens new perspectives for the biosynthesis of nanomaterials, which could become a potential biosorbent for the removal of toxic metals from polluted sites.

  1. The Intracellular Destiny of the Protein Corona: A Study on its Cellular Internalization and Evolution.

    Science.gov (United States)

    Bertoli, Filippo; Garry, David; Monopoli, Marco P; Salvati, Anna; Dawson, Kenneth A

    2016-11-22

    It has been well established that the early stages of nanoparticle-cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions, nanoparticles are typically internalized into the cell and trafficked along defined pathways such as, in many cases, the endolysosomal pathway. Indeed, if the original corona is partially retained on the nanoparticle surface, the biomolecules in this layer may play an important role in determining subsequent cellular processing. In this work, using a combination of organelle separation and fluorescence labeling of the initial extracellular corona, we clarify its intracellular evolution as nanoparticles travel within the cell. We show that specific proteins present in the original protein corona are retained on the nanoparticles until they accumulate in lysosomes, and, once there, they are degraded. We also report on how different bare surfaces (amino and carboxyl modified) affect the details of this evolution. One overarching discovery is that the same serum proteins can exhibit different intracellular processing when carried inside cells by nanoparticles, as components of their corona, compared to what is observed when they are transported freely from the extracellular medium.

  2. Intracellular Peptide Self-Assembly: A Biomimetic Approach for in Situ Nanodrug Preparation.

    Science.gov (United States)

    Du, Wei; Hu, Xiaomu; Wei, Weichen; Liang, Gaolin

    2018-04-18

    Most nanodrugs are preprepared by encapsulating or loading the drugs with nanocarriers (e.g., dendrimers, liposomes, micelles, and polymeric nanoparticles). However, besides the low bioavailability and fast excretion of the nanodrugs in vivo, nanocarriers often exhibit in vitro and in vivo cytotoxicity, oxidative stress, and inflammation. Self-assembly is a ubiquitous process in biology where it plays important roles and underlies the formation of a wide variety of complex biological structures. Inspired by some cellular nanostructures (e.g., actin filaments, microtubules, vesicles, and micelles) in biological systems which are formed via molecular self-assembly, in recent decades, scientists have utilized self-assembly of oligomeric peptide under specific physiological or pathological environments to in situ construct nanodrugs for lesion-targeted therapies. On one hand, peptide-based nanodrugs always have some excellent intrinsic chemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties. On the other hand, stimuli-regulated intracellular self-assembly of nanodrugs is quite an efficient way to accumulate the drugs in lesion location and can realize an in situ slow release of the drugs. In this review article, we provided an overview on recent design principles for intracellular peptide self-assembly and illustrate how these principles have been applied for the in situ preparation of nanodrugs at the lesion location. In the last part, we list some challenges underlying this strategy and their possible solutions. Moreover, we envision the future possible theranostic applications of this strategy.

  3. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  4. Corporate taxation and capital accumulation

    OpenAIRE

    Stephen Bond; Jing Xing

    2010-01-01

    We present new empirical evidence that aggregate capital accumulation is strongly influenced by the user cost of capital and, in particular, by corporate tax incentives summarised in the tax-adjusted user cost. We use sectoral panel data for the USA, Japan, Australia and ten EU countries over the period 1982-2007. Our panel combines data on capital stocks, value-added and relative prices from the EU KLEMS database with measures of effective corporate tax rates from the Oxford University Centr...

  5. Intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

    International Nuclear Information System (INIS)

    Liscum, L.; Ruggiero, R.M.; Faust, J.R.

    1989-01-01

    Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol

  6. WEALTH TAXATION AND WEALTH ACCUMULATION

    DEFF Research Database (Denmark)

    Jakobsen, Katrine Marie Tofthøj; Jakobsen, Kristian Thor; Kleven, Henrik

    Using administrative wealth records from Denmark, we study the effects of wealth taxes on wealth accumulation. Denmark used to impose one of the world's highest marginal tax rates on wealth, but this tax was drastically reduced and ultimately abolished between 1989 and 1997. Due to the specific d...... on wealth accumulation. Our simulations show that the long-run elasticity of wealth with respect to the net-of-tax return is sizeable at the top of distribution. Our paper provides the type of evidence needed to assess optimal capital taxation.......Using administrative wealth records from Denmark, we study the effects of wealth taxes on wealth accumulation. Denmark used to impose one of the world's highest marginal tax rates on wealth, but this tax was drastically reduced and ultimately abolished between 1989 and 1997. Due to the specific...... design of the wealth tax, these changes provide a compelling quasi-experiment for understanding behavioral responses among the wealthiest segments of the population. We find clear reduced-form effects of wealth taxes in the short and medium run, with larger effects on the very wealthy than...

  7. Evaluation of Maize Germplasm for Resistance to Aflatoxin Accumulation

    Directory of Open Access Journals (Sweden)

    Michael H. Blanco

    2012-03-01

    Full Text Available Aflatoxin contamination of maize grain threatens human food and animal feed safety. Breeding for reduced grain aflatoxin accumulation is one of the best strategies presently available to lower grain aflatoxin accumulation. Previously identified sources of germplasm with reduced grain aflatoxin accumulation are excessively tall and late maturing. The objective of this research was to screen germplasm and identify potential sources of aflatoxin resistance. KO679Y and CUBA117:S15-101-001-B-B-B-B inbreds were evaluated for aflatoxin accumulation alongside resistant and susceptible checks with both performing well. These two lines were also evaluated in various crosses. KO679Y performed especially well in crosses with Mp494 and Mp717, resulting in low ear rot and very low aflatoxin levels, but not well in other crosses. A breeding cross including CUBA117:S15-101-001-B-B-B-B as a parent accumulated low levels of aflatoxin both years it was evaluated. Lines resulting from these crosses are being advanced for further evaluation and improvement. KO679Y and CUBA117:S15-101-001-B-B-B-B may prove useful for breeders seeking germplasm sources for ear rot and mycotoxin reduction, especially KO679Y which matures a week earlier and is approximately 25% shorter than current lines resistant to grain aflatoxin accumulation.

  8. Tracking micro-optical resonances for identifying and sensing novel procaspase-3 protein marker released from cell cultures in response to toxins

    International Nuclear Information System (INIS)

    Chen, Ying-Jen; Vollmer, Frank; Xiang, Wei; Klucken, Jochen

    2016-01-01

    The response of cells to toxins is commonly investigated by detecting intracellular markers for cell death, such as caspase proteins. This requires the introduction of labels by the permeabilization or complete lysis of cells. Here we introduce a non-invasive tool for monitoring a caspase protein in the extracellular medium. The tool is based on highly sensitive optical micro-devices, referred to as whispering-gallery mode biosensors (WGMBs). WGMBs are functionalized with antibodies for the specific and label-free detection of procaspase-3 released from human embryonic kidney HEK293 and neuroglioma H4 cells after introducing staurosporine and rotenone toxins, respectively. Additional tests show that the extracellular accumulation of procaspase-3 is concomitant with a decrease in cell viability. The hitherto unknown release of procaspase-3 from cells in response to toxins and its accumulation in the medium is further investigated by Western blot, showing that the extracellular detection of procaspase-3 is interrelated with cytotoxicity of alpha-synuclein protein (aSyn) overexpressed in H4 cells. These studies provide evidence for procaspase-3 as a novel extracellular biomarker for cell death, with applications in cytotoxicity tests. Such WGMBs could be applied to further identify as-yet unknown extracellular biomarkers using established antibodies against intracellular antigens. (paper)

  9. A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation.

    Science.gov (United States)

    Capece, Tara; Walling, Brandon L; Lim, Kihong; Kim, Kyun-Do; Bae, Seyeon; Chung, Hung-Li; Topham, David J; Kim, Minsoo

    2017-11-06

    The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8 + T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8 + T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8 + T cells plays a key role in T cell activation and differentiation. © 2017 Capece et al.

  10. Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

    Directory of Open Access Journals (Sweden)

    Milos B. Rokic

    2018-04-01

    Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

  11. Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

    Directory of Open Access Journals (Sweden)

    Sha Liu

    Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

  12. Two-dimensional polyacrylamide gel electrophoresis of intracellular proteins

    International Nuclear Information System (INIS)

    Ojima, N.; Sakamoto, T.; Yamashita, M.

    1996-01-01

    Since two-dimensional electrophoresis was established by O'Farrell for analysis of intracellular proteins of Escherichia coli, it has been applied to separation of proteins of animal cells and tissues, and especially to identification of stress proteins. Using this technique, proteins are separated by isoelectric focusing containing 8 m urea in the first dimension and by SDS-PAGE in the second dimension. The gels are stained with Coomassie Blue R-250 dye, followed by silver staining. In the case of radio-labeled proteins, the gels are dried and then autoradiographed. In order to identify a specific protein separated by two-dimensional electrophoresis, a technique determining the N-terminal amino acid sequence of the protein has been developed recently. After the proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane, the membrane was stained for protein with Commassie Blue and a stained membrane fragment was applied to a protein sequencer. Our recent studies demonstrated that fish cells newly synthesized various proteins in response to heat shock, cold nd osmotic stresses. For example, when cellular proteins extracted from cold-treated rainbow trout cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold-treatment. N-Terminal sequence analysis showed that the cold-inducible protein was a homolog of mammalian valosin-containing protein and yeast cell division cycle gene product CDC48p. Furthermore, the sequence data were useful for preparing PCR primers and a rabbit antibody against a synthetic peptide to analyze a role for the protein in the function of trout cells and mechanisms for regulation

  13. Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate.

    Science.gov (United States)

    Welsh, D T

    2000-07-01

    The osmoadaptation of most micro-organisms involves the accumulation of K(+) ions and one or more of a restricted range of low molecular mass organic solutes, collectively termed 'compatible solutes'. These solutes are accumulated to high intracellular concentrations, in order to balance the osmotic pressure of the growth medium and maintain cell turgor pressure, which provides the driving force for cell extension growth. In this review, I discuss the alternative roles which compatible solutes may also play as intracellular reserves of carbon, energy and nitrogen, and as more general stress metabolites involved in protection of cells against other environmental stresses including heat, desiccation and freezing. Thus, the evolutionary selection for the accumulation of a specific compatible solute may not depend solely upon its function during osmoadaptation, but also upon the secondary benefits its accumulation provides, such as increased tolerance of other environmental stresses prevalent in the organism's niche or even anti-herbivory or dispersal functions in the case of dimethylsulfoniopropionate (DMSP). In the second part of the review, I discuss the ecological consequences of the release of compatible solutes to the environment, where they can provide sources of compatible solutes, carbon, nitrogen and energy for other members of the micro-flora. Finally, at the global scale the metabolism of specific compatible solutes (betaines and DMSP) in brackish water, marine and hypersaline environments may influence global climate, due to the production of the trace gases, methane and dimethylsulfide (DMS) and in the case of DMS, also couple the marine and terrestrial sulfur cycles.

  14. Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

    International Nuclear Information System (INIS)

    Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

    2014-01-01

    Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

  15. Intracellular calcium levels can regulate Importin-dependent nuclear import

    International Nuclear Information System (INIS)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-01-01

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery

  16. Intracellular transport of fat-soluble vitamins A and E.

    Science.gov (United States)

    Kono, Nozomu; Arai, Hiroyuki

    2015-01-01

    Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

  17. Exploring anti-bacterial compounds against intracellular Legionella.

    Directory of Open Access Journals (Sweden)

    Christopher F Harrison

    Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

  18. Advances in genetic manipulation of obligate intracellular bacterial pathogens

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    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  19. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    Science.gov (United States)

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  20. Intracellular calcium levels can regulate Importin-dependent nuclear import

    Energy Technology Data Exchange (ETDEWEB)

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  1. Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

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    Walmor eDe Mello

    2015-01-01

    Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

  2. Self-organization of intracellular gradients during mitosis

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    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  3. Overcoming drug resistance of MCF-7/ADR cells by altering intracellular distribution of doxorubicin via MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex.

    Science.gov (United States)

    Han, Min; Lv, Qing; Tang, Xin-Jiang; Hu, Yu-Lan; Xu, Dong-Hang; Li, Fan-Zhu; Liang, Wen-Quan; Gao, Jian-Qing

    2012-10-28

    Drug resistance is one of the critical reasons leading to failure in chemotherapy. Enormous studies have been focused on increasing intracellular drug accumulation through inhibiting P-glycoprotein (Pgp). Meanwhile, we found that major vault protein (MVP) may be also involved in drug resistance of human breast cancer MCF-7/ADR cells by transporting doxorubicin (DOX) from the action target (i.e. nucleus) to cytoplasma. Herein polyamidoamine (PAMAM) dendrimers was functionalized by a polysaccharide hyaluronic acid (HA) to effectively deliver DOX as well as MVP targeted small-interfering RNA (MVP-siRNA) to down regulate MVP expression and improve DOX chemotherapy in MCF-7/ADR cells. In comparison with DOX solution (IC50=48.5 μM), an enhanced cytotoxicity could be observed for DOX PAMAM-HA (IC50=11.3 μM) as well as enhanced tumor target, higher intracellular accumulation, increased blood circulating time and less in vivo toxicity. Furthermore, codelivery of siRNA and DOX by PAMAM-HA exhibited satisfactory gene silencing effect as well as enhanced stability and efficient intracellular delivery of siRNA, which allowed DOX access to nucleus and induced subsequent much more cytotoxicity than siRNA absent case as a result of MVP knockdown. This observation highlights a promising application of novel nanocarrier PAMAM-HA, which could co-deliver anticancer drug and siRNA, in reversing drug resistance by altering intracellular drug distribution. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection.

    Science.gov (United States)

    Kim, Su-Young; Park, Hye Yun; Jeong, Byeong-Ho; Jeon, Kyeongman; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Han, Seung-Jung; Shin, Sung Jae; Koh, Won-Jung

    2015-09-30

    Mycobacterium intracellulare is a major cause of Mycobacterium avium complex lung disease in many countries. Molecular studies have revealed several new Mycobacteria species that are closely related to M. intracellulare. The aim of this study was to re-identify and characterize clinical isolates from patients previously diagnosed with M. intracellulare lung disease at the molecular level. Mycobacterial isolates from 77 patients, initially diagnosed with M. intracellulare lung disease were re-analyzed by multi-locus sequencing and pattern of insertion sequences. Among the 77 isolates, 74 (96 %) isolates were designated as M. intracellulare based on multigene sequence-based analysis. Interestingly, the three remaining strains (4 %) were re-identified as "Mycobacterium indicus pranii" according to distinct molecular phylogenetic positions in rpoB and hsp65 sequence-based typing. In hsp65 sequevar analysis, code 13 was found in the majority of cases and three unreported codes were identified. In 16S-23S rRNA internal transcribed spacer (ITS) sequevar analysis, all isolates of both species were classified within the Min-A ITS sequevar. Interestingly, four of the M. intracellulare isolates harbored IS1311, a M. avium-specific element. Two of three patients infected with "M. indicus pranii" had persistent positive sputum cultures after antibiotic therapy, indicating the clinical relevance of this study. This analysis highlights the importance of precise identification of clinical isolates genetically close to Mycobacterium species, and suggests that greater attention should be paid to nontuberculous mycobacteria lung disease caused by "M. indicus pranii".

  5. Activation of a Neospora caninum EGFR-Like Kinase Facilitates Intracellular Parasite Proliferation

    Directory of Open Access Journals (Sweden)

    Xiaoxia Jin

    2017-10-01

    Full Text Available The Apicomplexan parasite Neospora caninum, an obligate intracellular protozoan, causes serious diseases in a number of mammalian species, especially in cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle worldwide which have a major economic impact on the cattle industry. However, the mechanism by which N. caninum proliferates within host cells is poorly understood. Epidermal growth factor receptor (EGFR is a protein kinase ubiquitously expressed, present on cell surfaces in numerous species, which has been confirmed to be essential in signal transduction involved in cell growth, proliferation, survival, and many other intracellular processes. However, the presence of EGFR in N. caninum and its role in N. caninum proliferation remain unclear. In the present study, we identified a putative EGFR-like kinase in N. caninum, which could be activated in tachyzoites by infection or treatment with rNcMIC3 [containing four epidermal growth factor (EGF domains] or human EGF. Blockade of EGFR-like in tachyzoites by AG1478 significantly reduced parasite proliferation in host cells. Our data suggested that the activation of tachyzoite EGFR-like might facilitate the intracellular proliferation of N. caninum.

  6. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

  7. Intracellular Zn(II) Intoxication Leads to Dysregulation of the PerR Regulon Resulting in Heme Toxicity in Bacillus subtilis

    Science.gov (United States)

    2016-01-01

    Transition metal ions (Zn(II), Cu(II)/(I), Fe(III)/(II), Mn(II)) are essential for life and participate in a wide range of biological functions. Cellular Zn(II) levels must be high enough to ensure that it can perform its essential roles. Yet, since Zn(II) binds to ligands with high avidity, excess Zn(II) can lead to protein mismetallation. The major targets of mismetallation, and the underlying causes of Zn(II) intoxication, are not well understood. Here, we use a forward genetic selection to identify targets of Zn(II) toxicity. In wild-type cells, in which Zn(II) efflux prevents intoxication of the cytoplasm, extracellular Zn(II) inhibits the electron transport chain due to the inactivation of the major aerobic cytochrome oxidase. This toxicity can be ameliorated by depression of an alternate oxidase or by mutations that restrict access of Zn(II) to the cell surface. Conversely, efflux deficient cells are sensitive to low levels of Zn(II) that do not inhibit the respiratory chain. Under these conditions, intracellular Zn(II) accumulates and leads to heme toxicity. Heme accumulation results from dysregulation of the regulon controlled by PerR, a metal-dependent repressor of peroxide stress genes. When metallated with Fe(II) or Mn(II), PerR represses both heme biosynthesis (hemAXCDBL operon) and the abundant heme protein catalase (katA). Metallation of PerR with Zn(II) disrupts this coordination, resulting in depression of heme biosynthesis but continued repression of catalase. Our results support a model in which excess heme partitions to the membrane and undergoes redox cycling catalyzed by reduced menaquinone thereby resulting in oxidative stress. PMID:27935957

  8. Subcellular neuropharmacology: the importance of intracellular targeting.

    Science.gov (United States)

    Miyashiro, Kevin Y; Bell, Thomas J; Sul, Jai-Yoon; Eberwine, James

    2009-04-01

    Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.

  9. Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

    Science.gov (United States)

    Wilke, Georgia; Ravindran, Soumya; Funkhouser-Jones, Lisa; Barks, Jennifer; Wang, Qiuling; VanDussen, Kelli L; Stappenbeck, Thaddeus S; Kuhlenschmidt, Theresa B; Kuhlenschmidt, Mark S; Sibley, L David

    2018-06-27

    Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti- Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and

  10. Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis

    International Nuclear Information System (INIS)

    Maaroufi, Younes; Lacroix, Marc; Lespagnard, Laurence; Journé, Fabrice; Larsimont, Denis; Leclercq, Guy

    2000-01-01

    Iodinated oestradiol-labeled oestrogen receptor (ER) isoforms devoid of amino-terminal ABC domains represent about two-thirds of the whole receptor population detected in cytosol samples from human breast cancers. This high frequency could not be ascribed to the expression of truncated mRNAs, or to the proteolysis of the native ER peptide at the time of homogenization or assay, suggesting an intracellular proteolysis. Free amino-terminal and ligand-binding domains maintained together within oligomeric structure(s); increase of ionic strength separated them. The amino-terminal region was consistently detected in the cell nucleus by specific immunohistochemistry leading to the concept of a potential intranuclear association between ER cleavage products and/or other regulatory proteins. We previously reported that about two-thirds of [ 125 I]oestradiol-labelled cytosolic ERs from breast cancer samples eluted as low-molecular-weight isoforms (≤ 37 kDa, size-exclusion fast pressure liquid chromatography [FPLC]). These isoforms failed to adsorb strongly to hydroxylapatite at high ionic strength, a property that was ascribed to receptors devoid of amino-terminal ABC domains. In view of recent data concerning intracellular proteolysis of several transcriptional regulators, the possibility of such behaviour for ER was assessed. The clinical significance of ER measurement in breast cancer cytosols is well established; approximately 50% of ER-positive cases respond to endocrine therapy. Whether such a poor correlation is related to a high proportion of cleaved ER is a question of prime importance. Failure of routine ER assays to discriminate between full-length and cleaved receptors led us to develop an oestradiol-binding assay based on hydroxylapatite adsorption. The aims of the present study were to demonstrate that hydroxylapatite adsorption assay easily identifies cleaved cytosolic ER forms and to assess the origin of such ER forms. Breast cancer cytosols classified as

  11. Intracellular trafficking of a pH-responsive drug metal complex.

    Science.gov (United States)

    Kheirolomoom, Azadeh; Ingham, Elizabeth S; Commisso, Joel; Abushaban, Neveen; Ferrara, Katherine W

    2016-12-10

    We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1

  12. Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

    Directory of Open Access Journals (Sweden)

    Beijing K. Huang

    2014-01-01

    Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

  13. Simple Recovery of Intracellular Gold Nanoparticles from Peanut Seedling Roots.

    Science.gov (United States)

    Raju, D; Mehta, Urmil J; Ahmad, Absar

    2015-02-01

    Fabrication of inorganic nanomaterials via a biological route witnesses the formation either extracellularly, intracellulary or both. Whereas extracellular formation of these nanomaterials is cherished owing to their easy and economical extraction and purification processes; the intracellular formation of nanomaterials, due to the lack of a proper recovery protocol has always been dreaded, as the extraction processes used so far were tedious, costly, time consuming and often resulting in very low recovery. The aim of the present study was to overcome the problems related with the extraction and recovery of intracellularly synthesized inorganic nanoparticles, and to devise a method to increasing the output, the shape, size, composition and dispersal of nanoparticles is not altered. Water proved to be much better system as it provided well dispersed, stable gold nanoparticles and higher recovery. This is the first report, where intracellular nanoparticles have been recovered using a very cost-effective and eco-friendly approach.

  14. Accumulation of cobalt by cephalopods

    International Nuclear Information System (INIS)

    Nakahara, Motokazu

    1981-01-01

    Accumulation of cobalt by cephalopod mollusca was investigated by radiotracer experiments and elemental analysis. In the radiotracer experiments, Octopus vulgaris took up cobalt-60 from seawater fairly well and the concentration of the nuclide in whole body attained about 150 times the level of seawater at 25th day at 20 0 C. Among the tissues and organs measured, branchial heart which is the specific organ of cephalopods showed the highest affinity for the nuclide. The organ accumulated about 50% of the radioactivity in whole body in spite of its little mass as 0.2% of total body weight. On the other hand, more than 90% of the radioactivity taken up from food (soft parts of Gomphina melanaegis labelled with cobalt-60 previously in an aquarium) was accumulated in liver at 3rd day after the single administration and then the radioactivity in the liver seemed to be distributed to other organs and tissues. The characteristic elution profiles of cobalt-60 was observed for each of the organs and tissues in Sephadex gel-filtration experiment. It was confirmed by the gel-filtration that most of cobalt-60 in the branchial heart was combined with the constituents of low molecular weights. The average concentration of stable cobalt in muscle of several species of cephalopods was 5.3 +- 3.0 μg/kg wet and it was almost comparable to the fish muscle. On the basis of soft parts, concentration of the nuclide closed association among bivalve, gastropod and cephalopod except squid that gave lower values than the others. (author)

  15. Control of the intracellular redox state by glucose participates in the insulin secretion mechanism.

    Directory of Open Access Journals (Sweden)

    Eduardo Rebelato

    Full Text Available BACKGROUND: Production of reactive oxygen species (ROS due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS. In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out. METHODOLOGY/PRINCIPAL FINDINGS: Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP. Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism. CONCLUSIONS: The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.

  16. Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

    International Nuclear Information System (INIS)

    Phair, R.D.; Hai, C.M.

    1986-01-01

    A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

  17. Technetium accumulation, fate, and behavior in plants

    International Nuclear Information System (INIS)

    Cataldo, D.A.; Wildung, R.E.; Garland, T.R.

    1978-01-01

    Technetium, a product of the nuclear fuel cycle, is highly soluble in water and mobile in soils as the pertechnetate ion (TcO - 4 ). Soluble ions in soil have the potential for competing with nutrient ions for membrane carrier sites involved in ion uptake by plants. A study was, therefore, undertaken to determine the availability, toxicity, and mechanism of pertechnetate uptake by soybean (Glycine max cv. Williams). Technetium was effectively accumulated by plants at soil concentrations of 0.01 to 0.1 μg/g and in nutrient culture at levels as low as 0.02 pg/ml. Plants grown on soils containing technetium at levels below 0.1 μg/g effectively removed up to 90% of the technetium from soil. Minimal mobilization of technetium from vegetative tissues to the seed occurred during senescence. Chemical analyses indicated that the xylem-mobile form of technetium was TcO 4 - . The uptake rate of technetium by intact plants was multiphasic over the concentration range of 0.01 to 10μM; this suggests active uptake and a specificity for technetium in the root absorption process. Because of the efficiency of technetium accumulation and the probability of its chemical toxicity, competition kinetic studies were undertaken to identify possible nutrient analogs. Nutrients effective in reducing technetium uptake included the Mn 2+ , SO 4 2- , H 2 PO 4 - , and MoO 4 2- ions

  18. Microbial cells as biosorbents for heavy metals: accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Strandberg, G.W.; Shumate, S.E. II; Parrott, J.R. Jr.

    1981-01-01

    Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (<10 s), and no response to environmental parameters could be detected. Metabolism was not required for metal uptake by either organism. Cell-bound uranium reached a concentration of 10 to 15% of the dry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P. aeruginosa cells within a given population possessed visible uranium deposits when examined by electron microscopy. Rates of uranium uptake by S. cerevisiae were increased by chemical pretreatment of the cells. Uranium could be removed chemically from S. cerevisiae cells, and the cells could then be reused as a biosorbent

  19. Tethering factors as organizers of intracellular vesicular traffic.

    Science.gov (United States)

    Yu, I-Mei; Hughson, Frederick M

    2010-01-01

    Intracellular trafficking entails the budding, transport, tethering, and fusion of transport vesicles and other membrane carriers. Here we review recent progress toward a mechanistic understanding of vesicle tethering. The known tethering factors are large complexes important for one or more intracellular trafficking pathways and are capable of interacting directly with many of the other principal components of the cellular trafficking machinery. Our review emphasizes recent developments in the in vitro reconstitution of vesicle tethering and the structural characterization of multisubunit tethering factors. The combination of these and other approaches has led to exciting progress toward understanding how these essential nanomachines work.

  20. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    Science.gov (United States)

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  1. Metal accumulation by stream bryophytes, related to chemical speciation

    Energy Technology Data Exchange (ETDEWEB)

    Tipping, E. [Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP (United Kingdom)], E-mail: et@ceh.ac.uk; Vincent, C.D.; Lawlor, A.J.; Lofts, S. [Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP (United Kingdom)

    2008-12-15

    Metal accumulation by aquatic bryophytes was investigated using data for headwater streams of differing chemistry. The Windermere Humic Aqueous Model (WHAM) was applied to calculate chemical speciation, including competitive proton and metal interactions with external binding sites on the plants. The speciation modelling approach gives smaller deviations between observed and predicted bryophyte contents of Cu, Zn, Cd and Pb than regressions based on total filtered metal concentrations. If all four metals, and Ni, are considered together, the WHAM predictions are superior at the 1% level. Optimised constants for bryophyte binding by the trace metals are similar to those for humic substances and simple carboxylate ligands. Bryophyte contents of Na, Mg and Ca are approximately explained by binding at external sites, while most of the K is intracellular. Oxide phases account for some of the Al, and most of the Mn, Fe and Co. - Speciation modelling can be used to interpret the accumulation of Ni, Cu, Zn, Cd and Pb by bryophytes, supporting its use to quantify trace metal bioavailability in the field.

  2. Rickettsia phylogenomics: unwinding the intricacies of obligate intracellular life.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available BACKGROUND: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs of open reading frames (ORFs will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs. METHODOLOGY/PRINCIPAL FINDINGS: We present 1823 representative (no gene duplications and 259 non-representative (at least one gene duplication rickettsial OGs. While the highly reductive (approximately 1.2 MB Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group, TG (typhus group, TRG (transitional group, and SFG (spotted fever group rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. CONCLUSION/SIGNIFICANCE: Collectively, we determined the relative conservation and

  3. Luminal uptake and intracellular transport of insulin in renal proximal tubules

    International Nuclear Information System (INIS)

    Hellfritzsch, M.; Christensen, E.I.; Sonne, O.

    1986-01-01

    It is generally accepted that proteins taken up from the renal tubular fluid are transported into lysosomes in proximal tubule cells. Recently, however, it has been postulated that insulin in isolated perfused rat kidneys did not accumulate in lysosomes but to a certain degree in the Golgi region. The present study was undertaken to investigate the intracellular handling of biologically unaltered insulin in rat renal proximal tubule cells. Rats were prepared for in vivo micropuncture and either a colloidal gold insulin complex or insulin monoiodinated in the A-14 position ( 125 I-insulin) was microinfused into proximal tubules. After 5, 10, 25 or 60 min the tubules were fixed by microinfusion of glutaraldehyde and processed for electron microscopy or electron microscope autoradiography. A qualitative analysis of tubules infused with colloidal gold insulin or 125 I-insulin showed that insulin was taken up by endocytosis and transported to lysosomes, and a quantitative autoradiographic analysis of the 125 I-insulin microinfused tubules showed that the grain density after five min was significantly increased for endocytic vacuoles and for lysosomes. After 60 min the grain density was still significant over lysosomes. The accumulation of grains was non-significant over all other areas analyzed at any time. This study shows that insulin is taken up from the luminal side of the proximal tubule by endocytosis and transported to the lysosomes. There was no significant transport to the Golgi region

  4. 31P-NMR differentiation between intracellular phosphate pools in Cosmarium (chlorophyta)

    International Nuclear Information System (INIS)

    Elgavish, A.; Elgavish, G.A.

    1980-01-01

    31 P nuclear magnetic resonance (NMR) spectroscopy of intact Cosmarium sp. cells is presented as a suitable tool for the differentiation of intracellular accumulation pools of polyphosphates. The cold trichloroacetic acid (TCA) insoluble fraction is shown to contain most of the total cellular phosphate in the phosphate rich Cosmarium cells. Moreover, evidence from a 31 P-NMR study and electron microscopic observations of cold TCA treated Cosmarium cells indicate that this fraction consists mostly of polyphosphates which seem to retain the native morphological structure observed in the untreated cells. The determination of orthophosphate in the hot water extract of Cosmarium cells did not measure the polyphosphate pools. Determination of total phosphorus content in the hot water extract rendered a value three times higher than the frequently used orthophosphate determination procedure. However, as revealed by the 31 P-NMR spectra and the chemical analyses of the extract and of the treated cells, even total phosphorus in the extract measured only 30% of the total cellular phosphorus. 31 P-NMR enabled the unequivocal chemical identification of the major phosphate compounds in the hot water extract (Surplus P) as orthophosphate and polyphosphates of about 10 phosphate units chainlength. More than 70% of the accumulation pool of polyphosphates was still in the cells after extraction. However, the electron microscopy study revealed that the native granular structure of polyphosphates had been destroyed by the hot water extraction procedure

  5. Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

    International Nuclear Information System (INIS)

    Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

    2010-01-01

    The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

  6. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    Science.gov (United States)

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellularaccumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  7. Association of postfire peat accumulation and microtopography in boreal bogs

    Energy Technology Data Exchange (ETDEWEB)

    Benscoter, B.W.; Vitt, D.H. [Southern Illinois Univ., Carbondale, IL (United States). Dept. of Plant Biology; Wieder, R.K. [Villanova Univ., Villanova, PA (United States). Dept. of Biology

    2005-09-01

    Fire impacts peatland species composition by differentially removing vegetation and resetting succession, which results in peat accumulation changes. A study of peat accumulation and microtopography in 2 burned bogs in Alberta was presented in this paper. Measurements of current and historic microtopography were made, and cores were collected along the gradient to identify the depth of peat accumulated since fires, as well as to assess its properties. It was observed that current microtopography was significant and correlated with the immediate post-fire surface relief. Differences in the magnitude of variability between sites suggested that differential rates of growth between features were exacerbated between sites and reflected in bog microtopography. Rates of organic matter accumulation ranged from 156 to 257 g/m{sup 2} per year, and were elevated but comparable to recent published rates. It was noted that organic matter content and accumulation rates were greater for hummocks than hollows at the Athabasca bog, but the difference between features diminished at Sinkhole Lake. It was concluded that the pattern and properties of peat accumulation and microtopography post-fire is topographical, and hence species dependent. Rates of change are dependent on fire severity and its effect on vegetation composition and succession. 33 refs., 4 figs.

  8. Biota-Sediment Accumulation Factor Data

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Biota-Sediment Accumulation Factor contains approximately 20,000 biota-sediment accumulation factors (BSAFs) from 20 locations (mostly Superfund sites) for...

  9. Differential inhibitory response to telcagepant on αCGRP induced vasorelaxation and intracellular Ca(2+) levels in the perfused and non-perfused isolated rat middle cerebral artery

    DEFF Research Database (Denmark)

    Erdling, André; Sheykhzade, Majid; Edvinsson, Lars

    2017-01-01

    BACKGROUND: Calcitonin gene-related peptide (CGRP) is one of the most potent endogenous vasodilators identified to date. The present study elucidates the differential interaction of CGRP, its receptor and the effect of the CGRP-receptor antagonist telcagepant on intracellular Ca(2+) -levels...... and tension in rat middle cerebral arteries (MCA) by pressurized arteriography, FURA-2/wire myography and immunohistochemistry. METHODS: A pressurized arteriograph system was used to evaluate changes in MCA tension when subjected to CGRP and/or telcagepant. Intracellular calcium levels were evaluated using......, while abluminal telcagepant inhibited the relaxation (10(-6) M). Using the FURA-2 method in combination with wire myography we observed that αCGRP reduced intracellular calcium levels and in parallel the vascular tone. Telcagepant (10(-6) M) inhibited both vasorelaxation and drop in intracellular...

  10. The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

    Science.gov (United States)

    Johannes, Ludger

    2002-07-01

    Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

  11. Charge accumulation in lossy dielectrics: a review

    DEFF Research Database (Denmark)

    Rasmussen, Jørgen Knøster; McAllister, Iain Wilson; Crichton, George C

    1999-01-01

    At present, the phenomenon of charge accumulation in solid dielectrics is under intense experimental study. Using a field theoretical approach, we review the basis for charge accumulation in lossy dielectrics. Thereafter, this macroscopic approach is applied to planar geometries such that the mat......At present, the phenomenon of charge accumulation in solid dielectrics is under intense experimental study. Using a field theoretical approach, we review the basis for charge accumulation in lossy dielectrics. Thereafter, this macroscopic approach is applied to planar geometries...

  12. Accumulation of hepatic Hsp70 and plasma cortisol in Oreochromis ...

    African Journals Online (AJOL)

    The hepatic isoforms Hsp70, Hsp74 and Hsp76 were identified and quantified from copper exposures. Long-term DDT exposure did not result in significant induction of hepatic Hsp70. An increase in plasma cortisol concentration was associated with a decrease in heat shock protein accumulation after cadmium exposure, ...

  13. Identification of a Paralog-Specific Notch1 Intracellular Domain Degron

    OpenAIRE

    Broadus, Matthew R.; Chen, Tony W.; Neitzel, Leif R.; Ng, Victoria H.; Jodoin, Jeanne; Lee, Laura A.; Salic, Adrian; Robbins, David J.; Capobianco, Anthony J.; Patton, James G.; Huppert, Stacey S.; Lee, Ethan

    2016-01-01

    Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box...

  14. Radiocaesium accumulation by different plant species

    International Nuclear Information System (INIS)

    Filiptsova, G.G.

    2000-01-01

    Using the model object influence of mineral nutritions level on radiocaesium accumulation by different plant species has been studied. It was shown the wheat roots accumulation the minimal value on radiocaesium on normal potassium level, the rye roots accumulation maximal level radiocaesium. (authors)

  15. 47 CFR 32.3100 - Accumulated depreciation.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Accumulated depreciation. 32.3100 Section 32... Accumulated depreciation. (a) This account shall include the accumulated depreciation associated with the... with depreciation amounts concurrently charged to Account 6561, Depreciation expense—telecommunications...

  16. Nickel-accumulating plant from Western Australia

    Energy Technology Data Exchange (ETDEWEB)

    Severne, B C; Brooks, R R

    1972-01-01

    A small shrub Hybanthus floribundus (Lindl.) F. Muell. Violaceae growing in Western Australia accumulates nickel and cobalt to a very high degree. Values of up to 23% nickel in leaf ash may represent the highest relative accumulation of a metal on record. The high accumulation of nickel poses interesting problems in plant physiology and plant biochemistry. 9 references, 2 figures, 1 table.

  17. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    Science.gov (United States)

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  18. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

  19. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  20. Intracellular pH gradients in migrating cells

    DEFF Research Database (Denmark)

    Martin, Christine; Pedersen, Stine Helene Falsig; Schwab, Albrecht

    2011-01-01

    might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of p...

  1. Structural rearrangement of the intracellular domains during AMPA receptor activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang

    2016-01-01

    -clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence...

  2. The interferon response to intracellular DNA: why so many receptors?

    Science.gov (United States)

    Unterholzner, Leonie

    2013-11-01

    The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

    Directory of Open Access Journals (Sweden)

    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  4. Engineering of obligate intracellular bacteria: progress, challenges and paradigms

    Science.gov (United States)

    Over twenty years have passed since the first report of genetic manipulation of an obligate intracellular bacterium. Through progress interspersed by bouts of stagnation, microbiologists and geneticists have developed approaches to genetically manipulate obligates. A brief overview of the current ge...

  5. Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms

    DEFF Research Database (Denmark)

    Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena

    2013-01-01

    these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...

  6. Dihydroceramide biology - Structure-specific metabolism and intracellular localization

    NARCIS (Netherlands)

    Kok, JW; NikolovaKarakashian, M; Klappe, K; Alexander, C; Merrill, AH

    1997-01-01

    This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DN-Cer), an intermediate in de novo sphingolipid biosynthesis, When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl-DH-Cer (C-6-NBD-DH-Cer) was incubated with HT29, NRK, BHK,

  7. CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

    Directory of Open Access Journals (Sweden)

    Samuel eGoodchild

    2012-06-01

    Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

  8. Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

    Directory of Open Access Journals (Sweden)

    Jinhua Li

    2016-02-01

    Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

  9. Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

    Science.gov (United States)

    Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

    2014-11-01

    TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Cytoplasmic tail of coronavirus spike protein has intracellular

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

  11. Facilitating Intracellular Drug Delivery by Ultrasound-Activated Microbubbles

    NARCIS (Netherlands)

    Lammertink, BHA

    2017-01-01

    The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model

  12. Energy Accumulation by Hydrogen Technologies

    Directory of Open Access Journals (Sweden)

    Jiřina Čermáková

    2012-01-01

    Full Text Available Photovoltaic power plants as a renewable energy source have been receiving rapidly growing attention in the Czech Republic and in the other EU countries. This rapid development of photovoltaic sources is having a negative effect on the electricity power system control, because they depend on the weather conditions and provide a variable and unreliable supply of electric power. One way to reduce this effect is by accumulating electricity in hydrogen. The aim of this paper is to introduce hydrogen as a tool for regulating photovoltaic energy in island mode. A configuration has been designed for connecting households with the photovoltaic hybrid system, and a simulation model has been made in order to check the validity of this system. The simulation results provide energy flows and have been used for optimal sizing of real devices. An appropriate system can deliver energy in a stand-alone installation.

  13. Electron-Positron Accumulator (EPA)

    CERN Multimedia

    Photographic Service

    1986-01-01

    After acceleration in the low-current linac LIL-W, the electrons and positrons are accumulated in EPA to obtain a sufficient intensity and a suitable time-structure, before being passed on to the PS for further acceleration to 3.5 GeV. Electrons circulate from right to left, positrons in the other direction. Dipole bending magnets are red, focusing quadrupoles blue, sextupoles for chromaticity-control orange. The vertical tube at the left of the picture belongs to an optical transport system carrying the synchrotron radiation to detectors for beam size measurement. Construction of EPA was completed in spring 1986. LIL-W and EPA were conceived for an energy of 600 MeV, but operation was limited to 500 MeV.

  14. Nickel accumulation by Hybanthus floribundus

    Energy Technology Data Exchange (ETDEWEB)

    Severne, B C

    1974-04-26

    Several ecotypes of Hybanthus floribundus are found across the southern part of Australia. However, the three nickel accumulating ecotypes are restricted to a broad belt in Western Australia. Nickel concentrations in this shrub were observed to decrease southwards (from 8000 to 1000 p.p.m.) as the annual rainfall increased from 7 inches to more than 30 inches. Studies have shown that nickel concentrations increase from the roots through the rootstock, into the stems and reach maximum towards the leaf tips. High nickel concentrations are also seen in seed capsules (1500 p.p.m.), seeds (2000 p.p.m.) and flowers. The maximum nickel concentration recorded is 1.6% (26% nickel in ash) in mature leaf tissue. 16 references, 2 tables.

  15. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  16. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-12-09

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

  17. Radionuclide accumulation peculiarities demonstrated by vegetable varieties

    International Nuclear Information System (INIS)

    Kruk, A.V.; Goncharenko, G.G.; Kilchevsky, A.V.

    2004-01-01

    This study focused on ecological and genetic aspects of radionuclide accumulation demonstrated by a number of vegetable varieties. The researches resulted in determining the cabbage varieties which were characterised by the minimal level of radionuclide accumulation. It was shown that the above varieties manifested the relation between radionuclide accumulation and morphobiological characteristics such as vegetation period duration and yield criteria. The study specified the genotypes with high ecological stability as regards to radionuclide accumulation: 'Beloruskaya 85' cabbage and 'Dokhodny' tomato showed the best response to Cs 137, while 'Beloruskaya 85', 'Rusinovka', 'Amager 611' cabbage varieties and 'Sprint' tomato showed the minimal level of Sr 90 accumulation. (authors)

  18. Guidelines for Waste Accumulation Areas (WAAs)

    International Nuclear Information System (INIS)

    1991-07-01

    The purpose of this document is to set conditions for establishing and maintaining areas for the accumulation of hazardous waste at LBL. Areas designed for accumulation of these wastes in quantities greater than 100 kg (220 lb) per month of solid waste or 55 gallons per month of liquid waste are called Waste Accumulation Areas (WAAs). Areas designed for accumulation of wastes in smaller amounts are called Satellite Accumulation Areas (SAAs). This document provides guidelines for employee and organizational responsibilities for WAAs; constructing a WAA; storing waste in a WAA; operating and maintaining a WAA, and responding to spills in a WAA. 4 figs

  19. Intracellular Na(+) and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons.

    Science.gov (United States)

    Wang, Yi-Chi; Yang, Jyh-Jeen; Huang, Rong-Chi

    2012-10-01

    Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na(+) and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K(+) to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K(+) to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K(+)-induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na(+) imaging with a Na(+)-sensitive fluorescent dye indicated saturating accumulation of intracellular Na(+) in response to pump blockade with zero K(+). The Na(+) ionophore monensin also induced Na(+) loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na(+) or by pump blockade. Conversely, Na(+) depletion with Na(+)-free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na(+) levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na(+) and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons.

  20. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Lojk J

    2015-02-01

    Full Text Available Jasna Lojk,1 Vladimir B Bregar,1 Maruša Rajh,1 Katarina Miš,2 Mateja Erdani Kreft,3 Sergej Pirkmajer,2 Peter Veranič,3 Mojca Pavlin1 1Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, 2Institute of Pathophysiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Abstract: Magnetic nanoparticles (NPs are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA in three cell types: Chinese Hamster Ovary (CHO, mouse melanoma (B16 cell line, and primary human myoblasts (MYO. We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better

  1. Screening of Phosphorus-Accumulating Fungi and Their Potential for Phosphorus Removal from Waste Streams.

    Science.gov (United States)

    Ye, Yulin; Gan, Jing; Hu, Bo

    2015-11-01

    While bacteria have been primarily studied for phosphorus (P) removal in wastewater treatment, fungi and their ability to accumulate intracellular polyphosphate are less investigated. P-accumulating fungal strains were screened from soybean plants and surrounding soil by flask cultivation with potato dextrose broth and KH2PO4 in this study. Mucor circinelloides was selected for its high efficiency in P removal efficiency and high cellular P content. Neisser staining and growth-curve analysis confirmed that M. circinelloides stored polyphosphate intracellularly by luxury phosphate uptake. The effect of culture medium compositions on P removal efficiency and cellular P content was also investigated. Monosaccharides (such as glucose and fructose) and organic nitrogen (N, such as urea, and peptone) promoted fungi growth and P accumulation. M. circinelloides also preferred organic phosphates. When glucose, urea, and phytic acid sodium salt were used as the carbon, N, and P source, respectively, the maximum utilization efficiency was 40.1% for P and 7.08% for cellular P content. In addition, the potential of M. circinelloides for P removal from waste streams was investigated. Compared with the non-inoculated control culture, inoculation with M. circinelloides improved the soluble P removal in treating wastewater centrate, screened manure, and digested manure.

  2. Nitrogen-deprivation elevates lipid levels in Symbiodinium spp. by lipid droplet accumulation: morphological and compositional analyses.

    Directory of Open Access Journals (Sweden)

    Pei-Luen Jiang

    Full Text Available Stable cnidarian-dinoflagellate (genus Symbiodinium endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B upon nitrogen (N-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503, indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG and cholesterol ester (CE were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs, a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm.

  3. Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells.

    Science.gov (United States)

    Nalaskowski, Marcus M; Ehm, Patrick; Rehbach, Christoph; Nelson, Nina; Täger, Maike; Modest, Kathrin; Jücker, Manfred

    2018-05-28

    The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1. In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells. Copyright © 2017. Published by Elsevier Inc.

  4. PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum

    OpenAIRE

    Yoshida, Ken-ichi; Takemoto, Yuki; Sotsuka, Takayuki; Tanaka, Kosei; Takenaka, Shinji

    2013-01-01

    Background Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-β-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding ...

  5. Bidens tripartite L.: A Cd-accumulator confirmed by pot culture and site sampling experiment

    International Nuclear Information System (INIS)

    Wei Shuhe; Niu Rongcheng; Srivastava, Mrittunjai; Zhou Qixing; Wu Zhijie; Sun Tieheng; Hu Yahu; Li Yunmeng

    2009-01-01

    Characteristics of accumulation and tolerance of cadmium (Cd) in Bidens tripartite L. were investigated to identify Cd-accumulating properties. In this study, pot culture experiment and site sampling experiments were conducted to assess whether this plant is a heavy metal hyperaccumulator or accumulator. The results indicated that the Cd enrichment factor (concentration in plant/soil) and Cd translocation factor (concentration in shoot/root) of B. tripartite was principally >1 in pot culture and concentration gradient experiments. Shoot biomass was not reduced significantly (p -1 , the threshold concentration for a Cd-hyperaccumulator. In the site sampling experiment, B. tripartite also showed Cd-accumulator properties. Based on these results, B. tripartite could be identified as a Cd-accumulator. Thus, B. tripartite should only be considered as a Cd-accumulator.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-15

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

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

    International Nuclear Information System (INIS)

    Wu Yun; Wang Wenxiong

    2011-01-01

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

  8. Copper accumulation by stickleback nests containing spiggin.

    Science.gov (United States)

    Pinho, G L L; Martins, C M G; Barber, I

    2016-07-01

    The three-spined stickleback is a ubiquitous fish of marine, brackish and freshwater ecosystems across the Northern hemisphere that presents intermediate sensitivity to copper. Male sticklebacks display a range of elaborate reproductive behaviours that include nest construction. To build the nests, each male binds nesting material together using an endogenous glycoprotein nesting glue, known as 'spiggin'. Spiggin is a cysteine-rich protein and, therefore, potentially binds heavy metals present in the environment. The aim of this study was to investigate the capacity of stickleback nests to accumulate copper from environmental sources. Newly built nests, constructed by male fish from polyester threads in laboratory aquaria, were immersed in copper solutions ranging in concentration from 21.1-626.6 μg Cu L(-1). Bundles of polyester threads from aquaria without male fish were also immersed in the same copper solutions. After immersion, nests presented higher amounts of copper than the thread bundles, indicating a higher capacity of nests to bind this metal. A significant, positive correlation between the concentration of copper in the exposure solution and in the exposed nests was identified, but there was no such relationship for thread bundles. Since both spiggin synthesis and male courtship behaviour are under the control of circulating androgens, we predicted that males with high courtship scores would produce and secrete high levels of the spiggin protein. In the present study, nests built by high courtship score males accumulated more copper than those built by low courtship score males. Considering the potential of spiggin to bind metals, the positive relationship between fish courtship and spiggin secretion seems to explain the higher amount of copper on the nests from the fish showing high behaviour scores. Further work is now needed to determine the consequences of the copper binding potential of spiggin in stickleback nests for the health and survival of

  9. Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency

    International Nuclear Information System (INIS)

    Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S.

    2006-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-Jκ, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway

  10. Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

    Science.gov (United States)

    Janero, D R; Burghardt, C; Feldman, D

    1988-10-01

    Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

  11. Comparison of accumulation of four metalloids in Allium sativum.

    Science.gov (United States)

    Ogra, Yasumitsu; Awaya, Yumi; Anan, Yasumi

    2015-05-01

    In this study, we evaluated the accumulation and metabolism of four metalloids: arsenic (As), selenium (Se), antimony (Sb), and tellurium (Te) in garlic to determine whether garlic can be used for the phytoremediation of those metalloids. Garlic was able to efficiently accumulate As and Se, the two-fourth-period metalloids. However, it was not able to accumulate Sb and Te, the two-fifth-period metalloids, because their bioaccumulation factors were below one. Speciation analyses revealed that four metalloids could be metabolized in garlic, although their metabolites could not be identified yet. Results also suggested that garlic was able to distinguish the metalloids in groups 15 and 16 and the fourth and fifth periods, i.e., As, Se, Sb, and Te. Therefore, garlic is one of the potential plants for the phytoremediation of the fourth-period metalloids.

  12. Caesium accumulation by microorganisms: uptake mechanisms, cation competition, compartmentalization and toxicity

    International Nuclear Information System (INIS)

    Avery, S.V.

    1995-01-01

    The continued release of caesium radioisotopes into the environment has led to a resurgence of interest in microbe-Cs interactions. Caesium exists almost exclusively as the monovalent cation Cs + in the natural environment. Although Cs + is a weak Lewis acid that exhibits a low tendency to form complexes with ligands, its chemical similarity to the biologically essential alkali cation K + facilitates high levels of metabolism-dependent intracellular accumulation. Microbial Cs + (K + ) uptake is generally mediated by monovalent cation transport systems located on the plasma membrane. These differ widely in specificity for alkali cations and consequently microorganisms display large differences in their ability to accumulate Cs + ; Cs + appears to have an equal or greater affinity than K + for transport in certain microorganisms. Microbial Cs + accumulation is markedly influenced by the presence of external cations, e.g. K + , Na + , NH 4 + and H + , and is generally accompanied by an approximate stoichiometric exchange for intracellular K + . However, stimulation of growth of K + -starved microbial cultures by Cs + is limited and it has been proposed that it is not the presence of Cs + in cells that is growth inhibitory but rather the resulting loss of K + . Increased microbial tolerance to Cs + may result from sequestration of Cs + in vacuoles or changes in the activity and/or specificity of transport systems mediating Cs + uptake. The precise intracellular target(s) for Cs + - induced toxicity has yet to be clearly defined, although certain internal structures, e.g. ribosomes, become unstable in the presence of Cs + and Cs + is known to substitute poorly for K + in the activation of many K + -requiring enzymes. (author)

  13. Hemochromatosis enhances tumor progression via upregulation of intracellular iron in head and neck cancer.

    Directory of Open Access Journals (Sweden)

    Michelle Lenarduzzi

    Full Text Available Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC, outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC.Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS, clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry.In a panel of HNSCC cell lines, hemochromatosis (HFE was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX, significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression.Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

  14. IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex

    Directory of Open Access Journals (Sweden)

    Khian Hong Pua

    2017-01-01

    Full Text Available Summary: Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA is a mixed polyketide and non-ribosomal peptide synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA. It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5′-monophosphate dehydrogenase 2 (IMPDH2 as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-β-synthase (CBS domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1. This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation. : Pua et al. identify IMPDH2 as an intracellular target of the PPIA-SFA complex and show that the CBS domains of IMPDH2 are required for cellular proliferation. Keywords: cyclophilin A, sanglifehrin A, inosine-5′-monophosphate dehydrogenase, cystathionine-β-synthase domains, protein-protein interactions

  15. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Directory of Open Access Journals (Sweden)

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  16. Regulation of dopamine transporter trafficking by intracellular amphetamine

    DEFF Research Database (Denmark)

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang

    2006-01-01

    -induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent...... alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic...... redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A...

  17. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements

    DEFF Research Database (Denmark)

    Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig

    2014-01-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells......, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped...... in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods....

  18. Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

    DEFF Research Database (Denmark)

    Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

    2011-01-01

    The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

  19. Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems.

    Science.gov (United States)

    Ngo, Anh H; Bose, Sohini; Do, Loi H

    2018-03-23

    This concept article focuses on the rapid growth of intracellular chemistry dedicated to the integration of small-molecule metal catalysts with living cells and organisms. Although biological systems contain a plethora of biomolecules that can deactivate inorganic species, researchers have shown that small-molecule metal catalysts could be engineered to operate in heterogeneous aqueous environments. Synthetic intracellular reactions have recently been reported for olefin hydrogenation, hydrolysis/oxidative cleavage, azide-alkyne cycloaddition, allylcarbamate cleavage, C-C bond cross coupling, and transfer hydrogenation. Other promising targets for new biocompatible reaction discovery will also be discussed, with a special emphasis on how such innovations could lead to the development of novel technologies and chemical tools. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Chelation of intracellular calcium blocks insulin action in the adipocyte

    International Nuclear Information System (INIS)

    Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

    1987-01-01

    The hypothesis that intracellular Ca 2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca 2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl 2 and the Ca 2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl 2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 μM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125 I=labeled insulin to adipocytes. These findings suggest that intracellular Ca 2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca 2+ -dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin

  1. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    Science.gov (United States)

    2016-05-26

    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...only allow entirely new investigations into the replication of these viruses, but also how this method can be applied to any virus with a known...localization, TurboFISH, hemorrhagic fever virus replication 1. Introduction RNA FISH was developed as a method to visualize cellular RNA by binding a

  2. Mycobacterium avium-intracellulare: a rare cause of subacromial bursitis.

    Science.gov (United States)

    Sinha, Raj; Tuckett, John; Hide, Geoff; Dildey, Petra; Karsandas, Alvin

    2015-01-01

    Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations.

  3. Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes

    DEFF Research Database (Denmark)

    Westgate, G E; Weaver, A C; Couchman, J R

    1985-01-01

    immunofluorescent staining for BPA is linear at the basement membrane zone (BMZ) of skin and many other epithelial tissues. At higher magnification however, we observed a punctate staining pattern for BPA which was regular in appearance and suggested localization of BPA to discrete structures at the BMZ. Subsequent...... intracellularly both in vivo and in vitro. We suggest that BPA is not normally a lamina lucida component, but that it may form part of a linkage between the cytoskeleton and the basement membrane....

  4. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Onkar Sharma

    2016-03-01

    Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

  5. Molecular evolution, intracellular organization, and the quinary structure of proteins.

    OpenAIRE

    McConkey, E H

    1982-01-01

    High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most ...

  6. Control of intracellular heme levels: Heme transporters and heme oxygenases

    OpenAIRE

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

  7. Fatty Acid Signaling: The New Function of Intracellular Lipases

    Directory of Open Access Journals (Sweden)

    Zuzana Papackova

    2015-02-01

    Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

  8. Estimating the biophysical properties of neurons with intracellular calcium dynamics.

    Science.gov (United States)

    Ye, Jingxin; Rozdeba, Paul J; Morone, Uriel I; Daou, Arij; Abarbanel, Henry D I

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V(t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  9. Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

    Science.gov (United States)

    Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

    2009-01-01

    BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

  10. Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

    Science.gov (United States)

    Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan

    2016-10-03

    The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

  11. Glucosylceramide accumulation is not confined to the lysosome in fibroblasts from patients with Gaucher disease.

    Science.gov (United States)

    Fuller, Maria; Rozaklis, Tina; Lovejoy, Melanie; Zarrinkalam, Krystyna; Hopwood, John J; Meikle, Peter J

    2008-04-01

    Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.

  12. Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

    Science.gov (United States)

    Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

    2016-05-01

    Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Effect of minerals on accumulation of Cs by fungus Saccaromyces cerevisiae

    International Nuclear Information System (INIS)

    Ohnuki, Toshihiko; Sakamoto, Fuminori; Yamasaki, Shinya; Kozai, Naofumi; Shiotsu, Hiroyuki; Utsunomiya, Satoshi; Watanabe, Naoko; Kozaki, Tamotsu

    2015-01-01

    The accumulation of Cs by unicellular fungus of Saccharomyces cerevisiae in the presence of minerals has been studied to elucidate the role of microorganisms in the migration of radioactive Cs in the environment. Two different types of experiments were employed: experiments using stable Cs to examine the effect of a carbon source on the accumulation of Cs, and accumulation experiments of radioactive Cs from agar medium containing 137 Cs and zeolite, vermiculite, phlogopite, smectite, mica, or illite as mineral supplements. In the former type of experiments, the Cs-accumulated cells were analyzed by scanning electron microscopy equipped with energy dispersive X-ray analysis (SEM-EDS). In the latter type, the radioactivity in the yeast cells was measured by an autoradiography technique. When a carbon source was present, higher amounts of Cs accumulated in the cells than in the resting condition without a carbon source. Analyses with SEM-EDS showed that no mineral formed on the cell surface. These results indicate that the yeast cells accumulate Cs by adsorption on the cell surface and intracellular accumulation. In the presence of minerals in the agar medium, the radioactivity in the yeast cells was in the order of mica > smectite, illite >> vermiculite, phlogopite, zeolite. This order is inversely correlated to the ratio of the concentration of radioactive Cs between the minerals and the medium solution. These results strongly suggest that the yeast accumulates radioactive Cs competitively with minerals. - Graphical abstract: Autoradiography analysis showed that presence of larger amounts of mineral of vermiculite collected in South Africa induced less accumulation of radioactive Cs in yeast cells from the medium. - Highlights: • Effect of minerals on the accumulation of radioactive Cs by yeast was studied. • Presence of minerals reduced accumulation of radioactive Cs by yeast. • The order of reduction is mica>smectite, illite>>vermiculite, phlogopite

  14. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Science.gov (United States)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  15. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    Energy Technology Data Exchange (ETDEWEB)

    Volland, Stefanie, E-mail: Stefanie.Volland@stud.sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria); Luetz, Cornelius, E-mail: cornelius.luetz@uibk.ac.at [Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck (Austria); Michalke, Bernhard, E-mail: bernhard.michalke@helmholtz-muenchen.de [Helmholtz Zentrum Muenchen, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstaedter Landstrasse 1, 85764 Neuherberg (Germany); Luetz-Meindl, Ursula, E-mail: ursula.luetz-meindl@sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria)

    2012-03-15

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 {mu}M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  16. Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

    International Nuclear Information System (INIS)

    Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

    2012-01-01

    Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

  17. ACCUMULATION AND CONSUMPTION IN MICROECONOMIC SYSTEM

    Directory of Open Access Journals (Sweden)

    Serghey A. Amelkin

    2004-12-01

    Full Text Available Two main processes are common for an economic system. They are consumption and accumulation. The first one is described by utility function, either cardinal or ordinal one. The mathematical model for accumulation process can be constructed using wealth function introduced within the frame of irreversible microeconomics. Characteristics of utility and wealth functions are compared and a problem of extreme performance of resources exchange process is solved for a case when both the consumption and accumulation exist.

  18. Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

    Science.gov (United States)

    Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

    2016-01-01

    The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

  19. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Directory of Open Access Journals (Sweden)

    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  20. Leading survey and research report for fiscal 1999. New technology based on functions involved in intracellular protein transport; 1999 nendo saibonai tanpakushitsu yuso kino riyo gijutsu kenkyu hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    An intercellular transport technology (artificial manipulation of an intracellular protein transport system in eucaryotes) is studied for the accumulation of cytotoxic proteins, whose expression has so far been difficult, and activated proteins, which have avoided decomposition, in appropriate intracellular minute organs. The aim is to construct a system to allow foreign proteins high in productivity and quality to express themselves for production in eucaryotes. Basic surveys were conducted of the intracellular biological functions of single-membrane organelles (endoplasmic reticulum, peroxisome, vacuole/lysosome, and Golgi body), the molecular mechanism of protein transport to each organelle, and protein activation and quality control, and element technologies were extracted. For the development of novel pharmaceuticals making use of the intracellular protein transport technology, an activated protein production system was built and a search was made for transport activity impeding substances. Research tasks relative to the development of the new technologies were isolated, such as the visualization of intercellular transport. A survey was made of the market for pharmaceuticals, cosmetics, enzymes, and visualizing equipment (fluorescence microscope provided with new functions), etc. (NEDO)

  1. Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation.

    Science.gov (United States)

    Abdallah, Basem M; Jafari, Abbas; Zaher, Walid; Qiu, Weimin; Kassem, Moustapha

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

    Science.gov (United States)

    Kozaki, Ikko; Shimizu, Kazunori; Honda, Hiroyuki

    2017-08-01

    Intracellular functional peptides that play a significant role inside cells have been receiving a lot of attention as regulators of cellular activity. Previously, we proposed a novel screening system for intracellular functional peptides; it combined a photo-cleavable peptide array system with cell-penetrating peptides (CPPs). Various peptides can be delivered into cells and intracellular functions of the peptides can be assayed by means of our system. The aim of the present study was to demonstrate that the proposed screening system can be used for assessing the intracellular activity of peptides. The cell death-inducing peptide (LNLISKLF) identified in a mitochondria-targeting domain (MTD) of the Noxa protein served as an original peptide sequence for screening of peptides with higher activity via modification of the peptide sequence. We obtained 4 peptides with higher activity, in which we substituted serine (S) at the fifth position with phenylalanine (F), valine (V), tryptophan (W), or tyrosine (Y). During analysis of the mechanism of action, the modified peptides induced an increase in intracellular calcium concentration, which was caused by the treatment with the original peptide. Higher capacity for cell death induction by the modified peptides may be caused by increased hydrophobicity or an increased number of aromatic residues. Thus, the present work suggests that the intracellular activity of peptides can be assessed using the proposed screening system. It could be used for identifying intracellular functional peptides with higher activity through comprehensive screening. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Gelsolin-Cu/ZnSOD interaction alters intracellular reactive oxygen species levels to promote cancer cell invasion

    KAUST Repository

    Tochhawng, Lalchhandami

    2016-07-07

    The actin-binding protein, gelsolin, is a well known regulator of cancer cell invasion. However, the mechanisms by which gelsolin promotes invasion are not well established. As reactive oxygen species (ROS) have been shown to promote cancer cell invasion, we investigated on the hypothesis that gelsolin-induced changes in ROS levels may mediate the invasive capacity of colon cancer cells. Herein, we show that increased gelsolin enhances the invasive capacity of colon cancer cells, and this is mediated via gelsolin\\'s effects in elevating intracellular superoxide (O2 .-) levels. We also provide evidence for a novel physical interaction between gelsolin and Cu/ZnSOD, that inhibits the enzymatic activity of Cu/ZnSOD, thereby resulting in a sustained elevation of intracellular O2 .-. Using microarray data of human colorectal cancer tissues from Gene Omnibus, we found that gelsolin gene expression positively correlates with urokinase plasminogen activator (uPA), an important matrix-degrading protease invovled in cancer invasion. Consistent with the in vivo evidence, we show that increased levels of O2 .- induced by gelsolin overexpression triggers the secretion of uPA. We further observed reduction in invasion and intracellular O2 .- levels in colon cancer cells, as a consequence of gelsolin knockdown using two different siRNAs. In these cells, concurrent repression of Cu/ ZnSOD restored intracellular O2 .- levels and rescued invasive capacity. Our study therefore identified gelsolin as a novel regulator of intracellular O2 .- in cancer cells via interacting with Cu/ZnSOD and inhibiting its enzymatic activity. Taken together, these findings provide insight into a novel function of gelsolin in promoting tumor invasion by directly impacting the cellular redox milieu.

  4. Characterization of accumulated precipitates during subsurface iron removal

    International Nuclear Information System (INIS)

    Halem, Doris van; Vet, Weren de; Verberk, Jasper; Amy, Gary; Dijk, Hans van

    2011-01-01

    Research highlights: → Accumulated iron was not found to clog the well or aquifer after 12 years of subsurface iron removal. → 56-100% of accumulated iron hydroxides were found to be crystalline. → Subsurface iron removal favoured certain soil layers, either due to hydraulics or mineralogy. → Other groundwater constituents, such as manganese and arsenic were found to co-accumulate with iron. - Abstract: The principle of subsurface iron removal for drinking water supply is that aerated water is periodically injected into the aquifer through a tube well. On its way into the aquifer, the injected O 2 -rich water oxidizes adsorbed Fe 2+ , creating a subsurface oxidation zone. When groundwater abstraction is resumed, the soluble Fe 2+ is adsorbed and water with reduced Fe concentrations is abstracted for multiple volumes of the injection water. In this article, Fe accumulation deposits in the aquifer near subsurface treatment wells were identified and characterized to assess the sustainability of subsurface iron removal regarding clogging of the aquifer and the potential co-accumulation of other groundwater constituents, such as As. Chemical extraction of soil samples, with Acid-Oxalate and HNO 3 , showed that Fe had accumulated at specific depths near subsurface iron removal wells after 12 years of operation. Whether it was due to preferred flow paths or geochemical mineralogy conditions; subsurface iron removal clearly favoured certain soil layers. The total Fe content increased between 11.5 and 390.8 mmol/kg ds in the affected soil layers, and the accumulated Fe was found to be 56-100% crystalline. These results suggest that precipitated amorphous Fe hydroxides have transformed to Fe hydroxides of higher crystallinity. These crystalline, compact Fe hydroxides have not noticeably clogged the investigated well and/or aquifer between 1996 and 2008. The subsurface iron removal wells even need less frequent rehabilitation, as drawdown increases more slowly than in

  5. DMPD: Intracellular DNA sensors in immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18573338 Intracellular DNA sensors in immunity. Takeshita F, Ishii KJ. Curr Opin Im...munol. 2008 Aug;20(4):383-8. Epub 2008 Jun 23. (.png) (.svg) (.html) (.csml) Show Intracellular DNA sensors ...in immunity. PubmedID 18573338 Title Intracellular DNA sensors in immunity. Authors Takeshita F, Ishii KJ. P

  6. DMPD: NOD-like receptors (NLRs): bona fide intracellular microbial sensors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18585455 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Shaw...tml) (.csml) Show NOD-like receptors (NLRs): bona fide intracellular microbial sensors. PubmedID 18585455 Ti...tle NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Authors

  7. New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis.

    Science.gov (United States)

    Oberbach, Andreas; Schlichting, Nadine; Feder, Stefan; Lehmann, Stefanie; Kullnick, Yvonne; Buschmann, Tilo; Blumert, Conny; Horn, Friedemann; Neuhaus, Jochen; Neujahr, Ralph; Bagaev, Erik; Hagl, Christian; Pichlmaier, Maximilian; Rodloff, Arne Christian; Gräber, Sandra; Kirsch, Katharina; Sandri, Marcus; Kumbhari, Vivek; Behzadi, Armirhossein; Behzadi, Amirali; Correia, Joao Carlos; Mohr, Friedrich Wilhelm; Friedrich, Maik

    2017-01-01

    In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may

  8. Rate of ice accumulation during ice storms

    Energy Technology Data Exchange (ETDEWEB)

    Feknous, N. [SNC-Lavalin, Montreal, PQ (Canada); Chouinard, L. [McGill Univ., Montreal, PQ (Canada); Sabourin, G. [Hydro-Quebec, Montreal, PQ (Canada)

    2005-07-01

    The rate of glaze ice accumulation is the result of a complex process dependent on numerous meteorological and physical factors. The aim of this paper was to estimate the distribution rate of glaze ice accumulation on conductors in southern Quebec for use in the design of mechanical and electrical de-icing devices. The analysis was based on direct observations of ice accumulation collected on passive ice meters. The historical database of Hydro-Quebec, which contains observations at over 140 stations over period of 25 years, was used to compute accumulation rates. Data was processed so that each glaze ice event was numbered in a chronological sequence. Each event consisted of the time series of ice accumulations on each of the 8 cylinders of the ice meters, as well as on 5 of its surfaces. Observed rates were converted to represent the average ice on a 30 mm diameter conductor at 30 m above ground with a span of 300 m. Observations were corrected to account for the water content of the glaze ice as evidenced by the presence of icicles. Results indicated that despite significant spatial variations in the expected severity of ice storms as a function of location, the distribution function for rates of accumulation were fairly similar and could be assumed to be independent of location. It was concluded that the observations from several sites could be combined in order to obtain better estimates of the distribution of hourly rates of ice accumulation. However, the rates were highly variable. For de-icing strategies, it was suggested that average accumulation rates over 12 hour periods were preferable, and that analyses should be performed for other time intervals to account for the variability in ice accumulation rates over time. In addition, accumulation rates did not appear to be highly correlated with average wind speed for maximum hourly accumulation rates. 3 refs., 2 tabs., 10 figs.

  9. Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

    Science.gov (United States)

    Nakaya, Yuki; Miyazawa, Takayuki

    2014-06-01

    Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry

  10. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    Science.gov (United States)

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus , the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus , ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus .

  11. 31P MR spectroscopic measurement of intracellular pH in normal human hearts

    International Nuclear Information System (INIS)

    Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min

    2002-01-01

    To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of γ-ATP after performing MRS with ECG-gated two-dimensional 31 P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled 31 P CSI was performed on a 1.5-T scanner using a 1 H 31 P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of α-, β-, γ-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and γ-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm (γ-ATP), -7.8 ppm (α-ATP), and -16.3 ppm (β-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/β-ATP ratio was 2.20±0.17 and the PDE/β-ATP ratio, 1.04±0.09 pHi readings were 7.31±0.23 (calculated by the shift of Pi) and 6.81±0.20 (calculated by the shift of γ-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method

  12. {sup 31}P MR spectroscopic measurement of intracellular pH in normal human hearts

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min [Kyungpook National Univ., Taegu (Korea, Republic of)] [and others

    2002-05-01

    To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of {gamma}-ATP after performing MRS with ECG-gated two-dimensional {sup 31}P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled {sup 31}P CSI was performed on a 1.5-T scanner using a {sup 1}H{sup 31}P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of {alpha}-, {beta}-, {gamma}-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and {gamma}-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm ({gamma}-ATP), -7.8 ppm ({alpha}-ATP), and -16.3 ppm ({beta}-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/{beta}-ATP ratio was 2.20{+-}0.17 and the PDE/{beta}-ATP ratio, 1.04{+-}0.09 pHi readings were 7.31{+-}0.23 (calculated by the shift of Pi) and 6.81{+-}0.20 (calculated by the shift of {gamma}-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method.

  13. Dynamics of inorganic nutrients in intertidal sediments: porewater, exchangeable and intracellular pools

    Directory of Open Access Journals (Sweden)

    Emilio eGarcia-Robledo

    2016-05-01

    Full Text Available The study of inorganic nutrients dynamics in shallow sediments usually focuses on two main pools: the porewater (PW nutrients and the exchangeable (EX ammonium and phosphate. Recently, it has been found that microphytobenthos (MPB and other microorganisms can accumulate large amounts of nutrients intracellularly (IC, highlighting the biogeochemical importance of this nutrient pool. Storing nutrients could support the growth of autotrophs when nutrients are not available, and could also provide alternative electron acceptors for dissimilatory processes such as nitrate reduction. Here, we studied the magnitude and relative importance of these three nutrient pools (PW, IC and EX and their relation to chlorophylls (used as a proxy for MPB abundance and organic matter (OM contents in an intertidal mudflat of Cadiz Bay (Spain. MPB was localized in the first 4 mm of the sediment and showed a clear seasonal pattern; highest chlorophylls content was found during autumn and lowest during spring-summer. The temporal and spatial distribution of nutrients pools and MPB were largely correlated. Ammonium was higher in the IC and EX fractions, representing on average 59 and 37% of the total ammonium pool, respectively. Similarly, phosphate in the IC and EX fractions accounted on average for 40 and 31% of the total phosphate pool, respectively. Nitrate in the PW was low, suggesting low nitrification activity and rapid consumption. Nitrate accumulated in the IC pool during periods of moderate MPB abundance, being up to 66% of the total nitrate pool, whereas it decreased when chlorophyll concentration peaked likely due to a high nitrogen demand. EX-Nitrate accounted for the largest fraction of total sediment nitrate, 66% on average. The distribution of EX-Nitrate was significantly correlated with chlorophyll and OM, which probably indicates a relation of this pool to an increased availability of sites for ionic adsorption. This EX-Nitrate pool could represent an

  14. Metabolic responses of primary and transformed cells to intracellular Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Nadine Gillmaier

    Full Text Available The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using (13C-labelled glucose or glutamine as carbon tracers. The (13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.

  15. Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

    2017-08-29

    Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

  16. An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Christopher C. Thompson

    2011-02-01

    Full Text Available Iron is an essential cofactor in a number of critical biochemical reactions, and as such, its acquisition, storage, and metabolism is highly regulated in most organisms. The obligate intracellular bacterium, Chlamydia trachomatis experiences a developmental arrest when iron within the host is depleted. The nature of the iron starvation response in Chlamydia is relatively uncharacterized because of the likely inefficient method of iron depletion, which currently relies on the compound deferoxamine mesylate (DFO. Inefficient induction of the iron starvation response precludes the identification of iron-regulated genes. This report evaluated DFO with another iron chelator, 2,2’-bipyridyl (Bpdl and presented a systematic comparison of the two across a range of criteria in a single-treatment time-of-infection regimen. We demonstrate that the membrane permeable Bpdl was superior to DFO in the inhibition of chlamydia development, the induction of aberrant morphology, and the induction of an iron starvation transcriptional response in both host and bacteria. Furthermore, iron starvation using Bpdl identified the periplasmic iron binding protein-encoding ytgA gene as iron- responsive. Overall, the data present a compelling argument for the use of Bpdl, rather than DFO, in future iron starvation studies of chlamydia and other intracellular bacteria.

  17. Studies on the intracellular localization of hHR23B

    International Nuclear Information System (INIS)

    Katiyar, Samiksha; Lennarz, William J.

    2005-01-01

    Yeast Rad23, originally identified as a DNA repair protein, has been proposed to participate in other cellular functions, i.e., the proteasome-degradation pathway, the process of spindle pole body duplication and as a component of the anaphase checkpoint. Two human homologs of yeast Rad23, hHR23A and hHR23B, exhibit high sequence homology with yRad23 and also have been shown to be involved in DNA repair and proteasome-dependent degradation. Previous studies on the intracellular localization of hHR23A and hHR23B revealed their predominant localization in the nucleus during interphase and in the cytoplasm during mitosis. We have analyzed the localization of hHR23B during all the phases of the cell cycle using immunofluorescence. Unlike previous studies, our results suggest localization of hHR23B in the nucleus as well as in the cytoplasm during G1 phase. The nuclear levels of hHR23B decrease during S-phase of the cell cycle. When the cell enters mitosis, hHR23B relocalizes in the cytoplasm without association with chromatin. These results indicate that the intracellular distribution hHR23B is cell cycle dependent

  18. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    Energy Technology Data Exchange (ETDEWEB)

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  19. Dual-modality single particle orientation and rotational tracking of intracellular transport of nanocargos.

    Science.gov (United States)

    Sun, Wei; Gu, Yan; Wang, Gufeng; Fang, Ning

    2012-01-17

    The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.

  20. Rab7b at the intersection of intracellular trafficking and cell migration.

    Science.gov (United States)

    Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

    2015-01-01

    Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

  1. The positron accumulator ring for the APS

    International Nuclear Information System (INIS)

    Crosbie, E.A.

    1989-01-01

    The Positron Accumulator Ring (PAR) is designed to accumulate and damp positrons from the 450-MeV linac during the 0.5-s cycle time of the injector synchrotron for the APS 7-GeV storage ring. During 0.4 s of each synchrotron cycle, up to 24 linac pulses are injected into the horizontal phase space of the PAR at a 60-Hz rate. Each injected pulse occupies about 1.3 of the circumference of the accumulator ring. After 0.1 s for longitudinal damping, the single accumulated bunch is transferred to one of the 353-MHz buckets of the injector synchrotron RF system. The bunch is accelerated to 7 GeV and transferred to the storage ring, while the PAR accumulates the next bunch of positrons. 2 refs., 3 figs., 2 tabs

  2. The positron accumulator ring for the APS

    International Nuclear Information System (INIS)

    Crosbie, E.A.

    1989-01-01

    The Positron Accumulator Ring (PAR) is designed to accumulate and damp positrons from the 450-MeV linac during the 0.5-s cycle time of the injector synchrotron for the APS 7-GeV storage ring. During 0.4 s of each synchrotron cycle, up to 24 linac pulses are injected into the horizontal phase space of the PAR at a 60-Hz rate. Each injected pulse occupies about 1/3 of the circumference of the accumulator ring. After 0.1 s for longitudinal damping, the single accumulated bunch is transferred to one of the 353-MHz buckets of the injector synchrotron RF system. The bunch is accelerated to 7 GeV and transferred to the storage ring, while the PAR accumulates the next bunch of positrons. 2 refs., 3 figs., 2 tabs

  3. Evidence Accumulation and Choice Maintenance Are Dissociated in Human Perceptual Decision Making.

    Directory of Open Access Journals (Sweden)

    Mads Lund Pedersen

    Full Text Available Perceptual decision making in monkeys relies on decision neurons, which accumulate evidence and maintain choices until a response is given. In humans, several brain regions have been proposed to accumulate evidence, but it is unknown if these regions also maintain choices. To test if accumulator regions in humans also maintain decisions we compared delayed and self-paced responses during a face/house discrimination decision making task. Computational modeling and fMRI results revealed dissociated processes of evidence accumulation and decision maintenance, with potential accumulator activations found in the dorsomedial prefrontal cortex, right inferior frontal gyrus and bilateral insula. Potential maintenance activation spanned the frontal pole, temporal gyri, precuneus and the lateral occipital and frontal orbital cortices. Results of a quantitative reverse inference meta-analysis performed to differentiate the functions associated with the identified regions did not narrow down potential accumulation regions, but suggested that response-maintenance might rely on a verbalization of the response.

  4. Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.

    Science.gov (United States)

    Marshall, Lee L; Stimpson, Scott E; Hyland, Ryan; Coorssen, Jens R; Myers, Simon J

    2014-04-01

    Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the SPTLC1 gene. The SPTLC1 protein is part of the SPT enzyme which is a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer's and Parkinson's disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases.

  5. The competence accumulation process in the technology transference strategy

    OpenAIRE

    Souza, André Silva de; Segatto-Mendes, Andréa Paula

    2008-01-01

    The present article evaluates and measures the technological competence accumulation in an automation area enterprise to distribution centers, Knapp Sudamérica Logistic and Automation Ltd, in the interval of the technology transference process previous period (1998-2001) and during the technology transference process (2002-2005). Therefore, based on an individual case study, the study identified the technology transference strategy and mechanism accorded between the head office and the branch...

  6. Paclitaxel loading in PLGA nanospheres affected the in vitro drug cell accumulation and antiproliferative activity

    Directory of Open Access Journals (Sweden)

    De Maria Ruggero

    2008-07-01

    Full Text Available Abstract Background PTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor® EL (polyethoxylated castor oil and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration. Methods In this paper PTX was loaded in PLGA NS. The activity of PTX-NS was assessed in vitro against thyroid, breast and bladder cancer cell lines in cultures. Cell growth was evaluated by MTS assay, intracellular NS uptake was performed using coumarin-6 labelled NS and the amount of intracellular PTX was measured by HPLC. Results NS loaded with 3% PTX (w/w had a mean size Conclusion These findings suggest that the greater biological effect of PTX-NS could be due to higher uptake of the drug inside the cells as shown by intracellular NS uptake and cell accumulation studies.

  7. Characterization of distinct Arctic aerosol accumulation modes and their sources

    Science.gov (United States)

    Lange, R.; Dall'Osto, M.; Skov, H.; Nøjgaard, J. K.; Nielsen, I. E.; Beddows, D. C. S.; Simo, R.; Harrison, R. M.; Massling, A.

    2018-06-01

    In this work we use cluster analysis of long term particle size distribution data to expand an array of different shorter term atmospheric measurements, thereby gaining insights into longer term patterns and properties of Arctic aerosol. Measurements of aerosol number size distributions (9-915 nm) were conducted at Villum Research Station (VRS), Station Nord in North Greenland during a 5 year record (2012-2016). Alongside this, measurements of aerosol composition, meteorological parameters, gaseous compounds and cloud condensation nuclei (CCN) activity were performed during different shorter occasions. K-means clustering analysis of particle number size distributions on daily basis identified several clusters. Clusters of accumulation mode aerosols (main size modes > 100 nm) accounted for 56% of the total aerosol during the sampling period (89-91% during February-April, 1-3% during June-August). By association to chemical composition, cloud condensation nuclei properties, and meteorological variables, three typical accumulation mode aerosol clusters were identified: Haze (32% of the time), Bimodal (14%) and Aged (6%). In brief: (1) Haze accumulation mode aerosol shows a single mode at 150 nm, peaking in February-April, with highest loadings of sulfate and black carbon concentrations. (2) Accumulation mode Bimodal aerosol shows two modes, at 38 nm and 150 nm, peaking in June-August, with the highest ratio of organics to sulfate concentrations. (3) Aged accumulation mode aerosol shows a single mode at 213 nm, peaking in September-October and is associated with cloudy and humid weather conditions during autumn. The three aerosol clusters were considered alongside CCN concentrations. We suggest that organic compounds, that are likely marine biogenic in nature, greatly influence the Bimodal cluster and contribute significantly to its CCN activity. This stresses the importance of better characterizing the marine ecosystem and the aerosol-mediated climate effects in the

  8. Characterization of accumulated precipitates during subsurface iron removal

    KAUST Repository

    Van Halem, Doris

    2011-01-01

    The principle of subsurface iron removal for drinking water supply is that aerated water is periodically injected into the aquifer through a tube well. On its way into the aquifer, the injected O2-rich water oxidizes adsorbed Fe 2+, creating a subsurface oxidation zone. When groundwater abstraction is resumed, the soluble Fe 2+ is adsorbed and water with reduced Fe concentrations is abstracted for multiple volumes of the injection water. In this article, Fe accumulation deposits in the aquifer near subsurface treatment wells were identified and characterized to assess the sustainability of subsurface iron removal regarding clogging of the aquifer and the potential co-accumulation of other groundwater constituents, such as As. Chemical extraction of soil samples, with Acid-Oxalate and HNO3, showed that Fe had accumulated at specific depths near subsurface iron removal wells after 12 years of operation. Whether it was due to preferred flow paths or geochemical mineralogy conditions; subsurface iron removal clearly favoured certain soil layers. The total Fe content increased between 11.5 and 390.8 mmol/kg ds in the affected soil layers, and the accumulated Fe was found to be 56-100% crystalline. These results suggest that precipitated amorphous Fe hydroxides have transformed to Fe hydroxides of higher crystallinity. These crystalline, compact Fe hydroxides have not noticeably clogged the investigated well and/or aquifer between 1996 and 2008. The subsurface iron removal wells even need less frequent rehabilitation, as drawdown increases more slowly than in normal production wells. Other groundwater constituents, such as Mn, As and Sr were found to co-accumulate with Fe. Acid extraction and ESEM-EDX showed that Ca occurred together with Fe and by X-ray Powder Diffraction it was identified as calcite. © 2010 Elsevier Ltd. All rights reserved.

  9. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  10. DspA/E contributes to apoplastic accumulation of ROS in nonhost A. thaliana

    Directory of Open Access Journals (Sweden)

    Alban eLaunay

    2016-04-01

    Full Text Available The bacterium Erwinia amylovora is responsible for the fire blight disease of Maleae, which provokes necrotic symptoms on aerial parts. The pathogenicity of this bacterium in hosts relies on its type three-secretion system (T3SS, a molecular syringe that allows the bacterium to inject effectors into the plant cell. E. amylovora-triggered disease in host plants is associated with the T3SS-dependent production of reactive oxygen species (ROS, although ROS are generally associated with resistance in other pathosystems. We showed previously that E. amylovora can multiply transiently in the nonhost plant Arabidopsis thaliana and that a T3SS-dependent production of intracellular ROS occurs during this interaction. In the present work we characterize the localization and source of hydrogen peroxide accumulation following E. amylovora infection. Transmission electron microscope (TEM analysis of infected tissues showed that hydrogen peroxide accumulation occurs in the cytosol, plastids, peroxisomes, and mitochondria as well as in the apoplast. Furthermore, TEM analysis showed that an E. amylovora dspA/E-deficient strain does not induce hydrogen peroxide accumulation in the apoplast. Consistently, a transgenic line expressing DspA/E accumulated ROS in the apoplast. The NADPH oxidase-deficient rbohD mutant showed a very strong reduction in hydrogen peroxide accumulation in response to E. amylovora inoculation. However, we did not find an increase in bacterial titers of E. amylovora in the rbohD mutant and the rbohD mutation did not suppress the toxicity of DspA/E when introgressed into a DspA/E-expressing transgenic line. Co-inoculation of E. amylovora with cycloheximide (CHX, which we found previously to suppress callose deposition and allow strong multiplication of E. amylovora in A. thaliana leaves, led to a strong reduction of apoplastic ROS accumulation but did not affect intracellular ROS. Our data strongly suggest that apoplastic ROS accumulation is

  11. Rapid granulation tissue regeneration by intracellular ATP delivery--a comparison with Regranex.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Howard

    Full Text Available This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

  12. Polymeric gel nanoparticle pH sensors for intracellular measurements

    OpenAIRE

    Almdal, Kristoffer; Andresen, Thomas Lars; Benjaminsen, Rikke Vicki; Christensen, Nynne Meyn; Henriksen, Jonas Rosager; Sun, Honghao

    2011-01-01

    Precise measurements of pH in cells and intracellular compartments are of importance to both the fundamental understanding of metabolism and to the development of drugs that are released from the endosomes-lysome pathway. We have developed polymer gel nanoparticles as carriers of covalently bound fluorophores for ratiometric measurements of pH. One pH insensitive fluorophore serves as a reference while one or more pH sensitive fluorophores serve to give the desired pH dependence of the output...

  13. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    Science.gov (United States)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  14. Dependence of cerebral arterial contractions on intracellularly stored Ca++.

    Science.gov (United States)

    Sasaki, T; Kassell, N F; Zuccarello, M

    1986-01-01

    The purpose of the present study was to evaluate the dependence of the arterial contractions induced by different vasoactive agents upon intracellularly stored calcium in canine versus monkey cerebral arteries. The potency for inducing contractions in Ca++-free media was in the order of 9,11-epithio-11,12-metano-thromboxane A2 (STXA2) greater than prostaglandin F2 alpha (PGF2 alpha) much greater than serotonin greater than K+ in canine basilar arteries, and STXA2 greater than PGF2 alpha much greater than serotonin = K+ in monkey basilar arteries.

  15. Oxygen effect and intracellular oxygen content (adaptation hypothesis)

    Energy Technology Data Exchange (ETDEWEB)

    Yarmonenko, S P; Ehpshtejn, I M [Akademiya Meditsinskikh Nauk SSSR, Moscow. Onkologicheskij Tsentr

    1977-01-01

    Experimental data indicating that a radiomodifying action of hypoxia is dependent on the ''prehistory'' of the irradiated object are considered. This dependence manifests itself in a decreased protective action of acute hypoxia on the hypoxia-adapted objects. To explain this a hypothesis is proposed connecting a degree of cell radiosensitivity modification, determined by the oxygen effect, with the intracellular oxygen content. The latter, in accord with current ideas, is regulated by variations in the diffusion resistance to oxygen shown by the cytoplasmic membranes depending on the energy level of the cell and the degree of its oxygenation.

  16. Oxygen effect and intracellular oxygen content (adaptation hypothesis)

    International Nuclear Information System (INIS)

    Yarmonenko, S.P.; Ehpshtejn, I.M.

    1977-01-01

    Experimental data indicating that a radiomodifying action of hypoxia is dependent on the ''prehistory'' of the irradiated object are considered. This dependence manifests itself in a decreased protective action of acute hypoxia on the hypoxia-adapted objects. To explain this a hypothesis is proposed connecting a degree of cell radiosensitivity modification, determined by the oxygen effect, with the intracellular oxygen content. The latter, in accord with current ideas, is regulated by variations in the diffusion resistance to oxygen shown by the cytoplasmic membranes depending on the energy level of the cell and the degree of its oxygenation

  17. A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity.

    Science.gov (United States)

    Frost, William N; Wang, Jean; Brandon, Christopher J

    2007-05-15

    Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations.

  18. Accumulation of metabolites during bacterial degradation of PAH-mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Vila, J.; Lopez, Z.; Bauza, J.I. [Universitat de Barcelona (Spain). Department de Microbiologia; Minguillon, C. [Parc Cientific de Barcelona (ES). Institut de Recerca de Barcelona (IRB-PCB); Grifoll, M.

    2003-07-01

    In a previous work we identified a number of metabolites accumulated during growth in pyrene by Mycobacterium sp. AP1, and proposed a metabolic pathway for pyrene utilization. In order to confirm and complete this pathway we have isolated and identified the pyrene-degrading strains Mycobacterium sp. PGP2, CP1 and CP2. During growth on pyrene, strains AP1, PGP2, CP1 and CP2 accumulated 4,5-cis-pyrene-dihydrodiol, 4,5-phenanthrene dicarboxylic acid, 4-phenanthrene carboxylic acid, 3,4-dihydroxy-3-hydrophenanthrene-4-carboxylic acid, phthalic acid, and 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid. Strains AP1, PGP2, CP1 and CP2 also grew on fluoranthene accumulating acenaphthenone, naphthalene-1,8-dicarboxylic acid, 9-fluorenone-1-carboxylic acid, Z-9-carboxymethylenefluorene-1-carboxylic acid and benzene-1,2,3-tricarboxylic acid. Similar metabolites were produced during growth onf fluoranthene by the Gram-positive strains CFt2 and CFt6, isolated by their capability of using this PAH as a sole source of carbon and energy. These fluoranthene-degrading strains also accumulated cis-1,9a-dihydroxy-1-hydrofluorene-9-one-8-carboxylic acid. In addition to pyrene and fluoranthene, all pyrene-degrading utilized phenanthrene as a sole source of carbon and energy, while the fluoranthene-degrading strains were unable to utilize pyrene or phenanthrene. Mycobacterium sp. AP1 acted on a wide range of PAHs, accumulating aromatic dicarboxylic acids, hydroxyacids, and ketones resulting from dioxygenation and ortho-cleavage, dioxygenation and meta-cleavage, and monooxygenation reactions. In cultures of strains AP1 and CP1 with a defined PAH-mixture only 20% removal of the parent compounds was observed. Analysis of acidic extracts showed the accumulation of the anticipated aromatic acids, suggesting that accumulation of acidic compounds could prevent further degradation of the mixture. Those results led us to isolation of strains DF11 and OH3, able to grow on the selected

  19. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    Science.gov (United States)

    Shah, Dhiral Ashwin

    functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. The same concept of naturally occurring protein-protein interactions can also be implemented to selectively bring intracellular protein targets in close proximity to proteasomal degradation machinery in cells and effect their depletion from the cellular compartments. This approach will be able to not only target entire pool of proteins to ubiquitination-mediated degradation, but also to specific sub-pools of posttranslationally modified proteins in the cell, provided peptides having distinct binding affinities are identified for posttranslational modifications. This system can then be tested for intracellular protein delivery using nanoparticle carriers to identify roles of different posttranslational modifications on the protein's activity. In future work, we propose to develop a cellular detection system, based on GFP complementation, which can be used to evaluate the efficiency of different protein delivery carriers to internalize proteins into the cell cytosol. We envision the application of nanoscale materials as intracellular protein delivery vehicles to target diverse cell signaling pathways at the posttranslational level, and subsequent metabolic manipulation, which may have interesting therapeutic properties and can potentially target stem cell fate.

  20. Development of a method to measure intracellular growth rate of parasitic acid-fast bacteria using radio-isotope and its improvement

    International Nuclear Information System (INIS)

    Nakata, Noboru; Fukutomi, Yasuo

    1999-01-01

    Development of measurement method for intracellular growth rate was attempted using gene-transfected acid-fast bacteria and Mycobacterium leprae. M. leprae was inoculated into a well, which was filled with fetus bovine serum containing a cover slip pasted with mouse monocyte-derived malignant cell lines, J774 and P388D1 and cultured for 3-4 hours. Then, the cells on the cover slip were mobilized with 0.1 N NaOH. The metabolic activity of M. leprae was assessed based on the β-oxidation activity of 14 C-palmitic acid. Then, it was investigated whether TNF is produced by the cell culture added with M. leprae or LPS. J774 cells abundantly produced TNF after sensitization with LPS and its production was depending on the amount of added bacteria, whereas TNF production after sensitization with LPS or M. leprae was little in P388D1 cells. Staining for acid-fast bacteria revealed that either of these cell lines has phagocytic activity for M. leprae. To identify the bacterial factor involved to the intracellular proliferation of acid-fast bacteria, transposon insertion mutagenesis was attempted to M. avium complex (MAC) and the degrees of drug-resistance in M. avium mino, M. intracellulare JATA-52 and 8 clinically isolated M. intracellulare strains were determined. M. intracellulare JATA-52 was resistant to kanamycin and plasmid pAL8 and pYT937 were both able to transform the strain with dose-dependency. Since M. intracellulare is pathogenic to human and the strain proliferates with a generation time shorter than that of M. tuberculosis, the former strain is thought suitable for the analysis of a mutated gene. Thus, it became possible to study transposition insertion mutagenesis in M. intracellulare. (M.N.)

  1. Development of a method to measure intracellular growth rate of parasitic acid-fast bacteria using radio-isotope and its improvement

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Noboru; Fukutomi, Yasuo [National Inst. of Infectious Diseases, Tokyo (Japan)

    1999-02-01

    Development of measurement method for intracellular growth rate was attempted using gene-transfected acid-fast bacteria and Mycobacterium leprae. M. leprae was inoculated into a well, which was filled with fetus bovine serum containing a cover slip pasted with mouse monocyte-derived malignant cell lines, J774 and P388D1 and cultured for 3-4 hours. Then, the cells on the cover slip were mobilized with 0.1 N NaOH. The metabolic activity of M. leprae was assessed based on the {beta}-oxidation activity of {sup 14}C-palmitic acid. Then, it was investigated whether TNF is produced by the cell culture added with M. leprae or LPS. J774 cells abundantly produced TNF after sensitization with LPS and its production was depending on the amount of added bacteria, whereas TNF production after sensitization with LPS or M. leprae was little in P388D1 cells. Staining for acid-fast bacteria revealed that either of these cell lines has phagocytic activity for M. leprae. To identify the bacterial factor involved to the intracellular proliferation of acid-fast bacteria, transposon insertion mutagenesis was attempted to M. avium complex (MAC) and the degrees of drug-resistance in M. avium mino, M. intracellulare JATA-52 and 8 clinically isolated M. intracellulare strains were determined. M. intracellulare JATA-52 was resistant to kanamycin and plasmid pAL8 and pYT937 were both able to transform the strain with dose-dependency. Since M. intracellulare is pathogenic to human and the strain proliferates with a generation time shorter than that of M. tuberculosis, the former strain is thought suitable for the analysis of a mutated gene. Thus, it became possible to study transposition insertion mutagenesis in M. intracellulare. (M.N.)

  2. Aflatoxin Accumulation in a Maize Diallel Cross

    Directory of Open Access Journals (Sweden)

    W. Paul Williams

    2015-06-01

    Full Text Available Aflatoxins, produced by the fungus Aspergillus flavus, occur naturally in maize. Contamination of maize grain with aflatoxin is a major food and feed safety problem and greatly reduces the value of the grain. Plant resistance is generally considered a highly desirable approach to reduction or elimination of aflatoxin in maize grain. In this investigation, a diallel cross was produced by crossing 10 inbred lines with varying degrees of resistance to aflatoxin accumulation in all possible combinations. Three lines that previously developed and released as sources of resistance to aflatoxin accumulation were included as parents. The 10 parental inbred lines and the 45 single crosses making up the diallel cross were evaluated for aflatoxin accumulation in field tests conducted in 2013 and 2014. Plants were inoculated with an A. flavus spore suspension seven days after silk emergence. Ears were harvested approximately 60 days later and concentration of aflatoxin in the grain determined. Parental inbred lines Mp717, Mp313E, and Mp719 exhibited low levels (3–12 ng/g of aflatoxin accumulation. In the diallel analysis, both general and specific combining ability were significant sources of variation in the inheritance of resistance to aflatoxin accumulation. General combining ability effects for reduced aflatoxin accumulation were greatest for Mp494, Mp719, and Mp717. These lines should be especially useful in breeding for resistance to aflatoxin accumulation. Breeding strategies, such as reciprocal recurrent selection, would be appropriate.

  3. A newly found cadmium accumulator-Taraxacum mongolicum

    International Nuclear Information System (INIS)

    Wei Shuhe; Zhou Qixing; Mathews, Shiny

    2008-01-01

    Identification of hyperaccumulator and accumulator is still key step of phytoextracting-contaminated soils by heavy metals. In a former published experiment, Taraxacum mongolicum showed basic characteristics of hyperaccumulators. In order to confirm if this plant was a Cd-hyperaccumulator, concentration gradient experiment and sample-analyzing experiments were designed and performed. The results showed that Cd enrichment factor and Cd transformation factor of T. mongolicum were all higher than 1 in concentration gradient experiment. The shoot biomasses did not reduced significantly (p -1 Cd spiked into soil. However, Cd concentration in shoot of T. mongolicum was not higher than 100 mg kg -1 the minimum a Cd-hyperaccumulator should have under the conditions of any concentration level of Cd spiked. Thus, T. mongolicum should be a Cd-accumulator. In the sample-analyzing experiments settled in a Pb-Zn mine area and Shenyang wastewater irrigation region, T. mongolicum also showed that Cd-accumulator characteristics. Based on these results, T. mongolicum could be identified as a Cd-accumulator, which may have important implication in plant physiology and gene engineering

  4. Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Alhonen-Hongisto, L; Seppänen, P; Jänne, J

    1980-01-01

    Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect. PMID:6786285

  5. Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Directory of Open Access Journals (Sweden)

    Anat Elmann

    2016-03-01

    Full Text Available Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures was determined by lactate dehydrogenase (LDH activity, intracellular ROS levels were detected using 2′,7′-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS. These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1 and p44/42 mitogen-activated protein kinases (MAPK, and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

  6. Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration

    International Nuclear Information System (INIS)

    Osawa, Hiroyuki; Ohnishi, Hirohide; Takano, Koji; Noguti, Takasi; Mashima, Hirosato; Hoshino, Hiroko; Kita, Hiroto; Sato, Kiichi; Matsui, Hirofumi; Sugano, Kentaro

    2006-01-01

    Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca 2+ ] i ). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca 2+ ] i , activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system

  7. Uranium accumulation by aquatic macrophyte, Pistia stratiotes

    International Nuclear Information System (INIS)

    Bhainsa, K.C.; D'Souza, S.F.

    2012-01-01

    Uranium accumulation by aquatic macrophyte, Pistia stratiotes from aqueous solution was investigated in laboratory condition. The objective was to evaluate the uranium accumulation potential and adopt the plant in uranium containing medium to improve its uptake capacity. The plant was found to tolerate and grow in the pH range of 3-7. Accumulation of uranium improved with increasing pH and the plant could remove 70% uranium from the medium (20 mg/L) within 24 hours of incubation at pH 5-6. Uptake of uranium on either side of this pH range decreased

  8. Geochemistry Model Validation Report: External Accumulation Model

    International Nuclear Information System (INIS)

    Zarrabi, K.

    2001-01-01

    The purpose of this Analysis and Modeling Report (AMR) is to validate the External Accumulation Model that predicts accumulation of fissile materials in fractures and lithophysae in the rock beneath a degrading waste package (WP) in the potential monitored geologic repository at Yucca Mountain. (Lithophysae are voids in the rock having concentric shells of finely crystalline alkali feldspar, quartz, and other materials that were formed due to entrapped gas that later escaped, DOE 1998, p. A-25.) The intended use of this model is to estimate the quantities of external accumulation of fissile material for use in external criticality risk assessments for different types of degrading WPs: U.S. Department of Energy (DOE) Spent Nuclear Fuel (SNF) codisposed with High Level Waste (HLW) glass, commercial SNF, and Immobilized Plutonium Ceramic (Pu-ceramic) codisposed with HLW glass. The scope of the model validation is to (1) describe the model and the parameters used to develop the model, (2) provide rationale for selection of the parameters by comparisons with measured values, and (3) demonstrate that the parameters chosen are the most conservative selection for external criticality risk calculations. To demonstrate the applicability of the model, a Pu-ceramic WP is used as an example. The model begins with a source term from separately documented EQ6 calculations; where the source term is defined as the composition versus time of the water flowing out of a breached waste package (WP). Next, PHREEQC, is used to simulate the transport and interaction of the source term with the resident water and fractured tuff below the repository. In these simulations the primary mechanism for accumulation is mixing of the high pH, actinide-laden source term with resident water; thus lowering the pH values sufficiently for fissile minerals to become insoluble and precipitate. In the final section of the model, the outputs from PHREEQC, are processed to produce mass of accumulation

  9. Manganese accumulation in the brain: MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Uchino, A.; Nomiyama, K.; Takase, Y.; Nakazono, T.; Nojiri, J.; Kudo, S. [Saga Medical School, Department of Radiology, Saga (Japan); Noguchi, T. [Kyushu University, Department of Clinical Radiology, Graduate School of Medicine, Fukuoka (Japan)

    2007-09-15

    Manganese (Mn) accumulation in the brain is detected as symmetrical high signal intensity in the globus pallidi on T1-weighted MR images without an abnormal signal on T2-weighted images. In this review, we present several cases of Mn accumulation in the brain due to acquired or congenital diseases of the abdomen including hepatic cirrhosis with a portosystemic shunt, congenital biliary atresia, primary biliary cirrhosis, congenital intrahepatic portosystemic shunt without liver dysfunction, Rendu-Osler-Weber syndrome with a diffuse intrahepatic portosystemic shunt, and patent ductus venosus. Other causes of Mn accumulation in the brain are Mn overload from total parenteral nutrition and welding-related Mn intoxication. (orig.)

  10. Heat and cold accumulators in vehicles

    Energy Technology Data Exchange (ETDEWEB)

    Kauranen, P.; Wikstroem, L. (VTT Technical Research Centre of Finland, Advanced Materials, Tampere (Finland)); Heikkinen, J. (VTT Technical Research Centre of Finland, Building Services and Indoor Environment, Espoo (Finland)); Laurikko, J.; Elonen, T. (VTT Technical Research Centre of Finland, Emission Control, Espoo (Finland)); Seppaelae, A. (Helsinki Univ. of Technology, Applied Thermodynamics, Espoo (Finland)). Email: ari.seppala@tkk.fi

    2009-07-01

    Phase Change Material (PCM) based heat and cold accumulators have been tailored for transport applications including a mail delivery van as well as the cold chains of foodstuff and blood products. The PCMs can store relative large amount of thermal energy in a narrow temperature interval as latent heat of fusion of their melting and crystallization processes. Compact heat and cold accumulators can be designed using PCMs. The aim of the project has been to reduce the exhaust gas and noise emissions and improve the fuel economy of the transport systems and to improve the reliability of the cold chains studied by storing thermal energy in PCM accumulators. (orig.)

  11. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    International Nuclear Information System (INIS)

    Sanson, Benoît; Wang, Tao; Sun, Jing; Wang, Liqun; Kaczocha, Martin; Ojima, Iwao; Deutsch, Dale; Li, Huilin

    2014-01-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels

  12. Imaging the intracellular degradation of biodegradable polymer nanoparticles

    Directory of Open Access Journals (Sweden)

    Anne-Kathrin Barthel

    2014-10-01

    Full Text Available In recent years, the development of smart drug delivery systems based on biodegradable polymeric nanoparticles has become of great interest. Drug-loaded nanoparticles can be introduced into the cell interior via endocytotic processes followed by the slow release of the drug due to degradation of the nanoparticle. In this work, poly(L-lactic acid (PLLA was chosen as the biodegradable polymer. Although common degradation of PLLA has been studied in various biological environments, intracellular degradation processes have been examined only to a very limited extent. PLLA nanoparticles with an average diameter of approximately 120 nm were decorated with magnetite nanocrystals and introduced into mesenchymal stem cells (MSCs. The release of the magnetite particles from the surface of the PLLA nanoparticles during the intracellular residence was monitored by transmission electron microscopy (TEM over a period of 14 days. It was demonstrated by the release of the magnetite nanocrystals from the PLLA surface that the PLLA nanoparticles do in fact undergo degradation within the cell. Furthermore, even after 14 days of residence, the PLLA nanoparticles were found in the MSCs. Additionally, the ultrastructural TEM examinations yield insight into the long term intercellular fate of these nanoparticles. From the statistical analysis of ultrastructural details (e.g., number of detached magnetite crystals, and the number of nanoparticles in one endosome, we demonstrate the importance of TEM studies for such applications in addition to fluorescence studies (flow cytometry and confocal laser scanning microscopy.

  13. Fluorescent nanosensors for intracellular measurements: synthesis, characterisation, calibration and measurement

    Directory of Open Access Journals (Sweden)

    Arpan Shailesh Desai

    2014-01-01

    Full Text Available Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer and a pH-insensitive reference fluorophore (internal standard immobilised in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesised using standard laboratory equipment and are detectable by non-invasive widely accessibly imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: 1 synthesis and characterisation of polyacrylamide and silica based nanosensors 2 nanosensor calibration and 3 performing measurements using fluorescence microscopy.

  14. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Energy Technology Data Exchange (ETDEWEB)

    Dobay, M. P. D., E-mail: maria.pamela.david@physik.uni-muenchen.de; Alberola, A. Piera; Mendoza, E. R.; Raedler, J. O., E-mail: joachim.raedler@physik.uni-muenchen.de [Ludwig-Maximilians University, Faculty of Physics, Center for NanoScience (Germany)

    2012-03-15

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  15. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    International Nuclear Information System (INIS)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-01-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  16. Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

    Science.gov (United States)

    Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

    2012-03-01

    Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

  17. Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

    Full Text Available ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1 is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

  18. Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

    Science.gov (United States)

    Li, Kefeng; Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Lulu; Song, Tao

    2017-09-01

    Magnetotactic bacteria (MTB) demonstrate photoresponse. However, little is known about the biological significance of this behaviour. Magnetosomes exhibit peroxidase-like activity and can scavenge reactive oxygen species (ROS). Magnetosomes extracted from the Magnetospirillum magneticum strain AMB-1 show enhanced peroxidase-like activity under illumination. The present study investigated the effects of light irradiation on nonmagnetic (without magnetosomes) and magnetic (with magnetosomes) AMB-1 cells. Results showed that light irradiation did not affect the growth of nonmagnetic and magnetic cells but significantly increased magnetosome synthesis and reduced intracellular ROS level in magnetic cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse the expression level of magnetosome formation-associated genes (mamA, mms6, mms13 and mmsF) and stress-related genes (recA, oxyR, SOD, amb0664 and amb2684). Results showed that light irradiation upregulated the expression of mms6, mms13 and mmsF. Furthermore, light irradiation upregulated the expression of stress-related genes in nonmagnetic cells but downregulated them in magnetic cells. Additionally, magnetic cells exhibited stronger phototactic behaviour than nonmagnetic ones. These results suggested that light irradiation could heighten the ability of MTB to eliminate intracellular ROS and help them adapt to lighted environments. This phenomenon may be related to the enhanced peroxidase-like activity of magnetosomes under light irradiation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Transient light-induced intracellular oxidation revealed by redox biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Kolossov, Vladimir L., E-mail: viadimer@illinois.edu [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  20. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  1. Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels.

    Science.gov (United States)

    Muro-Pastor, M I; Reyes, J C; Florencio, F J

    2001-10-12

    The regulatory circuits that control nitrogen metabolism are relatively well known in several bacterial model groups. However, much less is understood about how the nitrogen status of the cell is perceived in vivo. In cyanobacteria, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. In contrast, how NtcA activity is regulated is largely unknown. Assimilation of ammonium by most microorganisms occurs through the sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase. Interestingly, regulation of the expression of NtcA-dependent genes in the cyanobacterium Synechocystis sp. PCC 6803 is altered in mutants with modified levels of GS activity. Two types of mutants were analyzed: glnA null mutants that lack GS type I and gif mutants unable to inactivate GS in the presence of ammonium. Changes in the intracellular pools of 19 different amino acids and the keto acid 2-oxoglutarate were recorded in wild-type and mutant strains under different nitrogen conditions. Our data strongly indicate that the nitrogen status in cyanobacteria is perceived as changes in the intracellular 2-oxoglutarate pool.

  2. Optochemokine Tandem for Light-Control of Intracellular Ca2.

    Directory of Open Access Journals (Sweden)

    Katrin Feldbauer

    Full Text Available An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C, CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

  3. Transient light-induced intracellular oxidation revealed by redox biosensor

    International Nuclear Information System (INIS)

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-01-01

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition