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Sample records for active uptake mechanisms

  1. Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

    NASA Technical Reports Server (NTRS)

    Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

    1999-01-01

    The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

  2. Measuring the activities of higher organisms in activated sludge by means of mechanical shearing pretreatment and oxygen uptake rate.

    PubMed

    Hao, Xiaodi; Wang, Qilin; Cao, Yali; van Loosdrecht, Mark C M

    2010-07-01

    A pretreatment method was developed to assess the activities of higher organisms. The method is based on mechanical shearing to damage the large cells of the protozoan and metazoan community in activated sludge. The procedure was confirmed through experimentation to be effective in determining the activities of higher organisms by comparing oxygen uptake rates (OURs) before and after the higher organisms were eradicated. Shearing led to disintegration of flocs, which could be effectively reconstituted by centrifugation. The reconstitution of the sludge flocs was essential since otherwise the activity of the floc mass would be too high due to lack of diffusion limitation. Mechanical shearing had no influence on the morphology, quantity and specific activity of yeasts, and it was inferred that bacteria smaller than yeasts in size would also not be influenced by the applied shearing procedure. Moreover, the effect of filamentous organisms on the measured activities of higher organisms was experimentally demonstrated and analyzed, and determined to be so weak that it could be ignored. Based on these tests, five typical activated sludge processes were selected to measure the contribution of higher organisms to the original OUR. The measured activities of higher organisms ranged from 9.4 to 25.0% of the original OURs.

  3. Mechanisms of Ocean Heat Uptake

    NASA Astrophysics Data System (ADS)

    Garuba, Oluwayemi

    An important parameter for the climate response to increased greenhouse gases or other radiative forcing is the speed at which heat anomalies propagate downward in the ocean. Ocean heat uptake occurs through passive advection/diffusion of surface heat anomalies and through the redistribution of existing temperature gradients due to circulation changes. Atlantic meridional overturning circulation (AMOC) weakens in a warming climate and this should slow the downward heat advection (compared to a case in which the circulation is unchanged). However, weakening AMOC also causes a deep warming through the redistributive effect, thus increasing the downward rate of heat propagation compared to unchanging circulation. Total heat uptake depends on the combined effect of these two mechanisms. Passive tracers in a perturbed CO2 quadrupling experiments are used to investigate the effect of passive advection and redistribution of temperature anomalies. A new passive tracer formulation is used to separate ocean heat uptake into contributions due to redistribution and passive advection-diffusion of surface heating during an ocean model experiment with abrupt increase in surface temperature. The spatial pattern and mechanisms of each component are examined. With further experiments, the effects of surface wind, salinity and temperature changes in changing circulation and the resulting effect on redistribution in the individual basins are isolated. Analysis of the passive advection and propagation path of the tracer show that the Southern ocean dominates heat uptake, largely through vertical and horizontal diffusion. Vertical diffusion transports the tracer across isopycnals down to about 1000m in 100 years in the Southern ocean. Advection is more important in the subtropical cells and in the Atlantic high latitudes, both with a short time scale of about 20 years. The shallow subtropical cells transport the tracer down to about 500m along isopycnal surfaces, below this vertical

  4. Bulk chlorine uptake by polyamide active layers of thin-film composite membranes upon exposure to free chlorine-kinetics, mechanisms, and modeling.

    PubMed

    Powell, Joshua; Luh, Jeanne; Coronell, Orlando

    2014-01-01

    We studied the volume-averaged chlorine (Cl) uptake into the bulk region of the aromatic polyamide active layer of a reverse osmosis membrane upon exposure to free chlorine. Volume-averaged measurements were obtained using Rutherford backscattering spectrometry with samples prepared at a range of free chlorine concentrations, exposure times, and mixing, rinsing, and pH conditions. Our volume-averaged measurements complement previous studies that have quantified Cl uptake at the active layer surface (top ≈ 7 nm) and advance the mechanistic understanding of Cl uptake by aromatic polyamide active layers. Our results show that surface Cl uptake is representative of and underestimates volume-averaged Cl uptake under acidic conditions and alkaline conditions, respectively. Our results also support that (i) under acidic conditions, N-chlorination followed by Orton rearrangement is the dominant Cl uptake mechanism with N-chlorination as the rate-limiting step; (ii) under alkaline conditions, N-chlorination and dechlorination of N-chlorinated amide links by hydroxyl ion are the two dominant processes; and (iii) under neutral pH conditions, the rates of N-chlorination and Orton rearrangement are comparable. We propose a kinetic model that satisfactorily describes Cl uptake under acidic and alkaline conditions, with the largest discrepancies between model and experiment occurring under alkaline conditions at relatively high chlorine exposures.

  5. Peroxynitrite activates glucose uptake in 3T3-L1 adipocytes through a PI3-K-dependent mechanism.

    PubMed

    Guzman-Grenfell, Alberto M; Garcia-Macedo, Rebeca; Gonzalez-Martinez, Marco T; Hicks, Juan Jose; Medina-Navarro, Rafael

    2005-01-01

    Peroxynitrite, the product of the reaction between *NO and O2*-, is a strong oxidant and nitrating molecule, and it has been recently consideredas a component of some important signaling pathways. Herein, we report the effect of peroxynitrite on glucose uptake in 3T3-L1 adipocytes. Peroxynitrite stimulated glucose uptake and this effect was inhibited by citochalasin B, indicating the participation of facilitated GLUT transporters. Peroxynitrite-induced glucose uptake was not related to intracellular ATP, nor to external or internal calcium, but it was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. Additionally, we also found that peroxynitrite did not activate the insulin receptor nor the PI3-K downstream signaling protein kinase B (PKB/Akt). The dose-dependent inhibitory action of wortmannin suggests that peroxynitrite activates glucose transport without affecting GLUT transporters translocation.

  6. Change of crystallinity and mineral composition of fly ash with mechanical and chemical activation for the improvement of phosphate uptake.

    PubMed

    Liang, Zhu; He, XiaoJia; Ni, JinRen

    2010-10-01

    A detailed investigation of the development of the mineral composition and crystallinity of fly ash (FA) activated by an integrated process, as well as the relation between that development and phosphate uptake (PU) in solution, was conducted. This process, consisting of pretreatment (mechanical milling), alkali fusion (sodium hydroxide) at 550 °C and acid attack (3 mol L⁻¹ sulfuric acid), exhibited a remarkable activation effect. One-hour grinding could enhance PU from 0.67 to 1.66 mg PO₄³⁻-P g⁻¹ FA, and then under the optimum chemical conditions with the ratio of NaOH to FA of 0.5 g g⁻¹ and that of H₂SO₄ to FA of 3 mL g⁻¹, the PU was further improved to 7.14 mg g⁻¹. Results also indicated that the PU performance was closely linked with the crystallinity and mineral composition of FA, that is, the treated material with the lowest crystallinity and least crystal phase could achieve the highest PU. For the purpose of destroying original minerals in raw FA farthest and avoiding the production of new crystals, therefore, control on the ratio of NaOH to FA and that of acid to FA was very important during the chemical treatment.

  7. Mechanism of action of sarcoplasmic reticulum calcium-uptake activators--discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction.

    PubMed

    Berrebi-Bertrand, I; Lahouratate, P; Lahouratate, V; Camelin, J C; Guibert, J; Bril, A

    1997-08-01

    The Ca2+ uptake by the sarcoplasmic reticulum (SR) can be affected by direct modulation of the Ca2+ pump or by removing the inhibitory effect of dephosphorylated phospholamban. The effect of these mechanisms was assessed using ellagic acid and 1-(3,4-dimethoxyphenyl)-3-dodecanone. Both compounds (30 micromol/l) enhanced SR-Ca2+ uptake in rabbit cardiomyocytes by 65.3 +/- 13% and 44.3 +/- 6.7% for 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid, respectively (at pCa 6.2). A similar effect was observed in cardiac SR microsomes (59.5 +/- 7.4% and 45.1 +/- 6.7) with 30 micromol/l 1-(3,4-dimethodoxyphenyl)-3-dodecanone and ellagic acid, respectively. 1-(3,4-Dimethoxyphenyl)-3-dodecanone increased Ca2+ storage by cardiac SR microsomes mainly at high [Ca2+] with a 57% increase of Vmax, whereas ellagic acid increased Vmax to a smaller extent (22%) and stimulated Ca2+ uptake at lower [Ca2+] with a leftward-shift of the pCa/ATPase relationship by pCa 0.24. Ellagic acid also differed from 1-(3,4-dimethoxylphenyl)-3-dodecanone in that it produced a Ca2+ sensitizing effect only in cardiac SR microsomes (by pCa 0.3) whereas 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulated the ATPase, at saturating Ca2+, in both cardiac and skeletal muscle SR vesicles. It is suggested that 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulates directly the Ca2+-ATPase activity, in contrast to ellagic acid which enhances the cardiac SR-Ca2+ uptake by interacting with phospholamban, as confirmed by the lack of additive effect between ellagic acid and monoclonal antibodies raised against phospholamban. 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid constitute attractive pharmacological tools to investigate the functional consequences of enhancing SR Ca2+, uptake by affecting different mechanisms.

  8. Water uptake mechanism in crispy bread crust.

    PubMed

    van Nieuwenhuijzen, Neleke H; Meinders, Marcel B J; Tromp, R Hans; Hamer, Rob J; van Vliet, Ton

    2008-08-13

    Crispness is an important quality characteristic of dry solid food products such as crispy rolls. Its retention is directly related to the kinetics of water uptake by the crust. In this study, a method for the evaluation of the water sorption kinetics in bread crust is proposed. Two different sorption experiments were used: an oscillatory sorption test and a sorption test in which the air relative humidity (RH) was increased stepwise. These two experiments had different time scales, which made it possible to get a better understanding of the mechanisms involved. Results show that the adsorption and desorption dynamics of the oscillatory sorption test could be described by a single exponential in time. The water uptake rate ( k) was one of the fitting parameters. A maximum in the water uptake rate was found for a RH value between 50 and 70%. The rate parameters of the experiment where RH was increased stepwise were around a factor 10 lower than those derived from oscillatory sorption experiments. This is an important factor when designing experiments for the determination of water uptake rates. In addition, also a parameter describing the time dependence of the rate parameters of the oscillatory sorption experiment was calculated (C), again by fitting a single exponential to the rate parameters. C was in the same range as the rate parameter of the isotherm experiment. This indicates that different (relaxation) processes are acting at the same time in the bread crust during water uptake.

  9. Iron uptake mechanism in the chrysophyte microalga Dinobryon.

    PubMed

    Carmel, Nava; Tel-Or, Elisha; Chen, Yona; Pick, Uri

    2014-07-15

    The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.

  10. The mechanism of copper uptake by tyrosinase from Bacillus megaterium.

    PubMed

    Kanteev, Margarita; Goldfeder, Mor; Chojnacki, Michał; Adir, Noam; Fishman, Ayelet

    2013-12-01

    Tyrosinase belongs to the type 3 copper enzyme family, containing a dinuclear copper center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although copper ions are essential for the activity of tyrosinase, the mechanism of copper uptake is still unclear. We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison with CuB. Investigating copper accumulation in TyrBm, we found that the presence of copper has a more significant effect on the diphenolase activity. By decreasing the concentration of copper, we increased the diphenolase to monophenolase activity ratio twofold. Using a rational design approach, we identified five variants having an impact on copper uptake. We have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Further investigation of these variants revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, not only play a role in copper uptake, but are also important for enhancing the diphenolase activity. We propose a mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards L-dopa production.

  11. Potentiating the cellular targeting and anti-tumor activity of Dp44mT via binding to human serum albumin: two saturable mechanisms of Dp44mT uptake by cells.

    PubMed

    Merlot, Angelica M; Sahni, Sumit; Lane, Darius J R; Fordham, Ashleigh M; Pantarat, Namfon; Hibbs, David E; Richardson, Vera; Doddareddy, Munikumar R; Ong, Jennifer A; Huang, Michael L H; Richardson, Des R; Kalinowski, Danuta S

    2015-04-30

    Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) demonstrates potent anti-cancer activity. We previously demonstrated that 14C-Dp44mT enters and targets cells through a carrier/receptor-mediated uptake process. Despite structural similarity, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH) enter cells via passive diffusion. Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH. Chelator-HSA binding studies demonstrated the following order of relative affinity: Bp4eT≈PIH>Dp44mT. Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands. In contrast, HSA markedly stimulated Dp44mT uptake by cells, with two saturable uptake mechanisms identified. The first mechanism saturated at 5-10 µM (B(max):1.20±0.04 × 10⁷ molecules/cell; K(d):33±3 µM) and was consistent with a previously identified Dp44mT receptor/carrier. The second mechanism was of lower affinity, but higher capacity (B(max):2.90±0.12 × 10⁷ molecules/cell; K(d):65±6 µM), becoming saturated at 100 µM and was only evident in the presence of HSA. This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site. Significantly, the HSA-mediated increase in the targeting of Dp44mT to cancer cells potentiated apoptosis and could be important for enhancing efficacy.

  12. Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

    PubMed Central

    Luo, Shuanghui; Wang, Zhiying; Kansara, Viral; Pal, Dhananjay; Mitra, Ashim. K.

    2008-01-01

    The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [14C]ascorbic acid ([14C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di- substituted benzenes. Transepithelial transport of [14C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a Km of 83.2 μM and Vmax of 94.2 pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca2+/CaM mediated pathways. [14C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group i.e. halogen- and nitro- substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A−B) as well as basal to apical side (B−A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs. PMID:18417304

  13. Mechanisms and regulation of Na(+) uptake by freshwater fish.

    PubMed

    Kumai, Yusuke; Perry, Steve F

    2012-12-01

    Mechanisms of ion uptake by freshwater (FW) fish have received considerable attention over the past 80 years. Through an assortment of techniques incorporating whole animal physiology, electrophysiology and molecular biological approaches, three models have been proposed to account for Na(+) uptake. (1) Direct exchange of Na(+) and H(+) via one or more types of Na(+)/H(+) exchanger (slc9), (2) uptake of Na(+) through epithelial Na(+) channels energized by an electrical gradient created by H(+)-ATPase and (3) Na(+)/Cl(-) co-transport (slc12). While each mechanism is supported at least in part by theoretical or experimental data, there are several outstanding questions that have not yet been fully resolved. Furthermore, there are few details concerning how these Na(+) uptake mechanisms are fine tuned in response to the fluctuating FW environments. In this review, we summarize the current understanding of these three Na(+) uptake mechanisms and discuss their regulation by endocrine (cortisol and prolactin) and neurohumoral (catecholamines) factors.

  14. Is there a strategy I iron uptake mechanism in maize?

    PubMed

    Li, Suzhen; Zhou, Xiaojin; Chen, Jingtang; Chen, Rumei

    2016-03-28

    Iron is a metal micronutrient that is essential for plant growth and development. Graminaceous and nongraminaceous plants have evolved different mechanisms to mediate Fe uptake. Generally, strategy I is used by nongraminaceous plants like Arabidopsis, while graminaceous plants, such as rice, barley, and maize, are considered to use strategy II Fe uptake. Upon the functional characterization of OsIRT1 and OsIRT2 in rice, it was suggested that rice, as an exceptional graminaceous plant, utilizes both strategy I and strategy II Fe uptake systems. Similarly, ZmIRT1 and ZmZIP3 were identified as functional zinc and iron transporters in the maize genome, along with the determination of several genes encoding Zn and Fe transporters, raising the possibility that strategy I Fe uptake also occurs in maize. This mini-review integrates previous reports and recent evidence to obtain a better understanding of the mechanisms of Fe uptake in maize.

  15. Molecular mechanisms governing Arabidopsis iron uptake.

    PubMed

    Brumbarova, Tzvetina; Bauer, Petra; Ivanov, Rumen

    2015-02-01

    Plants are the principal source of dietary iron (Fe) for most of Earth's population and Fe deficiency can lead to major health problems. Developing strategies to improve plant Fe content is a challenge because Fe is essential and toxic and therefore regulating Fe uptake is crucial for plant survival. Acquiring soil Fe relies on complex regulatory events that occur in root epidermal cells. We review recent advances in elucidating many aspects of the regulation of Fe acquisition. These include the expanding protein network involved in FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT)-dependent gene regulation and novel findings on the intracellular trafficking of the Fe transporter IRON-REGULATED TRANSPORTER 1 (IRT1). We outline future challenges and propose strategies, such as exploiting natural variation, to further expand our knowledge.

  16. Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

    PubMed

    Berger, Jens; Hardt, Martin; Clauss, Wolfgang G; Fronius, Martin

    2010-07-01

    A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

  17. Characterization of a possible uptake mechanism of selective antibacterial peptides.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Castañón-González, Jorge Alberto; Buhse, Thomas; Sordo, Marili Leopold

    2013-01-01

    Selective antibacterial peptides containing less than 30 amino acid residues, cationic, with amphipathic properties, have been the subject of several studies due to their active participation and beneficial effects in strengthening the immune system of all living organisms. This manuscript reports the results of a comparison between the group of selective antibacterial peptides and another group called "cell penetrating peptides". An important number of the selective antibacterial peptides are cell penetrating peptides, suggesting that their toxicity is related to their uptake mechanism. The verification of this observation also includes the adaptation of a method previously published, called Polarity index, which reproduces and confirms the action of this new set of peptides. The efficiency of this method was verified based on four different databases, yielding a high score. The verification was based exclusively on the peptides already reported in the databases which have been experimentally verified.

  18. Mechanism of glutamate uptake in Zymomonas mobilis.

    PubMed Central

    Ruhrmann, J; Krämer, R

    1992-01-01

    The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values. PMID:1332937

  19. Nanodiamond internalization in cells and the cell uptake mechanism

    NASA Astrophysics Data System (ADS)

    Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

    2013-08-01

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

  20. Molecular mechanisms of foliar water uptake in a desert tree

    PubMed Central

    Yan, Xia; Zhou, Maoxian; Dong, Xicun; Zou, Songbing; Xiao, Honglang; Ma, Xiao-Fei

    2015-01-01

    Water deficits severely affect growth, particularly for the plants in arid and semiarid regions of the world. In addition to precipitation, other subsidiary water, such as dew, fog, clouds and small rain showers, may also be absorbed by leaves in a process known as foliar water uptake. With the severe scarcity of water in desert regions, this process is increasingly becoming a necessity. Studies have reported on physical and physiological processes of foliar water uptake. However, the molecular mechanisms remain less understood. As major channels for water regulation and transport, aquaporins (AQPs) are involved in this process. However, due to the regulatory complexity and functional diversity of AQPs, their molecular mechanism for foliar water uptake remains unclear. In this study, Tamarix ramosissima, a tree species widely distributed in desert regions, was investigated for gene expression patterns of AQPs and for sap flow velocity. Our results suggest that the foliar water uptake of T. ramosissima occurs in natural fields at night when the humidity is over a threshold of 85 %. The diurnal gene expression pattern of AQPs suggests that most AQP gene expressions display a circadian rhythm, and this could affect both photosynthesis and transpiration. At night, the PIP2-1 gene is also upregulated with increased relative air humidity. This gene expression pattern may allow desert plants to regulate foliar water uptake to adapt to extreme drought. This study suggests a molecular basis of foliar water uptake in desert plants. PMID:26567212

  1. Mechanically triggered solute uptake in soft contact lenses.

    PubMed

    Tavazzi, Silvia; Ferraro, Lorenzo; Fagnola, Matteo; Cozza, Federica; Farris, Stefano; Bonetti, Simone; Simonutti, Roberto; Borghesi, Alessandro

    2015-06-01

    Molecular arrangement plays a role in the diffusion of water and solutes across soft contact lenses. In particular, the uptake of solutes in hydrated contact lenses can occur as long as free water is available for diffusion. In this work, we investigated the effect of mechanical vibrations of low frequency (200 Hz) on the solute uptake. Hyaluronan, a polysaccharide of ophthalmic use, was taken as example of solute of interest. For a specific water-hydrated hydrogel material, differential scanning calorimetry experiments showed that a large fraction of the hydration water accounted for loosely-bound water, both before and after one week of daily-wear of the lenses. The size (of the order of magnitude of few hundreds of nanometers) of hyaluronan in aqueous solution was found to be less than the size of the pores of the lens observed by scanning electron microscopy. However, solute uptake in already-hydrated lenses was negligible by simple immersion, while a significant increase occurred under mechanical vibrations of 200 Hz, thus providing experimental evidence of mechanically triggered enhanced solute uptake, which is attributed to the release of interfacial loosely-bound water. Also other materials were taken into consideration. However, the effectiveness of mechanical vibrations for hyaluronan uptake is restricted to lenses containing interfacial loosely-bound water. Indeed, loosely-bound water is expected to be bound to the polymer with bonding energies of the order of magnitude of 10-100 J/g, which are compatible with the energy input supplied by the vibrations.

  2. Calcium uptake and bioelectrical activity of denervated and myotonic muscle

    PubMed Central

    Radu, H.; Gödri, I.; Albu, E.; Radu, A.; Robu, R.

    1970-01-01

    Calcium uptake on muscle microsomal fraction has been investigated in connection with bioelectrical activity in some muscle diseases. The findings showed a significant increase of calcium uptake in denervated muscle, which exhibited spontaneous bioelectrical activity (fibrillations). In myotonias, a low calcium uptake was peculiar to Steinert's disease but not to myotonia congenita. In other muscle diseases, such as progressive muscular dystrophy (Duchenne's type) or Charcot-Marie-Tooth's disease, the ability of muscle microsomal fraction to bind calcium was not changed. Starting with the key role of calcium in excitation-contraction coupling, the implications of calcium uptake disturbances in muscle electrogenesis are discussed. Images PMID:5431720

  3. Mechanisms of Hg(II) uptake and methylation in methylating bacteria

    SciTech Connect

    Morel, Francois M. M.

    2016-10-14

    The goal of this project was to understand the critical factors which control the availability and transport of Hg(II) into cells, a first step in the production of the neurotoxin, methylmercury. Specifically, this research focused on understanding the mechanism of bacterial mercury uptake and how mercury speciation affects the specificity and kinetics of mercury transport. Our research has shown that Hg(II) uptake in three different iron and sulfate-reducing proteobacteria occurs by the following mechanism (1) : Hg(II) uptake is an active transport process requiring energy, (2) it is dependent upon the structure of the Hg binding ligand, and (3) it is mediated by a heavy metal transporter such as one which transports the essential metal, Zn(II). In order to determine whether this mechanism extends to more diverse phylogenetic groups, we have begun examining Hg(II) uptake and bioavailability in two representative Hg methylating strains within the Firmicutes. These organisms have remarkably different membrane structures distinct from the Proteobacteria. Our results show low uptake rates in these two species of Firmicutes relative to the previously characterized Proteobacteria. This may explain the low methylation rates and yields observed in these organisms. Most surprisingly, however, these organisms appear to take up Hg(II) passively, as the addition of a protonophore failed to reduce Hg(II) uptake in these organisms. This is quite different to what has been observed previously for the Proteobacteria and suggests a different mechanism for Hg(II) uptake in the Firmicutes. We are continuing to understand and describe Hg(II) uptake in these organisms. A manuscript is expected to be submitted on this research in June 2016.

  4. Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.

    PubMed

    Lane, Darius J R; Chikhani, Sherin; Richardson, Vera; Richardson, Des R

    2013-06-01

    Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased (59)Fe uptake from transferrin by 1.5-2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased (59)Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated (59)Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of (59)Fe uptake from (59)Fe-citrate, only intracellular ascorbate is needed for transferrin (59)Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent (59)Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or (125)I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.

  5. Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes.

    PubMed

    Parks, Scott K; Tresguerres, Martin; Goss, Greg G

    2008-11-01

    Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.

  6. A Comparative Study of Iron Uptake Rates and Mechanisms amongst Marine and Fresh Water Cyanobacteria: Prevalence of Reductive Iron Uptake

    PubMed Central

    Lis, Hagar; Kranzler, Chana; Keren, Nir; Shaked, Yeala

    2015-01-01

    In this contribution, we address the question of iron bioavailability to cyanobacteria by measuring Fe uptake rates and probing for a reductive uptake pathway in diverse cyanobacterial species. We examined three Fe-substrates: dissolved inorganic iron (Fe') and the Fe-siderophores Ferrioxamine B (FOB) and FeAerobactin (FeAB). In order to compare across substrates and strains, we extracted uptake rate constants (kin = uptake rate/[Fe-substrate]). Fe' was the most bioavailable Fe form to cyanobacteria, with kin values higher than those of other substrates. When accounting for surface area (SA), all strains acquired Fe' at similar rates, as their kin/SA were similar. We also observed homogeneity in the uptake of FOB among strains, but with 10,000 times lower kin/SA values than Fe'. Uniformity in kin/SA suggests similarity in the mechanism of uptake and indeed, all strains were found to employ a reductive step in the uptake of Fe' and FOB. In contrast, different uptake pathways were found for FeAB along with variations in kin/SA. Our data supports the existence of a common reductive Fe uptake pathway amongst cyanobacteria, functioning alone or in addition to siderophore-mediated uptake. Cyanobacteria combining both uptake strategies benefit from increased flexibility in accessing different Fe-substrates. PMID:25768677

  7. The mechanism of zinc uptake by cultured rat liver cells.

    PubMed Central

    Taylor, J A; Simons, T J

    1994-01-01

    1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898

  8. Structure and uptake mechanism of bacteriocins targeting peptidoglycan renewal.

    PubMed

    Zeth, Kornelius

    2012-12-01

    Bacteriocins are narrow-spectrum protein antibiotics released to kill related bacteria of the same niche. Uptake of bacteriocins depends critically on the presence of an uptake receptor in the outer membrane, a translocation pore and an energy-dependent activating system of the inner membrane. Most bacteriocins act on the inner membrane as pore-forming toxins or they target cytoplasmic DNA/RNA and ribosomal synthesis respectively. Only two bacteriocins are known to become activated in the periplasmic space and to inhibit the renewal process of the peptidoglycan structure. In Escherichia coli, the Cma (colicin M) phosphatase is activated in the periplasmic space by the FkpA chaperone and subsequently degrades the C55-PP precursor unit of the peptidoglycan. Pst (pesticin) from Yersinia pestis carries a lysozyme homology domain to degrade peptidoglycan. Import of Pst is only achieved if the N-terminal translocation domain can span the outer membrane and if extensive unfolding of the protein during membrane passage is permitted. There is considerable plasticity in the import pathway since a chimaera comprising the activity domain replaced by T4 lysozyme is also translocated and active in killing those bacteria carrying the FyuA receptor.

  9. Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

    PubMed Central

    Resina, Sarah; Prevot, Paul; Thierry, Alain R.

    2009-01-01

    Background Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure. Methodology/Principal Findings To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels. Conclusions These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer. PMID:19557145

  10. Silicon uptake and transport is an active process in Cucumis sativus.

    PubMed

    Liang, Yongchao; Si, Jin; Römheld, Volker

    2005-09-01

    Cucumis sativus is a species known to accumulate high levels of silicon (Si) in the tops, though the mechanism for its high Si uptake is little understood. In a series of hydroponic experiments, we examined uptake and xylem loading of Si in C. sativus along with Vicia faba at three levels of Si (0.085, 0.17 and 1.70 mm). Measured Si uptake in C. sativus was more than twice as high as calculated from the rate of transpiration assuming no discrimination between silicic acid and water in uptake. Measured Si uptake in V. faba, however, was significantly lower than the calculated uptake. Concentration of Si in xylem exudates was several-fold higher in C. sativus, but was significantly lower in V. faba compared with the Si concentration in external solutions, regardless of Si levels. Silicon uptake was strongly inhibited by low temperature and 2,4-dinitrophenol, a metabolic inhibitor, in C. sativus but not in V. faba. It can be concluded that Si uptake and transport in C. sativus is active and independent of external Si concentrations, in contrast to the process in V. faba.

  11. Uptake as a Mechanism to Promote Student Learning

    ERIC Educational Resources Information Center

    Bell, Clare Valerie

    2013-01-01

    This study is a descriptive examination of uptake that occurred during classroom discourse in 33 Algebra I classrooms in nine U.S. states. Uptake refers to the act of taking up mathematical comments, questions, and constructions as objects of discourse. Uptake is important because it can be used for scaffolding authentic opportunities to learn and…

  12. Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction.

    PubMed

    Merry, Troy L; Lynch, Gordon S; McConell, Glenn K

    2010-12-01

    There is evidence that nitric oxide (NO) is required for the normal increases in skeletal muscle glucose uptake during contraction, but the mechanisms involved have not been elucidated. We examined whether NO regulates glucose uptake during skeletal muscle contractions via cGMP-dependent or cGMP-independent pathways. Isolated extensor digitorum longus (EDL) muscles from mice were stimulated to contract ex vivo, and potential NO signaling pathways were blocked by the addition of inhibitors to the incubation medium. Contraction increased (P < 0.05) NO synthase (NOS) activity (∼40%) and dichlorofluorescein (DCF) fluorescence (a marker of oxidant levels; ∼95%), which was prevented with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), and antioxidants [nonspecific antioxidant, N-acetylcysteine (NAC); thiol-reducing agent, DTT], respectively. L-NMMA and NAC both attenuated glucose uptake during contraction by ∼50% (P < 0.05), and their effects were not additive. Neither the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, which prevents the formation of cGMP, the cGMP-dependent protein (PKG) inhibitor Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3',5'-cyclic monophosphorothioate sodium salt nor white light, which breaks S-nitrosylated bonds, affects glucose uptake during contraction; however, DTT attenuated (P < 0.05) contraction-stimulated glucose uptake (by 70%). NOS inhibition and antioxidant treatment reduced contraction-stimulated increases in protein S-glutathionylation and tyrosine nitration (P < 0.05), without affecting AMPK or p38 MAPK phosphorylation. In conclusion, we provide evidence to suggest that NOS-derived oxidants regulate skeletal muscle glucose uptake during ex vivo contractions via a cGMP/PKG-, AMPK-, and p38 MAPK-independent pathway. In addition, it appears that NO and ROS may regulate skeletal muscle glucose uptake during contraction through a similar pathway.

  13. Nectar uptake in bats using a pumping-tongue mechanism

    PubMed Central

    Tschapka, Marco; Gonzalez-Terrazas, Tania P.; Knörnschild, Mirjam

    2015-01-01

    Many insects use nectar as their principal diet and have mouthparts specialized in nectarivory, whereas most nectar-feeding vertebrates are opportunistic users of floral resources and only a few species show distinct morphological specializations. Specialized nectar-feeding bats extract nectar from flowers using elongated tongues that correspond to two vastly different morphologies: Most species have tongues with hair-like papillae, whereas one group has almost hairless tongues that show distinct lateral grooves. Recent molecular data indicate a convergent evolution of groove- and hair-tongued bat clades into the nectar-feeding niche. Using high-speed video recordings on experimental feeders, we show distinctly divergent nectar-feeding behavior in clades. Grooved tongues are held in contact with nectar for the entire duration of visit as nectar is pumped into the mouths of hovering bats, whereas hairy tongues are used in conventional sinusoidal lapping movements. Bats with grooved tongues use a specific fluid uptake mechanism not known from any other mammal. Nectar rises in semiopen lateral grooves, probably driven by a combination of tongue deformation and capillary action. Extraction efficiency declined for both tongue types with a similar slope toward deeper nectar levels. Our results highlight a novel drinking mechanism in mammals and raise further questions on fluid mechanics and ecological niche partitioning. PMID:26601270

  14. Nectar uptake in bats using a pumping-tongue mechanism.

    PubMed

    Tschapka, Marco; Gonzalez-Terrazas, Tania P; Knörnschild, Mirjam

    2015-09-01

    Many insects use nectar as their principal diet and have mouthparts specialized in nectarivory, whereas most nectar-feeding vertebrates are opportunistic users of floral resources and only a few species show distinct morphological specializations. Specialized nectar-feeding bats extract nectar from flowers using elongated tongues that correspond to two vastly different morphologies: Most species have tongues with hair-like papillae, whereas one group has almost hairless tongues that show distinct lateral grooves. Recent molecular data indicate a convergent evolution of groove- and hair-tongued bat clades into the nectar-feeding niche. Using high-speed video recordings on experimental feeders, we show distinctly divergent nectar-feeding behavior in clades. Grooved tongues are held in contact with nectar for the entire duration of visit as nectar is pumped into the mouths of hovering bats, whereas hairy tongues are used in conventional sinusoidal lapping movements. Bats with grooved tongues use a specific fluid uptake mechanism not known from any other mammal. Nectar rises in semiopen lateral grooves, probably driven by a combination of tongue deformation and capillary action. Extraction efficiency declined for both tongue types with a similar slope toward deeper nectar levels. Our results highlight a novel drinking mechanism in mammals and raise further questions on fluid mechanics and ecological niche partitioning.

  15. Uptake of cationic dyes by sulfonated coal: Sorption mechanism

    SciTech Connect

    Mittal, A.K.; Venkobachar, C.

    1996-04-01

    Mechanistic aspects of sorption of Rhodamine B and Methylene Blue by sulfonated coal have been investigated. The coal surface before and after sulfonation has been characterized with the help of cation-exchange capacity measurements and infrared (IR) spectroscopy. These studies indicate that sulfuric acid treatment not only incorporates a SO{sub 3}H group on the coal surface but also oxidizes both aliphatic and aromatic fractions. The IR spectroscopy has been extensively applied to locate the active sites on the surface of the sorbent and the participating functional groups of the dye molecule. Graphical models of the sorbate-sorbent interaction have been proposed. These models are applied to explain the variation in the uptake potential of these dyes by sulfonated coal.

  16. Cardiac Uptake of Minocycline and Mechanisms for In Vivo Cardioprotection

    PubMed Central

    Romero-Perez, Diego; Fricovsky, Eduardo; Yamasaki, Katrina Go; Griffin, Michael; Barraza-Hidalgo, Maraliz; Dillmann, Wolfgang; Villarreal, Francisco

    2008-01-01

    Objectives The ability of minocycline to be transported into cardiac cells, concentrate in normal and ischemic myocardium and act as in vivo cardioprotector was examined. We also determined minocycline's capacity to act as a reducer of myocardial oxidative stress and matrix metalloproteinase (MMP) activity. Background The identification of compounds with the potential to reduce myocardial ischemic injury is of great interest. Tetracyclines (TTCs) are antibiotics with pleiotropic cytoprotective properties that accumulate in normal and diseased tissues. Minocycline is highly lipophilic and has shown promise as a possible cardioprotector. However, minocycline's potential as an in vivo cardioprotector as well as the means by which this action is attained are not well understood. Methods Rats were subjected to 45 min of ischemia and 48 h of reperfusion. Animals were treated 48 h before and 48 h after thoracotomy with either vehicle or 50 mg/kg/day minocycline. Tissue samples were used for biochemical assays and cultured cardiac cells for minocycline uptake experiments. Results Minocycline significantly reduced infarct size (∼33%), tissue MMP-9 activity and oxidative stress. Minocycline was concentrated ∼24-fold in normal (0.5 mM) and ∼50-fold in ischemic regions (1.1 mM) vs. blood. Neonatal rat cardiac fibroblasts, myocytes and adult fibroblasts demonstrate a time- and temperature-dependent uptake of minocycline to levels that approximate those of normal myocardium. Conclusions Given the high intracellular levels observed and results from the assessment of in vitro antioxidant and MMP inhibitor capacities, it is likely that minocycline acts to limit myocardial ischemic injury via mass action effects. PMID:18848143

  17. Multiple AMPK activators inhibit L-Carnitine uptake in C2C12 skeletal muscle myotubes.

    PubMed

    Shaw, Andy; Jeromson, Stewart; Watterson, Kenneth R; Pediani, John D; Gallagher, Iain; Whalley, Tim; Dreczkowski, Gillian; Brooks, Naomi; Galloway, Stuart; Hamilton, D Lee

    2017-03-15

    Mutations in the gene that encodes the principal L-Carnitine transporter, OCTN2, can lead to a reduced intracellular L-Carnitine pool and the disease Primary Carnitine Deficiency. L-Carnitine supplementation is used therapeutically to increase intracellular L-Carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake we hypothesised that AMPK activating compounds and insulin would increase L-Carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase L-Carnitine uptake at 100nM. However, L-Carnitine uptake was modestly increased at a dose of 150nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10mM, 5mM, 1mM, 0.5mM), A23187 (10μM)], inhibit mitochondrial function [Sodium Azide (75μM), Rotenone (1μM), Berberine (100μM), DNP (500μM)] or directly activate AMPK [AICAR (250μM)] were assessed for their ability to regulate L-Carnitine uptake. All compounds tested significantly inhibited L-Carnitine uptake. Inhibition by caffeine was not dantrolene (10μM) sensitive. Saturation curve analysis suggested that caffeine did not competitively inhibit L-Carnitine transport. However, the AMPK inhibitor Compound C (10μM) partially rescued the effect of caffeine suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits L-Carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.

  18. Active Sulforhodamine 101 Uptake into Hippocampal Astrocytes

    PubMed Central

    Schnell, Christian; Hagos, Yohannes; Hülsmann, Swen

    2012-01-01

    Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes. PMID:23189143

  19. Kinetic effect of Pd additions on the hydrogen uptake of chemically activated, ultramicroporous carbon

    SciTech Connect

    Bhat, Vinay V; Contescu, Cristian I; Gallego, Nidia C

    2010-01-01

    The effect of mixing chemically-activated ultramicroporous carbon (UMC) with Pd nanopowder is investigated. Results show that Pd addition doubles the rate of hydrogen uptake, but does not enhance the hydrogen capacity or improve desorption kinetics. The effect of Pd on the rate of hydrogen adsorption supports the occurrence of the hydrogen spillover mechanism in the Pd - UMC system.

  20. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms.

    PubMed

    Srinivasan, Padmanabhan; Kapadia, Rubina; Biswas, Arundhati; Said, Hamid M

    2014-11-01

    Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

  1. Increased skeletal muscle glucose uptake by rosemary extract through AMPK activation.

    PubMed

    Naimi, Madina; Tsakiridis, Theodoros; Stamatatos, Theocharis C; Alexandropoulos, Dimitris I; Tsiani, Evangelia

    2015-04-01

    Stimulation of the energy sensor AMP-activated kinase (AMPK) has been viewed as a targeted approach to increase glucose uptake by skeletal muscle and control blood glucose homeostasis. Rosemary extract (RE) has been reported to activate AMPK in hepatocytes and reduce blood glucose levels in vivo but its effects on skeletal muscle are not known. In the present study, we examined the effects of RE and the mechanism of regulation of glucose uptake in muscle cells. RE stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner. Maximum stimulation was seen with 5 μg/mL of RE for 4 h (184% ± 5.07% of control, p < 0.001), a response comparable to maximum insulin (207% ± 5.26%, p < 0.001) and metformin (216% ± 8.77%, p < 0.001) stimulation. RE did not affect insulin receptor substrate 1 and Akt phosphorylation but significantly increased AMPK and acetyl-CoA carboxylase phosphorylation. Furthermore, the RE-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C, but remained unchanged by the PI3K inhibitor, wortmannin. RE did not affect GLUT4 or GLUT1 glucose transporter translocation in contrast with a significant translocation of both transporters seen with insulin or metformin treatment. Our study is the first to show a direct effect of RE on muscle cell glucose uptake by a mechanism that involves AMPK activation.

  2. Cellular Uptake Mechanisms and Endosomal Trafficking of Supercharged Proteins

    PubMed Central

    Thompson, David B.; Villaseñor, Roberto; Dorr, Brent M.; Zerial, Marino; Liu, David R.

    2012-01-01

    Summary Supercharged proteins can deliver functional macromolecules into the cytoplasm of mammalian cells with potencies that exceed those of cationic peptides. The structural features of supercharged proteins that determine their delivery effectiveness and the intracellular fate of supercharged proteins once they enter cells have not yet been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge, and that scGFPs enter cells through multiple pathways including clathrin-dependent endocytosis and macropinocytosis. Supercharged proteins activate Rho and ERK1/2, and also alter the endocytic transport of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of supercharged proteins and implicate the modulation of endosomal trafficking as a determinant of cell-penetration and macromolecule-delivery efficiency. PMID:22840771

  3. Cr(VI) uptake mechanism of Bacillus cereus.

    PubMed

    Chen, Zhi; Huang, Zhipeng; Cheng, Yangjian; Pan, Danmei; Pan, Xiaohong; Yu, Meijuan; Pan, Zhiyun; Lin, Zhang; Guan, Xiong; Wu, Ziyu

    2012-04-01

    In this study, we investigated the Cr(VI) uptake mechanism in an indigenous Cr(VI)-tolerant bacterial strain -Bacillus cereus through batch and microscopic experiments. We found that both the cells and the supernatant collected from B. cereus cultivation could reduce Cr(VI). The valence state analysis revealed the complete transformation from Cr(VI) into Cr(III) by living B. cereus. Further X-ray absorption fine structure and Fourier transform infrared analyses showed that the reduced Cr(III) was coordinated with carboxyl and amido functional groups from either the cells or supernatant. Scanning electron microscopy and atomic force microscopy observation showed that noticeable Cr(III) precipitates were accumulated on bacterial surfaces. However, Cr(III) could also be detected in bacterial inner portions by using transmission electron microscopy thin section analysis coupled with energy dispersive X-ray spectroscopy. Through quantitative analysis of chromium distribution, we determined the binding ratio of Cr(III) in supernatant, cell debris and cytoplasm as 22%, 54% and 24%, respectively. Finally, we further discussed the role of bacterium-origin soluble organic molecules to the remediation of Cr(VI) pollutants.

  4. Uptake mechanism for iodine species to black carbon.

    PubMed

    Choung, Sungwook; Um, Wooyong; Kim, Minkyung; Kim, Min-Gyu

    2013-09-17

    Natural organic matter (NOM) plays an important role in determining the fate and transport of iodine species such as iodide (I(-)) and iodate (IO3(-)) in groundwater system. Although NOM exists as diverse forms in environments, prior iodine studies have mainly focused on uptake processes of iodide and iodate to humic materials. This study was conducted to determine the iodide and iodate uptake potential for a particulate NOM (i.e., black carbon [BC]). A laboratory-produced BC and commercial humic acid were used for batch experiments to compare their iodine uptake properties. The BC exhibited >100 times greater uptake capability for iodide than iodate at low pH of ~3, while iodide uptake was negligible for the humic acid. The uptake properties of both solids strongly depend on the initial iodine aqueous concentrations. After uptake reaction of iodide to the BC, X-ray absorption fine structure spectroscopy results indicated that the iodide was converted to electrophilic species, and iodine was covalently bound to carbon atom in polycyclic aromatic hydrocarbons present in the BC. The computed distribution coefficients (i.e., Kd values) suggest that the BC materials retard significantly the transport of iodide at low pH in environmental systems containing even a small amount of BC.

  5. Uptake Mechanism for Iodine Species to Black Carbon

    SciTech Connect

    Choung, Sungwook; Um, Wooyong; Kim, Min Kyung; Kim, Min-Gyu

    2013-08-13

    Natural organic matter (NOM) plays an important role in determining the fate and transport of iodine species such as iodide (I-) and iodate (IO3-) in groundwater system. Although NOM exists as diverse forms in environments, prior iodine studies have mainly focused on uptake processes of iodide and iodate to humic materials. This study was conducted to determine the iodide and iodate uptake potential for a particulate NOM (i.e., black carbon [BC]). A laboratory-produced BC and commercial humic acid were used for batch experiments to compare their iodine uptake properties. The BC exhibited >100 times greater uptake capability for iodide than iodate at low pH~3, while iodide uptake was negligible for the humic acid. The uptake properties of both solids strongly depend on the initial iodine aqueous concentrations. After uptake reaction of iodide to the BC, X-ray Absorption Fine Structure spectroscopy results indicated that the iodide was converted to electrophilic species, and iodine was covalently bound to carbon atom in polycyclic aromatic hydrocarbons present in the BC. The computed distribution coefficients (i.e., Kd values) suggest that the BC materials retard significantly the transport of iodide at low pH in environmental systems containing even a small amount of BC.

  6. Amino Acid Neurotransmitters; Mechanisms of Their Uptake into Synaptic Vesicles

    DTIC Science & Technology

    1991-08-01

    same neuron, at least from the cerebellar Golgi cell terminals. It should be kept in mind that the uptake of noradrenaline and dopamine in synaptic...vesicles prepared from rat brain is relatively non-specific. Noradrenaline containing vesicles can take up noradrenaline, dopamine and serotonin. In...also shown that the vesicles isolated from corpus striatum exhibited the same ratio of uptake of dopamine /noradrenaline as did vesicles from cerebral

  7. Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

    PubMed Central

    Avery, S V; Tobin, J M

    1992-01-01

    Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain. PMID:1335718

  8. Atypical Hydrogen Uptake on Chemically Activated, Ultramicroporous Carbon

    SciTech Connect

    Bhat, Vinay V; Contescu, Cristian I; Gallego, Nidia C; Baker, Frederick S

    2010-01-01

    Hydrogen adsorption at near-ambient temperatures on ultramicroporous carbon (UMC), derived through secondary chemical activation from a wood-based activated carbon was studied using volumetric and gravimetric methods. The results showed that physisorption is accompanied by a process of different nature that causes slow uptake at high pressures and hysteresis on desorption. In combination, this results in unusually high levels of hydrogen uptake at near-ambient temperatures and pressures (e.g. up to 0.8 wt % at 25 oC and 2 MPa). The heat of adsorption corresponding to the slow process leading to high uptake (17 20 kJ/mol) is higher than usually reported for carbon materials, but the adsorption kinetics is slow, and the isotherms exhibit pronounced hysteresis. These unusual properties were attributed to contributions from polarization-enhanced physisorption caused by traces of alkali metals residual from chemical activation. The results support the hypothesis that polarization-induced physisorption in high surface area carbons modified with traces of alkali metal ions is an alternate route for increasing the hydrogen storage capacity of carbon adsorbents.

  9. Mechanism of uptake of strontium isotopes in aspergillus lesions.

    PubMed

    Rawal, B D; Adiseshan, N

    1976-03-01

    Observations on experimental aspergillosis of chorioallantoic membranes confirmed that strontium-85 uptake in aspergillus lesions was directly due to infection by the fungus. Such uptake was not found in normal or in Toxoplasma gondii-infected control membranes. Further, the avidity of radionuclide uptake was proportional to the mycelial mass, as previously observed clinically. Investigations on 85Sr containing malt extract broth Aspergillus fumigatus cultures revealed that fungal hyphas did not contain the major proportion of radioactivity, but culture filtrates did, and suggested that a fungal metabolite may be responsible for radiostrontium binding. Subsequent radiochromatography of filtrates obtained from A. fumigatus cultures confirmed the existence of such a metabolite. Several clinical and laboratory observations support the concept that an aspergillus metabolite at foci of infection binds 85Sr and 87mSr.

  10. Calcium-Activated Phosphate Uptake in Contracting Corn Mitochondria 1

    PubMed Central

    Truelove, B.; Hanson, J. B.

    1966-01-01

    The phosphate inhibition of succinate-powered contraction in corn mitochondria can be reversed with calcium. Associated with this reversal is an accumulation of phosphate and calcium. Both ions are essential for accumulation, although strontium will partially substitute for calcium. Arsenate does not substitute for phosphate except in producing the inhibition of contraction. The antibiotics oligomycin and aurovertin do not block the phosphate inhibition of contraction or the calcium-activated phosphate uptake associated with the release of the inhibition. Dinitrophenol uncouples the phosphate uptake but permits full contraction. Calcium promotes inorganic phosphate accumulation in root tissue as well as in mitochondria. The results are discussed from the viewpoint of theories of calcium reaction with high energy intermediates of oxidative phosphorylation. It is concluded that calcium probably reacts with X∼P in corn mitochondria, rather than with X∼I as with animal mitochondria. PMID:16656343

  11. Saffron (Crocus sativus L.) increases glucose uptake and insulin sensitivity in muscle cells via multipathway mechanisms.

    PubMed

    Kang, Changkeun; Lee, Hyunkyoung; Jung, Eun-Sun; Seyedian, Ramin; Jo, MiNa; Kim, Jehein; Kim, Jong-Shu; Kim, Euikyung

    2012-12-15

    Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because of its various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elucidated the mechanism of the hypoglycemic actions of saffron through investigating its signaling pathways associated with glucose metabolism in C(2)C(12) skeletal muscle cells. Saffron strongly enhanced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA carboxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3-kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivity via both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt and mTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glucose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4 translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake and insulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mechanism of action of saffron and its potential as a therapeutic agent in diabetic patients.

  12. Cultivar variability of iron uptake mechanisms in rice (Oryza sativa L.).

    PubMed

    Pereira, Margarida P; Santos, Carla; Gomes, Ana; Vasconcelos, Marta W

    2014-12-01

    Rice (Oryza sativa L.) is the most important staple food in the world. It is rich in genetic diversity and can grow in a wide range of environments. Iron (Fe) deficiency is a major abiotic stress in crop production and in aerobic soils, where Fe forms insoluble complexes, and is not readily available for uptake. To cope with Fe deficiency, plants developed mechanisms for Fe uptake, and although rice was described as a Strategy II plant, recent evidence suggests that it is capable of utilizing mechanisms from both Strategies. The main objective of this work was to compare two cultivars, Bico Branco (japonica) and Nipponbare (tropical japonica), to understand if the regulation of Fe uptake mechanisms could be cultivar (cv.)dependent. Plants of both cultivars were grown under Fe-deficient and -sufficient conditions and physiological and molecular responses to Fe deficiency were evaluated. Bico Branco cv. developed more leaf chlorosis and was more susceptible to Fe deficiency, retaining more nutrients in roots, than Nipponbare cv., which translocated more nutrients to shoots. Nipponbare cv. presented higher levels of Fe reductase activity, which was significantly up-regulated by Fe deficiency, and had higher expression levels of the Strategy I-OsFRO2 gene in roots, while Bico Branco cv. induced more genes involved in Strategy II.These new findings show that rice cultivars have different responses to Fe deficiency and that the induction of Strategy I or II may be rice cultivar-dependent, although the utilization of the reduction mechanisms seems to be an ubiquitous advantage.

  13. The uptake mechanism and biocompatibility of graphene quantum dots with human neural stem cells

    NASA Astrophysics Data System (ADS)

    Shang, Weihu; Zhang, Xiaoyan; Zhang, Mo; Fan, Zetan; Sun, Ying; Han, Mei; Fan, Louzhen

    2014-05-01

    Cellular imaging after transplantation may provide important information to determine the efficacy of stem cell therapy. We have reported that graphene quantum dots (GQDs) are a type of robust biological labeling agent for stem cells that demonstrate little cytotoxicity. In this study, we examined the interactions of GQDs on human neural stem cells (hNSCs) with the aim to investigate the uptake and biocompatibility of GQDs. We examined the mechanism of GQD uptake by hNSCs and investigated the effects of GQDs on the proliferation, metabolic activity, and differentiation potential of hNSCs. This information is critical to assess the suitability of GQDs for stem cell tracking. Our results indicated that GQDs were taken up into hNSCs in a concentration- and time-dependent manner via the endocytosis mechanism. Furthermore, no significant change was found in the viability, proliferation, metabolic activity, and differentiation potential of hNSCs after treatment with GQDs. Thus, these data open a promising avenue for labeling stem cells with GQDs and also offer a potential opportunity to develop GQDs for biomedical applications.

  14. Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Kim, So Hui; Shin, Eun-Jung; Kim, Eun-Do; Bayaraa, Tsenguun; Frost, Susan Cooke; Hyun, Chang-Kee

    2007-11-01

    It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.

  15. Luminal and basolateral mechanisms involved in the renal tubular uptake of inorganic mercury

    SciTech Connect

    Zalups, R.K.; Minor, K.H.

    1995-09-01

    The present study provides evidence for the existence of both a luminal and a basolateral mechanism involved in the renal tubular uptake of inorganic mercury. The researchers compared the disposition of inorganic mercury in groups of surgical control rats, rats that underwent a unilateral ureteral ligation, and rats that underwent a bilateral ureteral ligation that were pretreated with either normal saline or a 7.5 mmol/kg intravenous dose of PAH 5 min prior to receiving a nontoxic 0.5-{mu}mol/kg intravenous dose of mercuric chloride. The {open_quotes}stop-flow{close_quotes} conditions induced by either unilateral or bilateral ureteral ligation caused a significant reduction in the uptake and content of mercury in the kidneys (whose ureter was ligated) both at 1 h and 24 h after the intravenous injection of the nontoxic dose of mercuric chloride. This decreased renal uptake of mercury was due specifically to decreased uptake of mercury in the renal cortex and outer stripe of the outer medulla. The amount of mercury has not taken up during ureteral ligation represents the portion of mercury that is presumably taken up by a luminal mechanism. Pretreatment with PAH also caused a significant reduction in the renal uptake of mercury in the cortex and outer stripe of the outer medulla. When either unilateral or bilateral ureteral ligation was combined with PAH pretreatment, an additive inhibitory effect occurred with respect to the renal uptake of mercury. In fact, the renal uptake of mercury was reduced by approximately 85% at 1 h after the injection of mercuric chloride. Since the luminal uptake of mercury was blocked by ureteral ligation, the effect of PAH on the renal uptake of mercury must have occurred at the basolateral membrane. Two distinct mechanisms are involved in mercury uptake, with one mechanism located on the luminal membrane and another located on the basolateral membrane. 22 refs., 11 figs., 2 tabs.

  16. Adsorption uptake of synthetic organic chemicals by carbon nanotubes and activated carbons

    NASA Astrophysics Data System (ADS)

    Brooks, A. J.; Lim, Hyung-nam; Kilduff, James E.

    2012-07-01

    Carbon nanotubes (CNTs) have shown great promise as high performance materials for adsorbing priority pollutants from water and wastewater. This study compared uptake of two contaminants of interest in drinking water treatment (atrazine and trichloroethylene) by nine different types of carbonaceous adsorbents: three different types of single walled carbon nanotubes (SWNTs), three different sized multi-walled nanotubes (MWNTs), two granular activated carbons (GACs) and a powdered activated carbon (PAC). On a mass basis, the activated carbons exhibited the highest uptake, followed by SWNTs and MWNTs. However, metallic impurities in SWNTs and multiple walls in MWNTs contribute to adsorbent mass but do not contribute commensurate adsorption sites. Therefore, when uptake was normalized by purity (carbon content) and surface area (instead of mass), the isotherms collapsed and much of the CNT data was comparable to the activated carbons, indicating that these two characteristics drive much of the observed differences between activated carbons and CNT materials. For the limited data set here, the Raman D:G ratio as a measure of disordered non-nanotube graphitic components was not a good predictor of adsorption from solution. Uptake of atrazine by MWNTs having a range of lengths and diameters was comparable and their Freundlich isotherms were statistically similar, and we found no impact of solution pH on the adsorption of either atrazine or trichloroethylene in the range of naturally occurring surface water (pH = 5.7-8.3). Experiments were performed using a suite of model aromatic compounds having a range of π-electron energy to investigate the role of π-π electron donor-acceptor interactions on organic compound uptake by SWNTs. For the compounds studied, hydrophobic interactions were the dominant mechanism in the uptake by both SWNTs and activated carbon. However, comparing the uptake of naphthalene and phenanthrene by activated carbon and SWNTs, size exclusion effects

  17. Adsorption uptake of synthetic organic chemicals by carbon nanotubes and activated carbons.

    PubMed

    Brooks, A J; Lim, Hyung-nam; Kilduff, James E

    2012-07-27

    Carbon nanotubes (CNTs) have shown great promise as high performance materials for adsorbing priority pollutants from water and wastewater. This study compared uptake of two contaminants of interest in drinking water treatment (atrazine and trichloroethylene) by nine different types of carbonaceous adsorbents: three different types of single walled carbon nanotubes (SWNTs), three different sized multi-walled nanotubes (MWNTs), two granular activated carbons (GACs) and a powdered activated carbon (PAC). On a mass basis, the activated carbons exhibited the highest uptake, followed by SWNTs and MWNTs. However, metallic impurities in SWNTs and multiple walls in MWNTs contribute to adsorbent mass but do not contribute commensurate adsorption sites. Therefore, when uptake was normalized by purity (carbon content) and surface area (instead of mass), the isotherms collapsed and much of the CNT data was comparable to the activated carbons, indicating that these two characteristics drive much of the observed differences between activated carbons and CNT materials. For the limited data set here, the Raman D:G ratio as a measure of disordered non-nanotube graphitic components was not a good predictor of adsorption from solution. Uptake of atrazine by MWNTs having a range of lengths and diameters was comparable and their Freundlich isotherms were statistically similar, and we found no impact of solution pH on the adsorption of either atrazine or trichloroethylene in the range of naturally occurring surface water (pH = 5.7-8.3). Experiments were performed using a suite of model aromatic compounds having a range of π-electron energy to investigate the role of π-π electron donor-acceptor interactions on organic compound uptake by SWNTs. For the compounds studied, hydrophobic interactions were the dominant mechanism in the uptake by both SWNTs and activated carbon. However, comparing the uptake of naphthalene and phenanthrene by activated carbon and SWNTs, size exclusion effects

  18. Parallel modulation of brown adipose tissue GDP-binding, substrate uptake and (Na(+)-K+)-ATPase activity in the rat.

    PubMed

    Zamora, F; Alemany, M; Arola, L

    1991-10-01

    Brown adipose tissue (Na(+)-K+)-ATPase activity, in vitro glucose uptake and 2-aminoisobutyric acid uptake, as well as mitochondrial GDP-binding and succinate dehydrogenase activity were determined in order to study the relationship between these parameters in control, cold acclimated and cafeteria-fed rats. GDP-binding, (Na(+)-K+)-ATPase and glucose uptake were increased in interscapular brown adipose tissue from cold-acclimated and cafeteria-fed rats, whereas 2-aminoisobutyric acid uptake was only increased in cafeteria-fed rats. GDP-binding and (Na(+)-K+)-ATPase activity showed a high correlation coefficient suggesting a parallel modulation of both systems, which would probably share a common regulation mechanism.

  19. IL-7 promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to support T-cell survival.

    PubMed

    Wofford, Jessica A; Wieman, Heather L; Jacobs, Sarah R; Zhao, Yuxing; Rathmell, Jeffrey C

    2008-02-15

    Lymphocyte homeostasis requires coordination of metabolic processes with cellular energetic and biosynthetic demands but mechanisms that regulate T-cell metabolism are uncertain. We show that interleukin-7 (IL-7) is a key regulator of glucose uptake in T lymphocytes. To determine how IL-7 affects glucose uptake, we analyzed IL-7 signaling mechanisms and regulation of the glucose transporter, Glut1. The IL-7 receptor (IL-7R) stimulated glucose uptake and cell-surface localization of Glut1 in a manner that required IL-7R Y449, which promoted rapid signal transducer and activator of transcription 5 (STAT5) activation and a delayed yet sustained activation of Akt. Each pathway was necessary for IL-7 to promote glucose uptake, as Akt1(-/-) T cells or PI3-kinase inhibition and RNAi of STAT5 led to defective glucose uptake in response to IL-7. STAT5 and Akt acted in a linear pathway, with STAT5-mediated transcription leading to Akt activation, which was necessary for STAT5 and IL-7 to promote glucose uptake and prevent cell death. Importantly, IL-7 required glucose uptake to promote cell survival. These data demonstrate that IL-7 promotes glucose uptake via a novel signaling mechanism in which STAT5 transcriptional activity promotes Akt activation to regulate Glut1 trafficking and glucose uptake that is critical for IL-7 to prevent T-cell death and maintain homeostasis.

  20. Study of glucose uptake activity of Helicteres isora Linn. fruits in L-6 cell lines

    PubMed Central

    Gupta, R. N.; Pareek, Anil; Suthar, Manish; Rathore, Garvendra S.; Basniwal, Pawan K.; Jain, Deepti

    2009-01-01

    The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization. H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine. Hot water extracts were analysed for glucose uptake activity and found to be significantly active at 200 μg/ml dose comparable with insulin and metformin. Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake. It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles. PMID:20336200

  1. Study of glucose uptake activity of Helicteres isora Linn. fruits in L-6 cell lines.

    PubMed

    Gupta, R N; Pareek, Anil; Suthar, Manish; Rathore, Garvendra S; Basniwal, Pawan K; Jain, Deepti

    2009-10-01

    The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization. H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine. Hot water extracts were analysed for glucose uptake activity and found to be significantly active at 200 mug/ml dose comparable with insulin and metformin. Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake. It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles.

  2. Cellular uptake and anticancer activity of carboxylated gallium corroles.

    PubMed

    Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H; Gray, Harry B; Termini, John; Lim, Punnajit

    2016-04-19

    We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax= 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 > 3 > 2 > 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging.

  3. Cellular uptake and anticancer activity of carboxylated gallium corroles

    PubMed Central

    Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H.; Gray, Harry B.; Termini, John; Lim, Punnajit

    2016-01-01

    We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50 values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50 values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax = 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 >> 3 > 2 >> 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging. PMID:27044076

  4. Sodium Stimulation of Uptake Hydrogenase Activity In Symbiotic Rhizobium1

    PubMed Central

    Kapulnik, Yoram; Phillips, Donald A.

    1986-01-01

    Initial observations showed a 100% increase in H2-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H2 evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na+ (29 versus 9 milligrams per kilogram) and less K+ (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na+ concentration of clay-grown plants with a much smaller reduction in K+ concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na+ strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na+ concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na+ increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.). PMID:16665057

  5. Microbial Enzyme Activity, Nutrient Uptake, and Nutrient Limitation in Forested Streams

    EPA Science Inventory

    We measured NH4 + and PO4 -3 uptake length (Sw), uptake velocity (Vf), uptake rate (U), biofilm enzyme activity (BEA), and channel geomorphology in streams draining forested catchments in the Northwestern (Northern California Coast Range and Cascade Mountains) and Southeastern (A...

  6. Biophysical response of living cells to boron nitride nanoparticles: uptake mechanism and bio-mechanical characterization

    NASA Astrophysics Data System (ADS)

    Rasel, Md. Alim Iftekhar; Li, Tong; Nguyen, Trung Dung; Singh, Sanjleena; Zhou, Yinghong; Xiao, Yin; Gu, YuanTong

    2015-11-01

    Boron nitride nanomaterials have attracted significant interest due to their superior chemical and physical properties. Despite these novel properties, investigation on the interaction between boron nitride nanoparticle (BN NP) and living systems has been limited. In this study, BN NP (100-250 nm) is assessed as a promising biomaterial for medical applications. The toxicity of BN NP is evaluated by assessing the cells behaviours both biologically (MTT assay, ROS detection etc.) and physically (atomic force microscopy). The uptake mechanism of BN NP is studied by analysing the alternations in cellular morphology based on cell imaging techniques. The results demonstrate in vitro cytocompatibility of BN NP with immense potential for use as an effective nanoparticle for various bio-medical applications.

  7. Mechanism of Iron Uptake by Peanut Plants 1

    PubMed Central

    Römheld, Volker; Marschner, Horst

    1983-01-01

    Iron deficiency in peanuts (Arachis hypogeae L.) caused an increase in release of caffeic acid, a higher rate of FeIII reduction, and increased rates of both FeIII chelate splitting and iron uptake. Experiments on FeIII reduction by phenolics (in vitro experiments) and by roots of Fe-deficient peanuts exclude the direct involvement of released phenolics in FeIII reduction by roots: FeIII reduction by phenolics had a pH optimum higher than 8.0 and was strongly dependent on the concentration and the stability of the supplied FeIII chelates. In contrast, FeIII reduction by roots of Fe-deficient peanuts had a pH optimum of about 5.0 and was less dependent on the stability of the supplied FeIII chelates. Furthermore, the observed release of phenolics into nutrient solution would have to be at least 200 times higher to attain the reduction rates of roots of Fe-deficient peanuts. The results of these experiments support the idea of an enzymic reduction of FeIII on the plasmalemma of cortical cells of roots. PMID:16662934

  8. Mechanism for uptake of silica particles by monocytic U937 cells.

    PubMed

    Hetland, G; Namork, E; Schwarze, P E; Aase, A

    2000-07-01

    We examined the mechanism for uptake by monocytic cells of particles found in the atmosphere of some industrial work places. As a model system, irregular crystalline silica particles (SPs), sphere-like cryptocrystalline microsilica particles (MPs) and carbon particles (CPs) were exposed to pro-monocytic U937 cells. Plasma-treated SP and MP, but not CP, activated the alternative complement pathway, but bound little C3b. However, all particles adsorbed serum IgG, IgA and IgM unspecifically. Phenotyping of U937 cells for complement receptors (CRs) and Fcgamma receptors (FcgammaRs) showed that interferon gamma (INFgamma) increased expression of FcgammaRI, CR3 (CD11b/CD18) and CR4 (CD11c/CD18) and that phorbol-12-myristate-13-acetate (PMA) increased expression of CR4. Scanning electron microscopy (SEM) demonstrated higher phagocytosis of plasma-treated SP than native SP by both PMA- and INFgamma-stimulated, but not unstimulated, cells. MP and CP could not be distinguished from cellular structures. Inhibition experiments in SEM revealed uptake of heparin-plasma-treated SP via FcgammaRI on INFgamma-stimulated U937 cells, but could not exclude possible participation of CR3. The results indicate that plasma-treated SPs bind Ig and are internalized by differentiated monocytic cells via FcgammaRI, which is known to trigger cellular production of toxic oxygen species that may induce pulmonary inflammation in vivo.

  9. Mechanisms Controlling Arsenic Uptake in Rice Grown in Mining Impacted Regions in South China

    PubMed Central

    Lu, Ying; Yan, Qiuyan; Shim, Hojae

    2014-01-01

    Foods produced on soils impacted by Pb-Zn mining activities are a potential health risk due to plant uptake of the arsenic (As) associated with such mining. A field survey was undertaken in two Pb-Zn mining-impacted paddy fields in Guangdong Province, China to assess As accumulation and translocation, as well as other factors influencing As in twelve commonly grown rice cultivars. The results showed that grain As concentrations in all the surveyed rice failed national food standards, irrespective of As speciation. Among the 12 rice cultivars, “SY-89” and “DY-162” had the least As in rice grain. No significant difference for As concentration in grain was observed between the rice grown in the two areas that differed significantly for soil As levels, suggesting that the amount of As contamination in the soil is not necessarily the overriding factor controlling the As content in the rice grain. The iron and manganese plaque on the root surface curtailed As accumulation in rice roots. Based on our results, the accumulation of As within rice plants was strongly associated with such soil properties such as silicon, phosphorus, organic matter, pH, and clay content. Understanding the factors and mechanisms controlling As uptake is important to develop mitigation measures that can reduce the amount of As accumulated in rice grains produced on contaminated soils. PMID:25251438

  10. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  11. Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

    PubMed

    Smith, Kelsy F; Bibb, Lori A; Schmitt, Michael P; Oram, Diana M

    2009-03-01

    Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

  12. WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

    PubMed Central

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-01-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

  13. Uptake, delivery, and anticancer activity of thymoquinone nanoparticles in breast cancer cells

    NASA Astrophysics Data System (ADS)

    Fakhoury, Isabelle; Saad, Walid; Bouhadir, Kamal; Nygren, Peter; Schneider-Stock, Regine; Gali-Muhtasib, Hala

    2016-07-01

    Thymoquinone (TQ) is a promising anticancer molecule but its development is hindered by its limited bioavailability. Drug encapsulation is commonly used to overcome low drug solubility, limited bioavailability, and nonspecific targeting. In this project, TQ nanoparticles (TQ-NP) were synthesized and characterized. The cytotoxicity of the NP was investigated in nontumorigenic MCF-10-A breast cells, while the uptake, distribution, as well as the anticancer potential were investigated in MCF-7 and MDA-MB-231 breast cancer cells. Flash Nanoprecipitation and dynamic light scattering coupled with scanning electron microscopy were used to prepare and characterize TQ-NP prior to measuring their anticancer potential by MTT assay. The uptake and subcellular intake of TQ-NP were evaluated by fluorometry and confocal microscopy. TQ-NP were stable with a hydrodynamic average diameter size around 100 nm. Entrapment efficiency and loading content of TQ-NP were high (around 80 and 50 %, respectively). In vitro, TQ-NP had equal or enhanced anticancer activity effects compared to TQ in MCF-7 and aggressive MDA-MB-231 breast cancer cells, respectively, with no significant cytotoxicity of the blank NP. In addition, TQ and TQ-NP were relatively nontoxic to MCF-10-A normal breast cells. TQ-NP uptake mechanism was both time and concentration dependent. Treatment with inhibitors of endocytosis suggested the involvement of caveolin in TQ-NP uptake. This was further confirmed by subcellular localization findings showing the colocalization of TQ-NP with caveolin and transferrin as well as with the early and late markers of endocytosis. Altogether, the results describe an approach for the enhancement of TQ anticancer activity and uncover the mechanisms behind cell-TQ-NP interaction.

  14. [Inflammasome: activation mechanisms].

    PubMed

    Suárez, Raibel; Buelvas, Neudo

    2015-03-01

    Inflammation is a rapid biologic response of the immune system in vascular tissues, directed to eliminate stimuli capable of causing damage and begin the process of repair. The macromolecular complexes known as "inflammasomes" are formed by a receptor, either NOD (NLR) or ALR, the receptor absent in melanoma 2 (AIM2). In addition, the inflammasome is formed by the speck-like protein associated to apoptosis (ASC) and procaspase-1, that may be activated by variations in the ionic and intracellular and extracellular ATP concentrations; and the loss of stabilization of the fagolisosomme by internalization of insoluble crystals and redox mechanisms. As a result, there is activation of the molecular platform and the processing of inflammatory prointerleukins to their active forms. There are two modalities of activation of the inflammasome: canonical and non-canonical, both capable of generating effector responses. Recent data associate NLRP 3, IL-1β and IL-18 in the pathogenesis of a variety of diseases, including atherosclerosis, type II diabetes, hyperhomocysteinemia, gout, malaria and hypertension. The inflammasome cascade is emerging as a new chemotherapeutic target in these diseases. In this review we shall discuss the mechanisms of activation and regulation of the inflammasome that stimulate, modulate and resolve inflammation.

  15. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  16. Mechanism of boron uptake by hydrocalumite calcined at different temperatures.

    PubMed

    Qiu, Xinhong; Sasaki, Keiko; Takaki, Yu; Hirajima, Tsuyoshi; Ideta, Keiko; Miyawaki, Jin

    2015-04-28

    Hydrocalumite (Ca-Al-layered double hydroxide (LDH)) was prepared and applied for the removal of borate. The properties of Ca-Al-LDH calcined at different temperatures were diverse, which affected the sorption density and mechanism of boron species. The sorption density increased with increase in calcined temperature and the sample calcined at 900°C (Ca-Al-LDH-900) showed the maximum sorption density in this work. The solid residues after sorption were characterized by (11)B NMR, (27)Al NMR, SEM, and XRD to investigate the sorption mechanism. Dissolution-reprecipitation was the main mechanism for sorption of borate in Ca-Al-LDH. For Ca-Al-LDH calcined at 300 and 500°C, regeneration occurred in a short time and the newly forming LDHs were decomposed to release Ca(2+) ions and formed ettringite with borate. Two stages occurred in the sorption of boron by Ca-Al-LDH calcined at 900°C. In the first stage, boron species adsorbed on the alumina gel resulting from the hydration of calcined products. In this stage, borate was included as an interlayer anion into the newly forming LDHs in the following stage, and then immobilized as HBO3(2-) into the interlayer, most the LDHs.

  17. UPTAKE OF NITRATE AND NITRITE BY DITYLUM BRIGHTWELLII-KINETICS AND MECHANISMS(1) (2).

    PubMed

    Eppley, R W; Coatsworth, J L

    1968-06-01

    Ditylum brightwellii grown on NO2 - as a nitrogen source took up and assimilated NO2 - only in the light, apparently via a photosynthetic nitrite reductase. Assimilation was inhibited by dichlorophenyldimethylurea (DCMU), KCN, partially by 2,4 dinitrophenol, and by NO3 -. Kinetics of inhibition of NO2 - assimilation by NO3 - appeared to be "competitive." D. brightwellii cells grown on NO2 - took up NO3 - in both light and dark and in both cases the uptake was inhibited by p-chloromercuribenzoate, but not by DCMU, KCN, or by NO2 -. Most of the NO3 - taken up in the dark was recovered unchanged from the cells. However only 40% of NO3 - taken up in light was recovered from the cells and no NO2 - was found. This suggests that a photosynthetic nitrate reduction mechanism was active in these cells. DCMU inhibited the light-induced NO3 - reduction. This mechanism of NO3 - reduction is distinct from that involving NADH nitrate reductase in D. brightwellii since the concentration of the latter enzyme is very low in cells grown on NO2 -. Saturation kinetics were observed for NO2 - and NO3 - uptake. Half-saturation concentrations (Ks values) were 4 and 2 μM, respectively. These values are compared with those obtained for NO2 - and NO3 - assimilation by other unicellular algae. The comparisons show lower Ks values in oceanic species compared with tide-pool or freshwater algae and they support the idea that Ks values for NO3 - assimilation may provide a key to understanding species succession when this is due to declining: nitrate concentrations in the sea.

  18. Fe Uptake Mechanism in Fe-Efficient Cucumber Roots 1

    PubMed Central

    Zocchi, Graziano; Cocucci, Sergio

    1990-01-01

    Fe-efficient plants respond to iron stress both by morphological and physiological modifications. In roots of a Fe-efficient plant (Cucumis sativus L.) grown in the presence or in the absence of iron, the capacity to acidify the external medium, change in the transmembrane electrical potential, and the ATPase activity have been determined. Roots from plants grown in the absence of iron showed a great capacity to acidify the external medium, a higher transmembrane electrical potential difference (−145 millivolts, versus −105 millivolts), and a higher ATPase activity (+30%). The administration of Fe2+, but not Fe3+, caused a block of the acidification capacity, a great decrease in the transmembrane electrical potential difference in root cells, and a large inhibition of the ATPase activity of isolated microsomal membrane vesicles. PMID:16667404

  19. Metal uptake and acute toxicity in zebrafish: common mechanisms across multiple metals.

    PubMed

    Alsop, Derek; Wood, Chris M

    2011-10-01

    Zebrafish larvae (Danio rerio) were used to examine the mechanisms of action and acute toxicities of metals. Larvae had similar physiological responses and sensitivities to waterborne metals as adults. While cadmium and zinc have previously been shown to reduce Ca(2+) uptake, copper and nickel also decreased Ca(2+) uptake, suggesting that the epithelial transport of all these metals is through Ca(2+) pathways. However, exposure to cadmium, copper or nickel for up to 48 h had little or no effect on total whole body Ca(2+) levels, indicating that the reduction of Ca(2+) uptake is not the acute toxic mechanism of these metals. Instead, mortalities were effectively related to whole body Na(+), which decreased up to 39% after 48 h exposures to different metals around their respective 96 h LC50s. Decreases in whole body K(+) were also observed, although they were not as pronounced or frequent as Na(+) losses. None of the metals tested inhibited Na(+) uptake in zebrafish (Na(+) uptake was in fact increased with exposure) and the observed losses of Na(+), K(+), Ca(2+) and Mg(2+) were proportional to the ionic gradients between the plasma and water, indicating diffusive ion loss with metal exposure. This study has shown that there is a common pathway for metal uptake and a common mechanism of acute toxicity across groups of metals in zebrafish. The disruption of ion uptake accompanying metal exposure does not appear to be responsible for the acute toxicity of metals, as has been previously suggested, but rather the toxicity is instead due to total ion loss (predominantly Na(+)).

  20. Copper speciation and isotopic fractionation in plants: uptake and translocation mechanisms.

    PubMed

    Ryan, Brooke M; Kirby, Jason K; Degryse, Fien; Harris, Hugh; McLaughlin, Mike J; Scheiderich, Kathleen

    2013-07-01

    The fractionation of stable copper (Cu) isotopes during uptake into plant roots and translocation to shoots can provide information on Cu acquisition mechanisms. Isotope fractionation ((65) Cu/(63) Cu) and intact tissue speciation techniques (X-ray absorption spectroscopy, XAS) were used to examine the uptake, translocation and speciation of Cu in strategy I (tomato-Solanum lycopersicum) and strategy II (oat-Avena sativa) plant species. Plants were grown in controlled solution cultures, under varied iron (Fe) conditions, to test whether the stimulation of Fe-acquiring mechanisms can affect Cu uptake in plants. Isotopically light Cu was preferentially incorporated into tomatoes (Δ(65) Cu(whole plant-solution ) = c. -1‰), whereas oats showed minimal isotopic fractionation, with no effect of Fe supply in either species. The heavier isotope was preferentially translocated to shoots in tomato, whereas oat plants showed no significant fractionation during translocation. The majority of Cu in the roots and leaves of both species existed as sulfur-coordinated Cu(I) species resembling glutathione/cysteine-rich proteins. The presence of isotopically light Cu in tomatoes is attributed to a reductive uptake mechanism, and the isotopic shifts within various tissues are attributed to redox cycling during translocation. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation are not redox selective.

  1. Phytotoxicity of salt and plant salt uptake: Modeling ecohydrological feedback mechanisms

    NASA Astrophysics Data System (ADS)

    Bauer-Gottwein, Peter; Rasmussen, Nikolaj F.; Feificova, Dagmar; Trapp, Stefan

    2008-04-01

    A new model of phytotoxicity of salt and plant salt uptake is presented and is coupled to an existing three-dimensional groundwater simulation model. The implementation of phytotoxicity and salt uptake relationships is based on experimental findings from willow trees grown in hydroponic solution. The data confirm an s-shaped phytotoxicity relationship as found in previous studies. Uptake data were explained assuming steady state salt concentration in plant roots, passive salt transport into the roots, and active enzymatic removal of salt from plant roots. On the one hand, transpiration strongly depends on groundwater salinity (phytotoxicity); on the other hand, transpiration significantly changes the groundwater salinity (uptake). This feedback loop generates interesting dynamic phenomena in hydrological systems that are dominated by transpiration and are influenced by significant salinity gradients. Generic simulations are performed for the Okavango island system and are shown to reproduce essential phenomena observed in nature.

  2. Uptake and transport mechanisms of decabromodiphenyl ether (BDE-209) by rice (Oryza sativa).

    PubMed

    Chow, Ka Lai; Man, Yu Bon; Tam, Nora Fung Yee; Liang, Yan; Wong, Ming Hung

    2015-01-01

    The extensive industrial use of brominated flame retardants has aroused rapidly growing public concerns about their ubiquity in the environment. The feasibility of uptake and translocation of decabromodiphenyl ether (BDE-209) by three rice cultivars, namely Fengmeizhan, Hefengzhan and Guangyinzhan, and the uptake mechanisms of BDE-209 into rice roots, were investigated by employing a partition-limited model. Uptake of BDE-209 by the rice cultivars (Fengmeizhan, Hefengzhan and Guangyinzhan) was examined by a 60-day cultivation in sterilized BDE-209 spiked sand, followed by Soxhlet extraction and gas chromatography-mass spectrometry (GC-MS) analysis. A partition-limited model was applied for estimating and describing the approach of the uptake of BDE-209 by rice in sand. The average quasi-equilibrium factor (αpt) of BDE-209 in root uptake in sand was 0.112×10(-3) for three rice cultivars in the present study (<1), implying a non-equilibrium movement of molecules and a dominated passive transport uptake. According to the results of sorption analysis of dead and fresh roots, apoplastic pathway likely dominated the transport of BDE-209 into roots cells.

  3. Mechanically Activated Ion Channels

    PubMed Central

    Ranade, Sanjeev S.; Syeda, Ruhma; Patapoutian, Ardem

    2015-01-01

    Mechanotransduction, the conversion of physical forces into biochemical signals, is an essential component of numerous physiological processes including not only conscious senses of touch and hearing, but also unconscious senses such as blood pressure regulation. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

  4. Mechanically Activated Ion Channels.

    PubMed

    Ranade, Sanjeev S; Syeda, Ruhma; Patapoutian, Ardem

    2015-09-23

    Mechanotransduction, the conversion of physical forces into biochemical signals, is essential for various physiological processes such as the conscious sensations of touch and hearing, and the unconscious sensation of blood flow. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels.

  5. Vitamin B12 uptake by intestinal microorganisms: mechanism and relevance to syndromes of intestinal bacterial overgrowth

    PubMed Central

    Giannella, R. A.; Broitman, S. A.; Zamcheck, N.

    1971-01-01

    The mechanism of bacterial uptake of vitamin B12, the spectrum of microorganisms capable of such uptake, and the factors involved were the subject of this study. Bacterial uptake of vitamin B12 was found to be at least a two stage process. A primary uptake phase which was rapid (1 min or less), pH dependent, nontemperature dependent, did not require viable organisms and was insensitive to either the metabolic inhibitor dinitrophenol or to the sulfhydryl inhibitor N-ethyl-maleimide. Protein denaturation (formalin treatment or autoclaving) abolished all B12 uptake. This primary uptake phase is thought to represent adsorption to binding or “receptor” sites on the cell wall. Second stage uptake was slower, pH and temperature dependent, required living bacteria, and was abolished by either dinitrophenol or N-ethyl-maleimide. This phase is dependent upon metabolic processes and may reflect transfer of B12 from surface “receptor” sites into the bacterial cell. Although differences among organisms were observed in total 1 hr uptake, number of surface “receptor” sites, and relative avidities for B12, all organisms except Streptococcus fecalis shared the two stage mechanism. Two Gram-positive organisms. Bacillus subtilis and Group A streptococcus, demonstrated the highest 1 hr vitamin B12 uptake values; Gram-negative bacteria required 2,000-10,000 the number of organisms for comparable uptake. Binding constants (Km) varied from 5.05 ±1.67 × 10-10M for B. subtilis to 6.18 ±3.08 × 10-9M for Klebsiella pneumoniae which approximate the Km for human intrinsic factor (0.38 × 10-10M). Competition between bacteria and intrinsic factor for vitamin B12 may be inferred from the similarity of these constants. These observations suggest that a variety of enteric and nonenteric organisms, not requiring exogenous B12, may play a role in the pathogenesis of the vitamin B12 malabsorption found in the intestinal bacterial overgrowth syndromes. PMID:4994753

  6. Gallium-based anti-infectives: targeting microbial iron-uptake mechanisms.

    PubMed

    Kelson, Andrew B; Carnevali, Maia; Truong-Le, Vu

    2013-10-01

    Microbes have evolved elaborate iron-acquisition systems to sequester iron from the host environment using siderophores and heme uptake systems. Gallium(III) is structurally similar to iron(III), except that it cannot be reduced under physiological conditions, therefore gallium has the potential to serve as an iron analog, and thus an anti-microbial. Because Ga(III) can bind to virtually any complex that binds Fe(III), simple gallium salts as well as more complex siderophores and hemes are potential carriers to deliver Ga(III) to the microbes. These gallium complexes represent a new class of anti-infectives that is different in mechanism of action from conventional antibiotics. Simple gallium salts such as gallium nitrate, maltolate, and simple gallium siderophore complexes such as gallium citrate have shown good antibacterial activities. The most studied complex has been gallium citrate, which exhibits broad activity against many Gram negative bacteria at ∼1-5μg/ml MICs, strong biofilm activity, low drug resistance, and efficacy in vivo. Using the structural features of specific siderophore and heme made by pathogenic bacteria and fungi, researchers have begun to evaluate new gallium complexes to target key pathogens. This review will summarize potential iron-acquisition system targets and recent research on gallium-based anti-infectives.

  7. The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the significance of oxygen uptake.

    PubMed

    Weiss, R H; Estabrook, R W

    1986-11-15

    The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes prepared from phenobarbital-treated rats resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide over a similar time course. Maximal activity was observed at pH 7-8. The addition of cumene hydroperoxide to boiled microsomes did not initiate oxygen uptake or produce thiobarbituric acid reactive products. Oxygen uptake was required for the formation of thiobarbituric acid reactive products, but not for the loss of hydroperoxide. The extent of oxygen uptake and thiobarbituric acid reactive product formation was linearly dependent on the concentration of cumene hydroperoxide and independent of the amount of microsomes. For each nanomole of cumene hydroperoxide utilized, 1.5 nmol of oxygen was consumed and 0.11 nmol of thiobarbituric acid reactive products was formed. In addition, a saturable reaction having a high affinity for cumene hydroperoxide was observed that was associated with little or no oxygen uptake and thiobarbituric acid reactive product formation. Butylated hydroxytoluene at substoichiometric concentrations inhibited the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation, indicating that cumene hydroperoxide-dependent lipid peroxidation may be an autocatalytic free radical process.

  8. Exercise and type 2 diabetes: molecular mechanisms regulating glucose uptake in skeletal muscle

    PubMed Central

    Goodyear, Laurie J.

    2014-01-01

    Exercise is a well-established tool to prevent and combat type 2 diabetes. Exercise improves whole body metabolic health in people with type 2 diabetes, and adaptations to skeletal muscle are essential for this improvement. An acute bout of exercise increases skeletal muscle glucose uptake, while chronic exercise training improves mitochondrial function, increases mitochondrial biogenesis, and increases the expression of glucose transporter proteins and numerous metabolic genes. This review focuses on the molecular mechanisms that mediate the effects of exercise to increase glucose uptake in skeletal muscle. PMID:25434013

  9. Structural specificity of mechanisms controlling the hepatic uptake and biliary output of methotrexate in the rat

    SciTech Connect

    Deutsch, J.C.; Kolhouse, J.F.

    1989-07-01

    Using an in vivo model with systemic administration of compounds, the hepatic uptake from blood and hepatic release into bile of (/sup 3/H)methotrexate ((/sup 3/H)MTX) are shown to involve structurally distinct and specific mechanisms. The hepatic uptake of (/sup 3/H)MTX from blood is shown to proceed through two separate mechanisms: one inhibitable by the bile salt cholic acid, and the other inhibitable by either unlabeled MTX or folic acid, but not the lipophilic antifol, trimetrexate. The biliary output of (/sup 3/H)MTX was shown to be related to the cholic acid-sensitive mechanism of hepatic uptake of (/sup 3/H)MTX. In contrast, the biliary output of (/sup 3/H)MTX was shown to be markedly stimulated by either unlabeled MTX or trimetrexate but not folic acid, demonstrating structural specificity for the biliary output of (/sup 3/H)MTX distinct from the structural specificity shown for the hepatic uptake of (/sup 3/H)MTX.

  10. Microbial iron uptake as a mechanism for dispersing iron from deep-sea hydrothermal vents.

    PubMed

    Li, Meng; Toner, Brandy M; Baker, Brett J; Breier, John A; Sheik, Cody S; Dick, Gregory J

    2014-01-01

    Deep-sea hydrothermal vents are a significant source of oceanic iron. Although hydrothermal iron rapidly precipitates as inorganic minerals on mixing with seawater, it can be stabilized by organic matter and dispersed more widely than previously recognized. The nature and source of this organic matter is unknown. Here we show that microbial genes involved in cellular iron uptake are highly expressed in the Guaymas Basin deep-sea hydrothermal plume. The nature of these microbial iron transporters, taken together with the low concentration of dissolved iron and abundance of particulate iron in the plume, indicates that iron minerals are the target for this microbial scavenging and uptake. Our findings indicate that cellular iron uptake is a major process in plume microbial communities and suggest new mechanisms for generating Fe-C complexes. This 'microbial iron pump' could represent an important mode of converting hydrothermal iron into bioavailable forms that can be dispersed throughout the oceans.

  11. The activation of cannabinoid receptors in striatonigral GABAergic neurons inhibited GABA uptake.

    PubMed

    Romero, J; de Miguel, R; Ramos, J A; Fernández-Ruiz, J J

    1998-01-01

    observed in synaptosome preparations obtained from the globus pallidus. In summary, the activation of CNRs located on striatonigral GABAergic neurons, which primarily access to GABA-B receptor synapses, was accompanied by a reduction in neurotransmitter uptake, thus prolonging the presence of GABA into the synaptic cleft. This mechanism might underly the CN-induced motor inhibition through the potentiation of the inhibitory effect of GABA on neuronal activity, in particular of nigrostriatal dopaminergic neurons.

  12. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle.

    PubMed Central

    Whitehead, J P; Soos, M A; Aslesen, R; O'rahilly, S; Jensen, J

    2000-01-01

    The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity. PMID:10903138

  13. Increased physical activity decreases hepatic free fatty acid uptake: a study in human monozygotic twins

    PubMed Central

    Hannukainen, Jarna C; Nuutila, Pirjo; Ronald, Borra; Kaprio, Jaakko; Kujala, Urho M; Janatuinen, Tuula; Heinonen, Olli J; Kapanen, Jukka; Viljanen, Tapio; Haaparanta, Merja; Rönnemaa, Tapani; Parkkola, Riitta; Knuuti, Juhani; Kalliokoski, Kari K

    2007-01-01

    Exercise is considered to be beneficial for free fatty acid (FFA) metabolism, although reports of the effects of increased physical activity on FFA uptake and oxidation in different tissues in vivo in humans have been inconsistent. To investigate the heredity-independent effects of physical activity and fitness on FFA uptake in skeletal muscle, the myocardium, and liver we used positron emission tomography (PET) in nine healthy young male monozygotic twin pairs discordant for physical activity and fitness. The cotwins with higher physical activity constituting the more active group had a similar body mass index but less body fat and 18 ± 10% higher V˙O2,max (P < 0.001) compared to the less active brothers with lower physical activity. Low-intensity knee-extension exercise increased skeletal muscle FFA and oxygen uptake six to 10 times compared to resting values but no differences were observed between the groups at rest or during exercise. At rest the more active group had lower hepatic FFA uptake compared to the less active group (5.5 ± 4.3 versus 9.0 ± 6.1 μmol (100 ml)−1 min−1, P = 0.04). Hepatic FFA uptake associated significantly with body fat percentage (P = 0.05). Myocardial FFA uptake was similar between the groups. In conclusion, in the absence of the confounding effects of genetic factors, moderately increased physical activity and aerobic fitness decrease body adiposity even in normal-weighted healthy young adult men. Further, increased physical activity together with decreased intra-abdominal adiposity seems to decrease hepatic FFA uptake but has no effects on skeletal muscle or myocardial FFA uptake. PMID:17053033

  14. A Transmissible Plant Shoot Factor Promotes Uptake Hydrogenase Activity in Rhizobium Symbionts 1

    PubMed Central

    Bedmar, Eulogio J.; Phillips, Donald A.

    1984-01-01

    Shoot/root grafting studies showed organ and host cultivar effects on net H2 evolution from Pisum sativum L. root nodules. Net H2 evolution from those nodules represents the sum of H2 formed by Rhizobium nitrogenase and H2 oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars `JI1205' or `Alaska' and `Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H2 uptake significantly (P ≤ 0.05) in Feltham First root nodules. The same plants also had less net H2 evolution at similar rates of C2H2 reduction than plants formed by grafting Feltham First shoots on Feltham First roots. Although JI1205 and Alaska shoots increased H2-uptake activity of Feltham First root nodules 28 days after the graft was made, intermediate to high levels of H2 uptake activity were still present in nodules on roots of both JI1205 and Alaska grafted to Feltham First shoots. These results indicate the presence of a transmissible shoot factor(s) which can increase uptake hydrogenase activity in a Rhizobium symbiont and show that root genotype also can influence that parameter. Parallel grafting experiments using the same pea cultivars in symbioses with R. leguminosarum strain 300, which lacks uptake hydrogenase activity, suggested that a transmissible shoot factor(s) altered H2 formation from nitrogenase by changing the electron allocation coefficient of that enzyme complex. The root and shoot factor(s) detected in this study had no permanent effect on strain 128C53. Bacterial cells isolated from Feltham First nodules with low H2 uptake activity formed root nodules on JI1205 and Alaska with high H2 uptake activity. Bacteroids isolated from nodules on intact JI1205, Alaska, or Feltham First plants with high, medium, or low H2 uptake activity, respectively, maintained those phenotypes during in vitro assays. PMID:16663677

  15. A transmissible plant shoot factor promotes uptake hydrogenase activity in Rhizobium symbionts.

    PubMed

    Bedmar, E J; Phillips, D A

    1984-07-01

    Shoot/root grafting studies showed organ and host cultivar effects on net H(2) evolution from Pisum sativum L. root nodules. Net H(2) evolution from those nodules represents the sum of H(2) formed by Rhizobium nitrogenase and H(2) oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars ;JI1205' or ;Alaska' and ;Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H(2) uptake significantly (P uptake activity of Feltham First root nodules 28 days after the graft was made, intermediate to high levels of H(2) uptake activity were still present in nodules on roots of both JI1205 and Alaska grafted to Feltham First shoots. These results indicate the presence of a transmissible shoot factor(s) which can increase uptake hydrogenase activity in a Rhizobium symbiont and show that root genotype also can influence that parameter.Parallel grafting experiments using the same pea cultivars in symbioses with R. leguminosarum strain 300, which lacks uptake hydrogenase activity, suggested that a transmissible shoot factor(s) altered H(2) formation from nitrogenase by changing the electron allocation coefficient of that enzyme complex.The root and shoot factor(s) detected in this study had no permanent effect on strain 128C53. Bacterial cells isolated from Feltham First nodules with low H(2) uptake activity formed root nodules on JI1205 and Alaska with high H(2) uptake activity. Bacteroids isolated from nodules on intact JI1205, Alaska, or Feltham First plants with high, medium, or low H(2) uptake activity, respectively, maintained those phenotypes during in vitro assays.

  16. Effect of Three Statins on Glucose Uptake of Cardiomyocytes and its Mechanism.

    PubMed

    Jiang, Zhenhuan; Yu, Bo; Li, Yang

    2016-08-11

    BACKGROUND The aim of this study was to investigate the effects of different statins on glucose uptake and to confirm its mechanism in primary cultured rat cardiomyocytes after administration of atorvastatin, pravastatin, and rosuvastatin. MATERIAL AND METHODS Primary cultured rat cardiomyocytes were randomly assigned to 5 groups: normal control group (OB), insulin group (S1), statin 1-μM (S2), 5-μM (S3), and 10-μM (S4) groups for 3 different statins. The 2-[3H]-DG uptake of each group was determined and the mRNA and protein expression levels of glucose transporter type 4 (GLUT4), insulin receptor substrate (IRs), and RhoA were assessed. RESULTS After treatment with different concentrations of statins and insulin, the 2-[3H]-DG uptake showed a significant negative correlation with the concentration of atorvastatin (P<0.05), and no significant correlation with pravastatin and rosuvastatin. The mRNA and protein expression levels of GLUT4 and IRs-1 in primary cultured cardiomyocytes were both significantly reduced by atorvastatin treatment (P<0.05). Pravastatin and rosuvastatin showed no significant effects on GLUT4 and IRs-1 expression. The mRNA and protein expression levels of RhoA both showed no significant difference when treated with the 3 statins. CONCLUSIONS These results confirm that atorvastatin can inhibit insulin-induced glucose uptake in primary cultured rat cardiomyocytes by regulating the PI3K/Akt insulin signal transduction pathway.

  17. Triphenyl phosphate enhances adipogenic differentiation, glucose uptake and lipolysis via endocrine and noradrenergic mechanisms.

    PubMed

    Cano-Sancho, German; Smith, Anna; La Merrill, Michele A

    2017-04-01

    The use of triphenyl phosphate (TPhP) as a flame retardant or plasticizer has increased during the last decade, resulting in widespread human exposure without commensurate toxicity assessment. The main objectives of this study were to assess the in vitro effect of TPhP and its metabolite diphenyl phosphate (DPhP) on the adipogenic differentiation of 3T3-L1 cells, as well as glucose uptake and lipolysis in differentiated 3T3-L1 adipocytes. TPhP increased pre-adipocyte proliferation and subsequent adipogenic differentiation of 3T3-L1 cells, coinciding with increased transcription in the CEBP and PPARG pathway. Treatment of mature adipocytes with TPhP increased the basal- and insulin stimulated- uptake of the glucose analog 2-[N (-7-nitrobenz-2-oxa1, 3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG). This effect was ablated by inhibition of PI3K, a member of the insulin signaling pathway. DPhP had no significant effect on cell proliferation and, compared to TPhP, a weaker effect on adipogenic differentiation and on 2-NBDG uptake. Both TPhP and DPhT significantly enhanced the isoproterenol-induced lipolysis, most likely by increasing the expression of lipolytic genes during and after differentiation. This study suggests that TPhP increases adipogenic differentiation, glucose uptake, and lipolysis in 3T3-L1 cells through endocrine and noradrenergic mechanisms.

  18. Medicago truncatula increases its iron-uptake mechanisms in response to volatile organic compounds produced by Sinorhizobium meliloti.

    PubMed

    Orozco-Mosqueda, Maria del Carmen; Macías-Rodríguez, Lourdes I; Santoyo, Gustavo; Farías-Rodríguez, Rodolfo; Valencia-Cantero, Eduardo

    2013-11-01

    Medicago truncatula represents a model plant species for understanding legume-bacteria interactions. M. truncatula roots form a specific root-nodule symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti. Symbiotic nitrogen fixation generates high iron (Fe) demands for bacterial nitrogenase holoenzyme and plant leghemoglobin proteins. Leguminous plants acquire Fe via "Strategy I," which includes mechanisms such as rhizosphere acidification and enhanced ferric reductase activity. In the present work, we analyzed the effect of S. meliloti volatile organic compounds (VOCs) on the Fe-uptake mechanisms of M. truncatula seedlings under Fe-deficient and Fe-rich conditions. Axenic cultures showed that both plant and bacterium modified VOC synthesis in the presence of the respective symbiotic partner. Importantly, in both Fe-rich and -deficient experiments, bacterial VOCs increased the generation of plant biomass, rhizosphere acidification, ferric reductase activity, and chlorophyll content in plants. On the basis of our results, we propose that M. truncatula perceives its symbiont through VOC emissions, and in response, increases Fe-uptake mechanisms to facilitate symbiosis.

  19. Sorptive uptake of selenium with magnetite and its supported materials onto activated carbon.

    PubMed

    Kwon, Jae H; Wilson, Lee D; Sammynaiken, R

    2015-11-01

    Kinetic and equilibrium uptake studies of selenite in aqueous solution with synthetic magnetite (Mag-P), commercial magnetite (Mag-C), goethite, activated carbon (AC), and a composite material containing 19% magnetite supported on activated carbon (CM-19) were investigated. Kinetic uptake studies used a one-pot setup at pH 5.26 at variable temperature. Sampling of unbound selenite in-situ was achieved with analytical detection by atomic absorbance. The sorptive uptake at equilibrium and kinetic conditions are listed in descending order: goethite>Mag-P>Mag-C>CM-19. Kinetic uptake parameters reveal that Mag-P showed apparent negative values for the activation energy (E(a)) and the enthalpy of activation (ΔH(‡)), in agreement with a multi-step process for the kinetic uptake of selenite. By contrast, Mag-C, CM-19, and goethite showed positive values for E(a) and ΔH(‡). The uptake properties of the various sorbent materials with selenite are in accordance with the formation of inner- and out-sphere complexes. Leaching of iron from the composite material (CM-19) was attenuated due to the stabilizing effect of the magnetite within the pore sites and the surface of AC. Supported iron oxide nanomaterial composites represent a unique sorbent material with tunable uptake properties toward inorganic selenite in aqueous solution.

  20. Thyroid function in fasting rats: variations in 131I uptake and transient decrease in peroxidase activity.

    PubMed

    Moura, E G; Ramos, C F; Nascimento, C C; Rosenthal, D; Breitenbach, M M

    1987-01-01

    Serum thyroxine and triiodothyronine, radioiodide thyroid uptake and thyroid peroxidase (TPO) activity were studied over a 2 to 5 day period in fasting rats treated (F+) or not (F-) with TSH. In F- rats, TPO activity was transiently decreased on the 3rd day, whereas in F+ it was always higher than in controls. On the 5th day, the 2 h thyroid uptake of 131I decreased in F-, while the 24 h uptake increased in both F- and F+. Serum T3 and T4 decreased in both fasting groups. Thus, not all effects of fasting on rat thyroid function are reverted by TSH administration, suggesting intrinsic impairment of glandular function.

  1. Growth and nitrogen uptake characteristics reveal outbreak mechanism of the opportunistic macroalga Gracilaria tenuistipitata.

    PubMed

    Wang, Chao; Lei, Anping; Zhou, Kai; Hu, Zhengyu; Hao, Wenlong; Yang, Junda

    2014-01-01

    Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3-) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH4+ than that with NO3- addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH4+ and NO3- uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3- uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g-1 DM h-1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3-, suggesting when both NH4+ and NO3- were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.

  2. Growth and Nitrogen Uptake Characteristics Reveal Outbreak Mechanism of the Opportunistic Macroalga Gracilaria tenuistipitata

    PubMed Central

    Wang, Chao; Lei, Anping; Zhou, Kai; Hu, Zhengyu; Hao, Wenlong; Yang, Junda

    2014-01-01

    Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3−) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH4+ than that with NO3− addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH4+ and NO3− uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3− uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g−1 DM h−1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3−, suggesting when both NH4+ and NO3− were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation. PMID:25299123

  3. Molecular Physiology of an Extra-renal Cl(-) Uptake Mechanism for Body Fluid Cl(-) Homeostasis.

    PubMed

    Wang, Yi-Fang; Yan, Jia-Jiun; Tseng, Yung-Che; Chen, Ruo-Dong; Hwang, Pung-Pung

    2015-01-01

    The development of an ion regulatory mechanism for body fluid homeostasis was an important trait for vertebrates during the evolution from aquatic to terrestrial life. The homeostatic mechanism of Cl(-) in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Unlike in mammals, in which the kidney plays a central role, in most fish species, the gill is responsible for the maintenance of Cl(-) homeostasis via Cl(-) transport uptake mechanisms. Previous studies in zebrafish identified Na(+)-Cl(-) cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl(-) uptake, similar to distal convoluted tubular cells in mammalian kidney. However, the mechanism by which basolateral ions exit from NCC cells is still unclear. Of the in situ hybridization signals of twelve members of the clc Cl(-) channel family, only that of clc-2c exhibited an ionocyte pattern in the gill and embryonic skin. Double in situ hybridization/immunocytochemistry confirmed colocalization of apical NCC2b with basolateral CLC-2c. Acclimation to a low Cl(-) environment increased mRNA expression of both clc-2c and ncc2b, and also the protein expression of CLC-2c in embryos and adult gills. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl(-) content in zebrafish embryos, a phenotype similar to that of ncc2b mutants; this finding suggests a role for CLC-2c in Cl(-) uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl(-) homeostasis. Further comparative genomic and phylogenetic analyses revealed that zebrafish CLC-2c is a fish-specific isoform that diverged from a kidney-predominant homologue, in the same manner as NCC2b and its counterparts (NCCs). Several lines of molecular and cellular physiological evidences demonstrated

  4. Regulation of AMPK Activation by CD36 Links Fatty Acid Uptake to β-Oxidation

    PubMed Central

    Sun, Jingyu; Pietka, Terri; Gross, Richard W.; Eckel, Robert H.; Su, Xiong; Stahl, Philip D.

    2015-01-01

    Increases in muscle energy needs activate AMPK and induce sarcolemmal recruitment of the fatty acid (FA) translocase CD36. The resulting rises in FA uptake and FA oxidation are tightly correlated, suggesting coordinated regulation. We explored the possibility that membrane CD36 signaling might influence AMPK activation. We show, using several cell types, including myocytes, that CD36 expression suppresses AMPK, keeping it quiescent, while it mediates AMPK activation by FA. These dual effects reflect the presence of CD36 in a protein complex with the AMPK kinase LKB1 (liver kinase B1) and the src kinase Fyn. This complex promotes Fyn phosphorylation of LKB1 and its nuclear sequestration, hindering LKB1 activation of AMPK. FA interaction with CD36 dissociates Fyn from the protein complex, allowing LKB1 to remain cytosolic and activate AMPK. Consistent with this, CD36−/− mice have constitutively active muscle and heart AMPK and enhanced FA oxidation of endogenous triglyceride stores. The molecular mechanism described, whereby CD36 suppresses AMPK, with FA binding to CD36 releasing this suppression, couples AMPK activation to FA availability and would be important for the maintenance of cellular FA homeostasis. Its dysfunction might contribute to the reported association of CD36 variants with metabolic complications of obesity in humans. PMID:25157091

  5. Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

    PubMed

    González-Barriga, Anchel; Nillessen, Bram; Kranzen, Julia; van Kessel, Ingeborg D G; Croes, Huib J E; Aguilera, Begoña; de Visser, Peter C; Datson, Nicole A; Mulders, Susan A M; van Deutekom, Judith C T; Wieringa, Bé; Wansink, Derick G

    2017-04-04

    Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

  6. Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells

    NASA Astrophysics Data System (ADS)

    Sun, Xin-Yuan; Gan, Qiong-Zhi; Ouyang, Jian-Ming

    2017-02-01

    Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

  7. Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells

    PubMed Central

    Sun, Xin-Yuan; Gan, Qiong-Zhi; Ouyang, Jian-Ming

    2017-01-01

    Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones. PMID:28150811

  8. Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells.

    PubMed

    Sun, Xin-Yuan; Gan, Qiong-Zhi; Ouyang, Jian-Ming

    2017-02-02

    Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

  9. Effects of xylitol on carbohydrate digesting enzymes activity, intestinal glucose absorption and muscle glucose uptake: a multi-mode study.

    PubMed

    Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

    2015-03-01

    The present study investigated the possible mechanism(s) behind the effects of xylitol on carbohydrate digesting enzymes activity, muscle glucose uptake and intestinal glucose absorption using in vitro, ex vivo and in vivo experimental models. The effects of increasing concentrations of xylitol (2.5%-40% or 164.31 mM-2628.99 mM) on alpha amylase and alpha glucosidase activity in vitro and intestinal glucose absorption and muscle glucose uptake were investigated under ex vivo conditions. Additionally, the effects of an oral bolus dose of xylitol (1 g per kg BW) on gastric emptying and intestinal glucose absorption and digesta transit in the different segments of the intestinal tract were investigated in normal and type 2 diabetic rats at 1 hour after dose administration, when phenol red was used as a recovery marker. Xylitol exhibited concentration-dependent inhibition of alpha amylase (IC₅₀ = 1364.04 mM) and alpha glucosidase (IC₅₀ = 1127.52 mM) activity in vitro and small intestinal glucose absorption under ex vivo condition. Xylitol also increased dose dependent muscle glucose uptake with and without insulin, although the uptake was not significantly affected by the addition of insulin. Oral single bolus dose of xylitol significantly delayed gastric emptying, inhibited intestinal glucose absorption but increased the intestinal digesta transit rate in both normal and diabetic rats compared to their respective controls. The data of this study suggest that xylitol reduces intestinal glucose absorption via inhibiting major carbohydrate digesting enzymes, slowing gastric emptying and fastening the intestinal transit rate, but increases muscle glucose uptake in normal and type 2 diabetic rats.

  10. A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells.

    PubMed

    Nabokina, Svetlana M; Said, Hamid M

    2012-08-01

    All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [(3)H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na(+)-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent K(m) = 0.157 ± 0.028 μM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca(2+)/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

  11. Ion Uptake Determination of Dendrochronologically-Dated Trees Using Neutron Activation Analysis

    SciTech Connect

    Kenan Unlu; P.I. Kuniholm; D.K.H. Schwarz; N.O. Cetiner; J.J. Chiment

    2009-03-30

    Uptake of metal ions by plan roots is a function of the type and concentration of metal in the soil, the nutrient biochemistry of the plant, and the immediate environment of the root. Uptake of gold (Au) is known to be sensitive to soil pH for many species. Soil acidification due to acid precipitation following volcanic eruptions can dramatically increase Au uptake by trees. Identification of high Au content in tree rings in dendrochronologically-dated, overlapping sequences of trees allows the identification of temporally-conscribed, volcanically-influenced periods of environmental change. Ion uptake, specifically determination of trace amounts of gold, was performed for dendrochronologically-dated tree samples utilizing Neutron Activation Analysis (NAA) technique. The concentration of gold was correlated with known enviironmental changes, e.g. volcanic activities, during historic periods.

  12. Southern Ocean heat and carbon uptake: mechanisms, recent trends, and future changes

    NASA Astrophysics Data System (ADS)

    Froelicher, T. L.

    2015-12-01

    The Southern Ocean's dominant influence on the global heat balance and nutrient and carbon cycles stems from the fact that it is the primary gateway through which Earth's cold, centuries old and nutrient rich deep and bottom waters interact with the atmosphere. The westerly winds in the Southern Hemisphere drive a strongly divergent surface flow that draws up water from below in a wide ring circling the Antarctic continent. In the first part of the talk, we assess the uptake, transport, and storage of oceanic anthropogenic carbon and heat in the Southern Ocean over the period 1861-2005 in a new set of carbon-climate Earth System Models. Simulations show that the Southern Ocean south of 30°S, covering only 30% of the global surface ocean area, accounts for more than 40% of global anthropogenic carbon uptake. Furthermore, the Southern Ocean takes up three quarters of the total excess heat generated by the increasing levels of greenhouse gases in the atmosphere. Anthropogenic carbon and heat storage show a common broad-scale pattern of change, but ocean heat storage is more structured than ocean carbon storage suggesting that different mechanisms are important. The Southern Ocean, however, remains the region where models differ the most in the representation of anthropogenic carbon and, in particular, heat uptake. While the Southern Ocean carbon uptake has increased considerably in recent decades, as expected based on the substantial increase in atmospheric CO2, there is considerable concern that this sink will saturate or even reverse in response to warming, changing ocean circulation and chemistry. In the second part of the talk, novel multi-millennial global warming simulations with a comprehensive Earth System Model under a 1% yr-1 atmospheric CO2 increase to 2xCO2 and constant forcing thereafter scenario will be used to explore future long-term changes in the Southern Ocean carbon uptake. We show that after full equilibration of the model with doubling of

  13. Calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles isolated from control and selenium deficient lambs.

    PubMed

    Tripp, M J; Whanger, P D; Schmitz, J A

    1993-06-01

    The calcium uptake and ATPase activity were studied using fragmented sarcoplasmic reticulum (FSR) vesicles from normal and selenium (vitamin E)--deficient lambs. The latter group was suffering from white muscle disease (WMD). The calcium uptake of FSR vesicles from muscle of WMD lambs was reduced 10-fold as compared to those from normal lambs. An inverse relationship was found with the calcium uptake ability of the FSR vesicles and the severity of WMD. ATPase activity was nonsignificantly lower in vesicles from WMD lambs. The most active FSR vesicles from both normal and WMD lambs banded at 27% when purified on linear sucrose density gradients. The number of protein bands appearing in acrylamide gels of the purified vesicles appeared to be directly proportional to the severity of WMD. The 75Se cosedimented with the calcium uptake and ATPase activity when FSR vesicles from a lamb injected with 75Se-selenite were subjected to linear sucrose density gradient centrifugation, suggesting that selenium is incorporated into these vesicles. Injection of selenium into WMD lambs resulted in significantly greater calcium uptake activity in vesicles 18 and 38 days later as compared with untreated WMD lambs. Injection of selenium in WMD lambs resulted in a marked decrease in plasma CPK activity and a significant increase of glutathione peroxidase activity in the blood.

  14. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  15. Dual Mechanisms of Ion Uptake in Relation to Vacuolation in Corn Roots

    PubMed Central

    Torii, Kenji; Laties, George G.

    1966-01-01

    Absorption isotherms for chloride and rubidium ions have been determined through a wide concentration range for nonvacuolate root tips, and for vacuolate subapical sections of corn root. In the range 0 to 0.5 mm, chloride absorption is hyperbolic with concentration in both tips and proximal sections. At high concentrations, 1 to 50 mm, a second multiple-hyperbolic isotherm for chloride is noted in vacuolate tissue, while the isotherm for nonvacuolate tips rises exponentially. A linear to exponentially rising isotherm is taken to signify diffusive permeation. The same distinction between tip and subapical tissue characterizes Rb absorption. Rb uptake is indifferent to the nature of the counterion at all concentrations in the tip, while the counterion exerts a predictable influence on Rb absorption in proximal tissue. The effect of a poorly absorbable anion on Rb uptake is greater in the high concentration range. Evidence is presented for the metabolic nature of ion transport into nonvacuolate root tips. Verification is offered that ion uptake is mediated by dual mechanisms, and the thesis is developed that the high-affinity (low Ks) system mediates ion passage through the plasma membrane while the low-affinity (high Ks) system implements transport through the tonoplast. PMID:16656332

  16. Dual mechanisms of ion uptake in relation to vacuolation in corn roots.

    PubMed

    Torii, K; Laties, G G

    1966-05-01

    Absorption isotherms for chloride and rubidium ions have been determined through a wide concentration range for nonvacuolate root tips, and for vacuolate subapical sections of corn root. In the range 0 to 0.5 mm, chloride absorption is hyperbolic with concentration in both tips and proximal sections. At high concentrations, 1 to 50 mm, a second multiple-hyperbolic isotherm for chloride is noted in vacuolate tissue, while the isotherm for nonvacuolate tips rises exponentially. A linear to exponentially rising isotherm is taken to signify diffusive permeation.The same distinction between tip and subapical tissue characterizes Rb absorption. Rb uptake is indifferent to the nature of the counterion at all concentrations in the tip, while the counterion exerts a predictable influence on Rb absorption in proximal tissue. The effect of a poorly absorbable anion on Rb uptake is greater in the high concentration range. Evidence is presented for the metabolic nature of ion transport into nonvacuolate root tips. Verification is offered that ion uptake is mediated by dual mechanisms, and the thesis is developed that the high-affinity (low K(s)) system mediates ion passage through the plasma membrane while the low-affinity (high K(s)) system implements transport through the tonoplast.

  17. On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells.

    PubMed Central

    Gesek, F A; Friedman, P A

    1992-01-01

    PTH stimulates transcellular Ca2+ absorption in renal distal convoluted tubules. The effect of PTH on membrane voltage, the ionic basis of the change in voltage, and the relations between voltage and calcium entry were determined on immortalized mouse distal convoluted tubule cells. PTH (10(-8) M) significantly increased 45Ca2+ uptake from basal levels of 2.81 +/- 0.16 to 3.88 +/- 0.19 nmol min-1 mg protein-1. PTH-induced 45Ca2+ uptake was abolished by the dihydropyridine antagonist, nifedipine (10(-5) M). PTH did not affect 22Na+ uptake. Intracellular calcium activity ([Ca2+]i) was measured in cells loaded with fura-2. Control [Ca2+]i averaged 112 +/- 21 nM. PTH increased [Ca2+]i over the range of 10(-11) to 10(-7) M. Maximal stimulation to 326 +/- 31 nM was achieved at 10(-8) M PTH. Resting membrane voltage measured with the potential sensitive dye DiO6(3) averaged -71 +/- 2 mV. PTH hyperpolarized cells by 19 +/- 4 mV. The chloride-channel blocker NPPB prevented PTH-induced hyperpolarization. PTH decreased and NPPB increased intracellular chloride, measured with the fluorescent dye SPQ. Chloride permeability was estimated by measuring the rate of 125I- efflux. PTH increased 125I- efflux and this effect was blocked by NPPB. Clamping voltage with K+/valinomycin; depolarizing membrane voltage by reducing extracellular chloride; or addition of NPPB prevented PTH-induced calcium uptake. In conclusion, PTH increases chloride conductance in distal convoluted tubule cells leading to decreased intracellular chloride activity, membrane hyperpolarization, and increased calcium entry through dihydropyridine-sensitive calcium channels. PMID:1522230

  18. Active uptake and extravesicular storage of m-iodobenzylguanidine in human neuroblastoma SK-N-SH cells

    SciTech Connect

    Smets, L.A.; Loesberg, C.; Janssen, M.; Metwally, E.A.; Huiskamp, R.

    1989-06-01

    Radioiodinated m-iodobenzylguanidine (MIBG), an analogue of the neurotransmitter norepinephrine (NE), is increasingly used in the diagnosis and treatment of neural crest tumors. Active uptake and subsequent retention of MIBG and NE was studied in human neuroblastoma SK-N-SH cells. Neuron-specific uptake of (125I)MIBG and (3H)NE saturated at extracellular concentration of 10(-6) M and exceeded by 20-30-fold that by passive diffusion alone. A minimum of 50% of accumulated MIBG remained permanently stored but the SK-N-SH cells were incapable of retaining recaptured (3H)NE. (125I)MIBG was displaced from intracellular binding sites by unlabeled MIBG with 10-fold higher potency than by unlabeled NE. MIBG stored in SK-N-SH cells was insensitive to depletion by the inhibitor of granular uptake reserpine (RSP) and was not precipitated in a granular fraction by differential centrifugation. Only few electron-dense granules were found in these cells by electron microscopy. In contrast, MIBG storage in PC-12 pheochromocytoma cells which contained many storage granules, was sensitive to RSP and part of accumulated drug was recovered in a granular fraction. Accordingly, storage of MIBG in the SK-N-SH neuroblastoma cells is predominantly extravesicular and thus essentially different from that of biogenic amines in normal adrenomedullary tissue or in pheochromocytoma tumors, while sharing with these tissues a common mechanism of active uptake.

  19. Effect of Three Statins on Glucose Uptake of Cardiomyocytes and its Mechanism

    PubMed Central

    Jiang, Zhenhuan; Yu, Bo; Li, Yang

    2016-01-01

    Background The aim of this study was to investigate the effects of different statins on glucose uptake and to confirm its mechanism in primary cultured rat cardiomyocytes after administration of atorvastatin, pravastatin, and rosuvastatin. Material/Methods Primary cultured rat cardiomyocytes were randomly assigned to 5 groups: normal control group (OB), insulin group (S1), statin 1-μM (S2), 5-μM (S3), and 10-μM (S4) groups for 3 different statins. The 2-[3H]-DG uptake of each group was determined and the mRNA and protein expression levels of glucose transporter type 4 (GLUT4), insulin receptor substrate (IRs), and RhoA were assessed. Results After treatment with different concentrations of statins and insulin, the 2-[3H]-DG uptake showed a significant negative correlation with the concentration of atorvastatin (P<0.05), and no significant correlation with pravastatin and rosuvastatin. The mRNA and protein expression levels of GLUT4 and IRs-1 in primary cultured cardiomyocytes were both significantly reduced by atorvastatin treatment (P<0.05). Pravastatin and rosuvastatin showed no significant effects on GLUT4 and IRs-1 expression. The mRNA and protein expression levels of RhoA both showed no significant difference when treated with the 3 statins. Conclusions These results confirm that atorvastatin can inhibit insulin-induced glucose uptake in primary cultured rat cardiomyocytes by regulating the PI3K/Akt insulin signal transduction pathway. PMID:27510725

  20. Cooperation of taurine uptake and dopamine D1 receptor activation facilitates the induction of protein synthesis-dependent late LTP.

    PubMed

    Suárez, Luz M; Bustamante, Julián; Orensanz, Luís M; Martín del Río, Rafael; Solís, José M

    2014-04-01

    Co-activation of NMDA and dopamine receptors is required for the induction of the late phase of LTP (L-LTP) that is dependent on new protein synthesis. Other neuromodulatory substances may also contribute to this process. Here, we examined whether taurine is one of the neuromodulators contributing to L-LTP induction, since it is known that taurine uptake induces a long-lasting synaptic potentiation dependent on protein synthesis, and taurine uptake inhibition blocks L-LTP induced by tetanization. Experiments were conducted using rat hippocampal slices where field synaptic potentials were evoked and recorded in CA3-CA1 synapses. Taurine (1 mM) applied 10 min before a high frequency stimulation (HFS) train converted a transitory early-LTP (E-LTP) into an L-LTP dependent on protein synthesis. This taurine effect was blocked by a taurine uptake inhibitor. A facilitation of L-LTP induction was also obtained by pre-application of SKF38393, a D1/D5 dopamine receptor (D1R) agonist. In this case, LTP facilitation was not affected by the taurine uptake inhibitor. Nevertheless, when taurine and SKF38393 were simultaneously pre-applied at a concentration that individually did not modify E-LTP, they produced a synergistic mechanism that facilitated the induction of L-LTP with a sole HFS train. This facilitation of L-LTP was blocked by inhibiting either taurine uptake or D1R activation. Taurine and SKF38393 activated different signaling pathways to transform E-LTP into L-LTP. Taurine-induced L-LTP facilitation required MAPK activation, while D1R-agonist-induced facilitation depended mainly on PKA activation and partially on MAPK activation. On the other hand, the synergistic mechanisms induced by the cooperative action of taurine and SKF38393 were impaired by inhibitors against MAPK, PKA and PI3-K. This pharmacological profile resembles that displayed by L-LTP induced by three HFS trains at 10-min intervals. These results indicate that taurine uptake is necessary and

  1. In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals

    NASA Astrophysics Data System (ADS)

    Holland, Jason P.; Giansiracusa, Jeffrey H.; Bell, Stephen G.; Wong, Luet-Lok; Dilworth, Jonathan R.

    2009-04-01

    The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [60/62/64Cu(II)ATSM] and [60/62/64Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO2-dependent in vitro cellular uptake and retention of [64Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k1 = 9.8 ± 0.59 × 10-4 s-1 and k2 = 2.9 ± 0.17 × 10-3 s-1), intracellular reduction (k3 = 5.2 ± 0.31 × 10-2 s-1), reoxidation (k4 = 2.2 ± 0.13 mol-1 dm3 s-1) and proton-mediated ligand dissociation (k5 = 9.0 ± 0.54 × 10-5 s-1). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm

  2. On the Water Uptake and CCN Activation of Tropospheric Organic Aerosols

    NASA Astrophysics Data System (ADS)

    Rastak, Narges; Pajunoja, Aki; Acosta Navarro, Juan-Camilo; Leong, Yu Jun; Cerully, Kate M.; Nenes, Athanasios; Kirkevåg, Alf; Topping, David; Virtanen, Annele; Riipinen, Ilona

    2016-04-01

    Aerosol particles introduce high uncertainties to radiative climate forcing. If exposed to a given relative humidity (RH), aerosol particles containing soluble material can absorb water and grow in size (hygroscopic growth). If RH is increased further beyond supersaturation (RH >100%) the particles can act as cloud condensation nuclei (CCN). Aerosol particles interactions with water vapour determine to a large extent their influence on climate. Organic aerosols (OA) contribute a large fraction (20-90%) of atmospheric submicron particulate mass, on the other hand they often consist of thousands of compounds with different properties. One of these properties is solubility, which affects the hygroscopic growth and cloud condensation nucleus (CCN) activation of the organic particles. We investigate the hygroscopic behaviour of complex organic aerosols accounting for the distribution of solubilities present in these mixtures. We use the SPARC method to estimate the solubility distributions of isoprene (IP) and monoterpene (MT) SOA based on their chemical composition, as predicted by the Master Chemical Mechanism (MCM). Combining these solubility distributions with the adsorption theory along with the non-ideal behaviour of organic mixtures, we predict the expected hygroscopic growth factors (HGFs), CCN activation behaviour and the related hygroscopicity parameters kappa for these mixtures. The predictions are compared to laboratory measurements as well as field data from MT- and IP-dominated measurement sites. The predicted solubility distributions do a good job in explaining the water uptake of these two mixture types at high relative humidities (RH around 90%), as well as their CCN activation - including the potential differences between the kappa values derived from HGF vs. CCN data. At lower relative humidities, however, the observed water uptake is higher than predicted on solubility alone, particularly for the MT-derived SOA. The data from the low RHs are further

  3. Does FDG uptake measure proliferative activity of human cancer cells? In vitro comparison with DNA flow cytometry and tritiated thymidine uptake.

    PubMed

    Higashi, K; Clavo, A C; Wahl, R L

    1993-03-01

    The relationship between 3H-2-fluoro-2-deoxy-D-glucose (FDG) uptake and the proliferative rate of a human ovarian adenocarcinoma cell line (HTB77IP3) was examined in vitro. HTB77IP3 cells were plated and allowed to grow through lag, exponential and plateau phases. Proliferative rate assessed by DNA flow cytometry and 3H-thymidine incorporation was highest in the lag phase and fell significantly as the cells progressed from the exponential through plateau phases. By DNA flow cytometry, the proliferation index (% of S+G2/M phase cells) fell from 65% to 23%. Thymidine uptake per cell also declined, by 82%, from lag to plateau phase. By contrast, 3H-FDG uptake per cell was largely unchanged as the cells progressed through the cell growth cycle. Total 3H-FDG uptake was strongly correlated with the number of viable cancer cells present (r = 0.957). Total thymidine uptake, however, substantially underestimated the number of viable cancer cells present. These in vitro differences in tracer uptake suggest that in this adenocarcinoma cell line, FDG measures a substantially different parameter (viable cell number) than thymidine (proliferative rate) and that these differences may result in disparate findings on PET imaging of cancers using these two tracers. Our data for this in vitro system indicate that FDG uptake does not relate to the proliferative activity of cancer cells. However, FDG uptake is strongly related to the number of viable tumor cells.

  4. Mechanisms of cellular uptake, intracellular transportation, and degradation of CIGB-300, a Tat-conjugated peptide, in tumor cell lines.

    PubMed

    Benavent Acero, Fernando R; Perera Negrin, Yasser; Alonso, Daniel F; Perea, Silvio E; Gomez, Daniel E; Farina, Hernán G

    2014-06-02

    CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate's phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.

  5. Study of 223Ra uptake mechanism by Fe3O4 nanoparticles: towards new prospective theranostic SPIONs

    NASA Astrophysics Data System (ADS)

    Mokhodoeva, Olga; Vlk, Martin; Málková, Eva; Kukleva, Ekaterina; Mičolová, Petra; Štamberg, Karel; Šlouf, Miroslav; Dzhenloda, Rustam; Kozempel, Ján

    2016-10-01

    The use of superparamagnetic iron oxide nanoparticles ( SPIONs) and radiolabelled nanoparticles ( NPs) has grown considerably over the recent years, and the SPIONs labelled with medicinal radionuclides offer new opportunities in multimodal diagnostics and in the drug-delivery systems for targeted alpha-particle therapy (TAT) driven by magnetic field gradient or by biologically active moieties bound on NPs shell. However, the mechanisms of NPs radiolabelling are not studied substantially and still remain unclear, even though the way of label attachment directly implies the stability of the label-nanoparticle construct. Since the 223Ra was the first clinically approved alpha-emitter, it is a promising nuclide for further development of its targeted carriers. We report here on the study of 223Ra uptake by the Fe3O4 SPIONs, together with an attempt to propose the 223Ra uptake mechanism by the Fe3O4 NPs in the presence of a phosphate buffer a typical formulation medium, under the pseudo-equilibrium conditions. Further, the in vitro stability tests of the prepared [223Ra]Fe3O4 NPs were performed to estimate the 223Ra label stability. The potential use of 223Ra-labelled SPIONs in theranostic applications is also discussed.

  6. Fetal programming of perivenous glucose uptake reveals a regulatory mechanism governing hepatic glucose output during refeeding.

    PubMed

    Murphy, Helena C; Regan, Gemma; Bogdarina, Irina G; Clark, Adrian J L; Iles, Richard A; Cohen, Robert D; Hitman, Graham A; Berry, Colin L; Coade, Zoe; Petry, Clive J; Burns, Shamus P

    2003-06-01

    Increased hepatic gluconeogenesis maintains glycemia during fasting and has been considered responsible for elevated hepatic glucose output in type 2 diabetes. Glucose derived periportally via gluconeogenesis is partially taken up perivenously in perfused liver but not in adult rats whose mothers were protein-restricted during gestation (MLP rats)-an environmental model of fetal programming of adult glucose intolerance exhibiting diminished perivenous glucokinase (GK) activity. We now show that perivenous glucose uptake rises with increasing glucose concentration (0-8 mmol/l) in control but not MLP liver, indicating that GK is flux-generating. The data demonstrate that acute control of hepatic glucose output is principally achieved by increasing perivenous glucose uptake, with rising glucose concentration during refeeding, rather than by downregulation of gluconeogenesis, which occurs in different hepatocytes. Consistent with these observations, glycogen synthesis in vivo commenced in the perivenous cells during refeeding, MLP livers accumulating less glycogen than controls. GK gene transcription was unchanged in MLP liver, the data supporting a recently proposed posttranscriptional model of GK regulation involving nuclear-cytoplasmic transport. The results are pertinent to impaired regulation of hepatic glucose output in type 2 diabetes, which could arise from diminished GK-mediated glucose uptake rather than increased gluconeogenesis.

  7. Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media.

    PubMed

    Albro, Michael B; Li, Roland; Banerjee, Rajan E; Hung, Clark T; Ateshian, Gerard A

    2010-08-26

    Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602-614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose-dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R(2)=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix.

  8. Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media

    PubMed Central

    Albro, Michael B.; Li, Roland; Banerjee, Rajan E.; Hung, Clark T.; Ateshian, Gerard A.

    2010-01-01

    Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602–614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose–dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R2=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix. PMID:20553797

  9. PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI

    PubMed Central

    Toh, Sue-Anne; Millar, John S.; Billheimer, Jeffrey; Fuki, Ilia; Naik, Snehal U.; Macphee, Colin; Walker, Max; Rader, Daniel J.

    2011-01-01

    PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/3H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change 3H-tracer plasma appearance, but surprisingly decreased fecal 3H-free sterol excretion by 43% (P < 0.01) over 48 h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with 3H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived 3H-CE uptake by adipose tissue (P < 0.005) with concomitant 22% decrease in HDL derived 3H-CE uptake by the liver (P < 0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived 3H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands. PMID:21291868

  10. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  11. A minimal model for the mitochondrial rapid mode of Ca²+ uptake mechanism.

    PubMed

    Bazil, Jason N; Dash, Ranjan K

    2011-01-01

    Mitochondria possess a remarkable ability to rapidly accumulate and sequester Ca²⁺. One of the mechanisms responsible for this ability is believed to be the rapid mode (RaM) of Ca²⁺ uptake. Despite the existence of many models of mitochondrial Ca²⁺ dynamics, very few consider RaM as a potential mechanism that regulates mitochondrial Ca²⁺ dynamics. To fill this gap, a novel mathematical model of the RaM mechanism is developed herein. The model is able to simulate the available experimental data of rapid Ca²⁺ uptake in isolated mitochondria from both chicken heart and rat liver tissues with good fidelity. The mechanism is based on Ca²⁺ binding to an external trigger site(s) and initiating a brief transient of high Ca²⁺ conductivity. It then quickly switches to an inhibited, zero-conductive state until the external Ca²⁺ level is dropped below a critical value (∼100-150 nM). RaM's Ca²⁺- and time-dependent properties make it a unique Ca²⁺ transporter that may be an important means by which mitochondria take up Ca²⁺ in situ and help enable mitochondria to decode cytosolic Ca²⁺ signals. Integrating the developed RaM model into existing models of mitochondrial Ca²⁺ dynamics will help elucidate the physiological role that this unique mechanism plays in mitochondrial Ca²⁺-homeostasis and bioenergetics.

  12. Effects of high NH+4 on K+ uptake, culm mechanical strength and grain filling in wheat

    PubMed Central

    Kong, Lingan; Sun, Mingze; Wang, Fahong; Liu, Jia; Feng, Bo; Si, Jisheng; Zhang, Bin; Li, Shengdong; Li, Huawei

    2014-01-01

    It is well established that a high external NH+4 concentration depresses many processes in plant development, but the underlying mechanisms are still not well understood. To determine whether the negative effects of high levels of NH+4 are related to competitive cation uptake, wheat was grown in a field with moderate (18 g N m−2) and high (30 g N m−2) supplies of NH+4 in the presence or absence of additional K+ (6 g K2O m−2) to examine culm mechanical strength, the main components of the vascular bundle, nitrogen (N) remobilization and the grain-filling rate. The results indicated that an excessive supply of NH+4 significantly decreased culm mechanical strength, the cellulose and lignin contents of vascular bundles, the N remobilization efficiency (NRE) and the grain-filling rate compared with a moderate level of NH+4. The additional provision of K+ considerably alleviated these negative effects of high NH+4, resulting in a 19.41–26.95% increase in culm mechanical strength during grain filling and a 34.59% increase in the NRE. An assay using the scanning ion-selective electrode technique (SIET) showed that the net rate of transmembrane K+ influx decreased by 84.62%, and measurements using flame photometry demonstrated that the K+ content decreased by 36.13% in wheat plants subjected to high NH+4. This study indicates that the effects of high NH+4 on culm mechanical strength, cellulose and lignin contents, the NRE and the grain-filling rate are probably associated with inhibition of K+ uptake in wheat. PMID:25566278

  13. Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.

    PubMed

    Kleemann, Robert; Grell, Matthias; Mischke, Ralf; Zimmermann, Gudrun; Bernhagen, Jürgen

    2002-03-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target

  14. OsPT2, a phosphate transporter, is involved in the active uptake of selenite in rice.

    PubMed

    Zhang, Lianhe; Hu, Bin; Li, Wei; Che, Ronghui; Deng, Kun; Li, Hua; Yu, Feiyan; Ling, Hongqing; Li, Youjun; Chu, Chengcai

    2014-03-01

    • Selenite is a predominant form of selenium (Se) available to plants, especially in anaerobic soils, but the molecular mechanism of selenite uptake by plants is not well understood. • ltn1, a rice mutant previously shown to have increased phosphate (Pi) uptake, was found to exhibit higher selenite uptake than the wild-type in both concentration- and time-dependent selenite uptake assays. Respiratory inhibitors significantly inhibited selenite uptake in the wildtype and the ltn1 mutant, indicating that selenite uptake was coupled with H(+) and energy-dependent. Selenite uptake was greatly enhanced under Pi-starvation conditions, suggesting that Pi transporters are involved in selenite uptake. • OsPT2, the most abundantly expressed Pi transporter in the roots, is also significantly up-regulated in ltn1 and dramatically induced by Pi starvation. OsPT2-overexpressing and knockdown plants displayed significantly increased and decreased rates of selenite uptake, respectively, suggesting that OsPT2 plays a crucial role in selenite uptake. Se content in rice grains also increased significantly in OsPT2-overexpressing plants. • These data strongly demonstrate that selenite and Pi share similar uptake mechanisms and that OsPT2 is involved in selenite uptake, which provides a potential strategy for breeding Se-enriched rice varieties.

  15. Rapid adaptation of activated sludge bacteria into a glycogen accumulating biofilm enabling anaerobic BOD uptake.

    PubMed

    Hossain, Md Iqbal; Paparini, Andrea; Cord-Ruwisch, Ralf

    2017-03-01

    Glycogen accumulating organisms (GAO) are known to allow anaerobic uptake of biological oxygen demand (BOD) in activated sludge wastewater treatment systems. In this study, we report a rapid transition of suspended activated sludge biomass to a GAO dominated biofilm by selective enrichment using sequences of anaerobic loading followed by aerobic exposure of the biofilm to air. The study showed that within eight weeks, a fully operational, GAO dominated biofilm had developed, enabling complete anaerobic BOD uptake at a rate of 256mg/L/h. The oxygen uptake by the biofilm directly from the atmosphere had been calculated to provide significant energy savings. This study suggests that wastewater treatment plant operators can convert activated sludge systems readily into a "passive aeration" biofilm that avoids costly oxygen transfer to bulk wastewater solution. The described energy efficient BOD removal system provides an opportunity to be coupled with novel nitrogen removal processes such as anammox.

  16. Mechanically Active Electrospun Materials

    NASA Astrophysics Data System (ADS)

    Robertson, Jaimee M.

    Electrospinning, a technique used to fabricate small diameter polymer fibers, has been employed to develop unique, active materials falling under two categories: (1) shape memory elastomeric composites (SMECs) and (2) water responsive fiber mats. (1) Previous work has characterized in detail the properties and behavior of traditional SMECs with isotropic fibers embedded in an elastomer matrix. The current work has two goals: (i) characterize laminated anisotropic SMECs and (ii) develop a fabrication process that is scalable for commercial SMEC manufacturing. The former ((i)) requires electrospinning aligned polymer fibers. The aligned fibers are similarly embedded in an elastomer matrix and stacked at various fiber orientations. The resulting laminated composite has a unique response to tensile deformation: after stretching and releasing, the composite curls. This curling response was characterized based on fiber orientation. The latter goal ((ii)) required use of a dual-electrospinning process to simultaneously electrospin two polymers. This fabrication approach incorporated only industrially relevant processing techniques, enabling the possibility of commercial application of a shape memory rubber. Furthermore, the approach had the added benefit of increased control over composition and material properties. (2) The strong elongational forces experienced by polymer chains during the electrospinning process induce molecular alignment along the length of electrospun fibers. Such orientation is maintained in the fibers as the polymer vitrifies. Consequently, residual stress is stored in electrospun fiber mats and can be recovered by heating through the polymer's glass transition temperature. Alternatively, the glass transition temperature can be depressed by introducing a plasticizing agent. Poly(vinyl acetate) (PVAc) is plasticized by water, and its glass transition temperature is lowered below room temperature. Therefore, the residual stress can be relaxed at room

  17. Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout (Oncorhynchus mykiss): evidence for a Na+ coupled uptake mechanism.

    PubMed

    Rubino, Julian G; Zimmer, Alex M; Wood, Chris M

    2015-05-01

    In vitro gut sac experiments were performed on freshwater and 60% seawater acclimated trout (Oncorhynchus mykiss) under treatments designed to discern possible mechanisms of intestinal ammonia transport. Seawater acclimation increased ammonia flux rate into the serosal saline (Jsamm) in the anterior intestine, however it did not alter Jsamm in the mid- or posterior intestine suggesting similar mechanisms of ammonia handling in freshwater and seawater fish. Both fluid transport rate (FTR) and Jsamm were inhibited in response to basolateral ouabain treatment, suggesting a linkage of ammonia uptake to active transport, possibly coupled to fluid transport processes via solvent drag. Furthermore, decreases in FTR and Jsamm caused by low Na(+) treatment indicated a Na(+) linked transport mechanism. Mucosal bumetanide (10(-4) M) had no impact on FTR, yet decreased Jsamm in the anterior and mid-intestine, suggesting NH4(+) substitution for K(+) on an apical NKCC, and at least a partial uncoupling of ammonia transport from fluid transport. Additional treatments (amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), phenamil, bafilomycin, 4',6-diamidino-2-phenylindole (DAPI), high sodium) intended to disrupt alternative routes of Na(+) uptake yielded no change in FTR or Jsamm, suggesting the absence of direct competition between Na(+) and ammonia for transport. Finally, [(14)C]methylamine permeability (PMA) measurements indicated the likely presence of an intestinal Rh-mediated ammonia transport system, as increasing NH4Cl (0, 1, 5 mmol l(-1)) concentrations reduced PMA, suggesting competition for transport through Rh proteins. Overall, the data presented in this paper provide some of the first insights into mechanisms of teleost intestinal ammonia transport.

  18. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

    PubMed Central

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  19. Active auditory mechanics in mosquitoes.

    PubMed Central

    Göpfert, M. C.; Robert, D.

    2001-01-01

    In humans and other vertebrates, hearing is improved by active contractile properties of hair cells. Comparable active auditory mechanics is now demonstrated in insects. In mosquitoes, Johnston's organ transduces sound-induced vibrations of the antennal flagellum. A non-muscular 'motor' activity enhances the sensitivity and tuning of the flagellar mechanical response in physiologically intact animals. This motor is capable of driving the flagellum autonomously, amplifying sound-induced vibrations at specific frequencies and intensities. Motor-related electrical activity of Johnston's organ strongly suggests that mosquito hearing is improved by mechanoreceptor motility. PMID:11270428

  20. The specific requirement for sodium chloride for the active uptake of l-glutamate by Halobacterium salinarium

    PubMed Central

    Stevenson, J.

    1966-01-01

    1. Uptake of l-glutamate by Halobacterium salinarium is dependent on high concentrations of sodium chloride in the environment. When the sodium chloride is replaced by isomolar concentrations of potassium chloride, sodium acetate or potassium acetate, only negligible uptake occurs. 2. Most of the glutamate taken up can be shown to be in the cells in the free state and at a concentration of at least 50 times that in the medium. Sodium chloride is therefore required for an active transport of the glutamate into the cells. 3. The question whether sodium chloride is essential for the actual migration of glutamate across the cell envelope or for the mechanism supplying energy for this migration is discussed on the basis of experiments on endogenous respiration and with inhibitors. PMID:5947144

  1. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization

    PubMed Central

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  2. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

    PubMed

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  3. In vitro Characterization of Uptake Mechanism of L-[methyl-3H]-methionine in Hepatocellular Carcinoma

    PubMed Central

    Kuang, Yu; Wang, Fangjing; Corn, David J.; Tian, Haibin; Lee, Zhenghong

    2015-01-01

    Purpose Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-11C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-11C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-11C]-Met in HCC cells. Procedures A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-3H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Results Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na+ dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-3H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of

  4. Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

    PubMed

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Pertíñez, Samuel Núñez; Busto, Jon V; Martín, César; Alagia, Adele; Goñi, Félix M; Eritja, Ramón; Alkorta, Itziar

    2017-01-01

    The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

  5. The Janus kinase 2 inhibitor fedratinib inhibits thiamine uptake: a putative mechanism for the onset of Wernicke's encephalopathy.

    PubMed

    Zhang, Qiang; Zhang, Yan; Diamond, Sharon; Boer, Jason; Harris, Jennifer J; Li, Yu; Rupar, Mark; Behshad, Elham; Gardiner, Christine; Collier, Paul; Liu, Phillip; Burn, Timothy; Wynn, Richard; Hollis, Gregory; Yeleswaram, Swamy

    2014-10-01

    The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

  6. Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation.

    PubMed

    Naimi, Madina; Vlavcheski, Filip; Murphy, Brennan; Hudlicky, Tomas; Tsiani, Evangelia

    2017-01-01

    Compounds that increase the activity of the energy sensor AMP-activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [(3) H]-2-deoxy-d-glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post-hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μmol/L CA a response (226 ± 9.62% of control, P=.001), similar to maximum insulin (201 ± 7.86% of control, P=.001) and metformin (213 ± 10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.

  7. Mechanisms of slower nitric oxide uptake by red blood cells and other hemoglobin-containing vesicles.

    PubMed

    Azarov, Ivan; Liu, Chen; Reynolds, Hannah; Tsekouras, Zaharo; Lee, Janet S; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2011-09-23

    Nitric oxide (NO) acts as a smooth muscle relaxation factor and plays a crucial role in maintaining vascular homeostasis. NO is scavenged rapidly by hemoglobin (Hb). However, under normal physiological conditions, the encapsulation of Hb inside red blood cells (RBCs) significantly retards NO scavenging, permitting NO to reach the smooth muscle. The rate-limiting factors (diffusion of NO to the RBC surface, through the RBC membrane or inside of the RBC) responsible for this retardation have been the subject of much debate. Knowing the relative contribution of each of these factors is important for several reasons including optimization of the development of blood substitutes where Hb is contained within phospholipid vesicles. We have thus performed experiments of NO uptake by erythrocytes and microparticles derived from erythrocytes and conducted simulations of these data as well as that of others. We have included extracellular diffusion (that is, diffusion of the NO to the membrane) and membrane permeability, in addition to intracellular diffusion of NO, in our computational models. We find that all these mechanisms may modulate NO uptake by membrane-encapsulated Hb and that extracellular diffusion is the main rate-limiting factor for phospholipid vesicles and erythrocytes. In the case of red cell microparticles, we find a major role for membrane permeability. These results are consistent with prior studies indicating that extracellular diffusion of several gas ligands is also rate-limiting for erythrocytes, with some contribution of a low membrane permeability.

  8. Esterification of Ginsenoside Rh2 Enhanced Its Cellular Uptake and Antitumor Activity in Human HepG2 Cells.

    PubMed

    Chen, Fang; Deng, Ze-Yuan; Zhang, Bing; Xiong, Zeng-Xing; Zheng, Shi-Lian; Tan, Chao-Li; Hu, Jiang-Ning

    2016-01-13

    Our previous research had indicated that the octyl ester derivative of ginsenoside Rh2 (Rh2-O) might have a higher bioavailability than Rh2 in the Caco-2 cell line. The aim of this study was to investigate the cellular uptake and antitumor effects of Rh2-O in human HepG2 cells as well as its underlying mechanism compared with Rh2. Results showed that Rh2-O exhibited a higher cellular uptake (63.24%) than Rh2 (36.76%) when incubated with HepG2 cells for 24 h. Rh2-O possessed a dose- and time-dependent inhibitory effect against the proliferation of HepG2 cells. The IC50 value of Rh2-O for inhibition of HepG2 cell proliferation was 20.15 μM, which was roughly half the value of Rh2. Rh2-O induced apoptosis of HepG2 cells through a mitochondrial-mediated intrinsic pathway. In addition, the accumulation of ROS was detected in Rh2-O-treated HepG2 cells, which participated in the apoptosis of HepG2 cells. Conclusively, the findings above all suggested that Rh2-O as well as Rh2 inducing HepG2 cells apoptosis might involve similar mechanisms; however, Rh2-O had better antitumor activities than Rh2, probably due to its higher cellular uptake.

  9. Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.

    PubMed

    Siska, Peter J; Kim, Bumki; Ji, Xiangming; Hoeksema, Megan D; Massion, Pierre P; Beckermann, Kathryn E; Wu, Jianli; Chi, Jen-Tsan; Hong, Jiyong; Rathmell, Jeffrey C

    2016-11-01

    T and B lymphocytes undergo metabolic re-programming upon activation that is essential to allow bioenergetics, cell survival, and intermediates for cell proliferation and function. To support changes in the activity of signaling pathways and to provide sufficient and necessary intracellular metabolites, uptake of extracellular nutrients increases sharply with metabolic re-programming. One result of increased metabolic activity can be reactive oxygen species (ROS), which can be toxic when accumulated in excess. Uptake of cystine allows accumulation of cysteine that is necessary for glutathione synthesis and ROS detoxification. Cystine uptake is required for T cell activation and function but measurements based on radioactive labeling do not allow analysis on single cell level. Here we show the critical role for cystine uptake in T cells using a method for measurement of cystine uptake using a novel CystineFITC probe. T cell receptor stimulation lead to upregulation of the cystine transporter xCT (SLC7a11) and increased cystine uptake in CD4+ and CD8+ human T cells. Similarly, lipopolysaccharide stimulation increased cystine uptake in human B cells. The CystineFITC probe was not toxic and could be metabolized to prevent cystine starvation induced cell death. Furthermore, blockade of xCT or competition with natural cystine decreased uptake of CystineFITC. CystineFITC is thus a versatile tool that allows measurement of cystine uptake on single cell level and shows the critical role for cystine uptake for T cell ROS regulation and activation.

  10. Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

    SciTech Connect

    Muñoz, Alexandra; Costa, Max

    2012-04-01

    Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses — most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties.

  11. Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

    PubMed Central

    Muñoz, Alexandra; Costa, Max

    2012-01-01

    Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses - most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980’s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties. PMID:22206756

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

    PubMed

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

    2016-01-01

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

  13. Assessment of glutamine synthetase activity by [13N]ammonia uptake in living rat brain.

    PubMed

    Momosaki, Sotaro; Ito, Miwa; Tonomura, Misato; Abe, Kohji

    2015-01-01

    Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

  14. All Physical Activity May Not Be Associated with a Lower Likelihood of Adolescent Smoking Uptake

    PubMed Central

    Audrain-McGovern, Janet; Rodriguez, Daniel

    2015-01-01

    Objective Research has documented that physical activity is associated with a lower risk of adolescent smoking uptake, yet it is unclear whether this relationship exists for all types of physical activity. We sought to determine whether certain types of physical activity are associated with a decreased or an increased risk of adolescent smoking uptake. Methods In this longitudinal cohort study, adolescents (n=1,356) were surveyed every six months for four years (age 14 – 18 years old). Smoking and physical activity were measured at each of the eight time-points. Physical activity that was negatively associated with smoking across the eight waves was considered positive physical activities (i.e., PPA; linked to not smoking such as racquet sports, running, and swimming laps). Physical activity that was positively associated with smoking across the eight waves were considered negative physical activities (i.e., NPA; linked to smoking such as skating, walking, bicycling, sport fighting, and competitive wrestling). Results Associative Processes Latent Growth Curve Modeling revealed that each 30-minute increase in NPA per week at baseline was associated with a 4-fold increased odds of smoking progression (OR=4.10, 95% CI=2.14, 7.83). By contrast, each 30-minute increase in PPA at baseline was associated with a 51% decrease in the odds of smoking progression (OR=.49, 95% CI=.25, .93). Conclusions The type of physical activity that an adolescent engages appears to be important for the uptake of cigarette smoking among adolescents. These associative relationships warrant consideration in interventions to increase overall physical activity and those promoting physical activity to prevent smoking uptake. PMID:26280377

  15. Ethanolic extract of Allium cepa stimulates glucose transporter typ 4-mediated glucose uptake by the activation of insulin signaling.

    PubMed

    Gautam, Sudeep; Pal, Savita; Maurya, Rakesh; Srivastava, Arvind K

    2015-02-01

    The present work was undertaken to investigate the effects and the molecular mechanism of the standardized ethanolic extract of Allium cepa (onion) on the glucose transport for controlling diabetes mellitus. A. cepa stimulates glucose uptake by the rat skeletal muscle cells (L6 myotubes) in both time- and dose-dependent manners. This effect was shown to be mediated by the increased translocation of glucose transporter typ 4 protein from the cytoplasm to the plasma membrane as well as the synthesis of glucose transporter typ 4 protein. The effect of A. cepa extract on glucose transport was stymied by wortmannin, genistein, and AI½. In vitro phosphorylation analysis revealed that, like insulin, A. cepa extract also enhances the tyrosine phosphorylation of the insulin receptor-β, insulin receptor substrate-1, and the serine phosphorylation of Akt under both basal and insulin-stimulated conditions without affecting the total amount of these proteins. Furthermore, it is also shown that the activation of Akt is indispensable for the A. cepa-induced glucose uptake in L6 myotubes. Taken together, these findings provide ample evidence that the ethanolic extract of A. cepa stimulates glucose transporter typ 4 translocation-mediated glucose uptake by the activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt dependent pathway.

  16. The effect of nitrite on aerobic phosphate uptake and denitrifying activity of phosphate-accumulating organisms.

    PubMed

    Yoshida, Y; Takahashi, K; Saito, T; Tanaka, K

    2006-01-01

    An anaerobic/aerobic/anoxic/aerobic sequencing batch reactor (SBR) was operated with municipal wastewater to investigate the effect of nitrite on biological phosphorus removal (BPR). When nitrite accumulated, aerobic phosphate uptake activity significantly decreased and, in case of hard exposure to nitrite, BPR severely deteriorated. The interesting observation was that the relative anoxic activity of phosphate accumulating organisms (PAOs) increased after nitrite exposure. Moreover batch tests of aerobic phosphate uptake in the presence/absence of nitrite indicated that PAOs with the higher relative anoxic activity are less sensitive to nitrite exposure. From these results, we concluded that BPR is sensitive to nitrite exposure, but BPR containing PAOs with the higher relative anoxic activity is possibly more stable against nitrite than BPR containing PAOs with the lower relative anoxic activity.

  17. Investigation of biomimetic shear stress on cellular uptake and mechanism of polystyrene nanoparticles in various cancer cell lines.

    PubMed

    Kang, Taehee; Park, Chulhun; Lee, Beom-Jin

    2016-12-01

    Cancer cells in the tumor microenvironment are affected by fluid shear stress generated by blood flow in the vascular microenvironment and interstitial flows in the tumor microenvironment. Thus, we investigated how fluidic shear stress affects cellular uptake as well as the endocytosis mechanism of nanoparticles using a biomimetic microfluidic system that mimics the human dynamic environment. Positively charged amino-modified polystyrene nanoparticles (PSNs) at 100 μg/mL were delivered to cancer cells under static and biomimetic dynamic conditions (0.5 dyne/cm(2)). Additionally, the experiment was done in the presence of endocytosis inhibitors specific for one of the endocytosis pathways. To evaluate cellular uptake of cationic PSNs, the fluorescence intensity of cationic PSNs in cancer cells was measured by flow cytometer and fluorescence images were taken using confocal laser scanning microscopy. Cancer cells in dynamic conditions exhibited higher cellular uptake of PSNs and showed different cellular uptake mechanisms compared with those in static conditions. From these results, it suggested that biomimetic dynamic conditions stimulated specific endocytosis and prompted cellular uptake. It was also important to consider fluidic shear stress as one of the critical factors because cellular uptake and drug delivery could play a key role in cancer cells and metastasis.

  18. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review.

    PubMed

    Huvenne, Hanneke; Smagghe, Guy

    2010-03-01

    RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans' SID-1 protein and an 'alternative' endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.

  19. Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis

    PubMed Central

    Whitson, Jeremy A.; Sell, David R.; Goodman, Michael C.; Monnier, Vincent M.; Fan, Xingjun

    2016-01-01

    Purpose Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain >1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens. Methods Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Wild-type (WT) lenses could accumulate GSH from γ-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had >1 mM GSH, whereas aqueous had <20 μM GSH. Testing physiologically relevant GSH concentrations for uptake in vivo, we found that both LEGSKO and WT lenses could obtain GSH from the vitreous but not from the aqueous. Vitreous rapidly accumulated GSH from the circulation, and depletion of circulating GSH reduced vitreous but not aqueous GSH. Conclusions The above data provide, for the first time, evidence for the existence of dual mechanisms of GSH uptake into the lens, one mechanism being a passive, high-flux transport through the vitreous exposed side of the lens versus an active, carrier-mediated uptake mechanism at the anterior of the lens. PMID:27472077

  20. Albumin and Uptake of Drugs in Cells: Additional Validation Exercises of a Recently Published Equation that Quantifies the Albumin-Facilitated Uptake Mechanism(s) in Physiologically Based Pharmacokinetic and Pharmacodynamic Modeling Research.

    PubMed

    Poulin, Patrick; Haddad, Sami

    2015-12-01

    The impact of albumin concentration on the uptake of drugs in cells might involve mechanisms going beyond the free drug concentration hypothesis. Proceeding from the assumption that both the unbound and protein-bound drug fractions can be available for uptake, several authors have argued that the uptake of highly bound drugs in cells might be driven mainly by the albumin-facilitated uptake mechanism(s). Hence, a novel approach quantifying the additional contribution of the protein-bound drug complex and pH gradient effect in diverse in vitro-to-in vivo extrapolation (IVIVE) procedures of drug uptake and clearance has been proposed and extensively validated by Poulin et al. (2015. J Pharm Sci. Epub ahead of print); this approach consisted of replacing the unbound fraction in plasma (fup ) with an adjusted fup value (fup-adjusted ). After a second review of literature, the objective of the present study was to perform further validation exercises of the concept of fup-adjusted by using additional case examples of IVIVEs that covered diverse drug properties and experimental settings with varied albumin concentrations (e.g., perfused liver, isolated and suspended hepatocytes, and cultured cells overexpressing transporters). Again, the novel IVIVE method based on fup-adjusted was the best-performing prediction method of the uptake rate (or clearance) as a function of protein binding compared with the conventional method based on the fup theory (absolute average fold error of 1.4 vs. 7.4). Therefore, the present study confirms the utility of fup-adjusted compared with fup in IVIVE procedures for drugs highly bound to albumin, and the improvement was observed particularly in the higher range of albumin concentrations. From these findings, we may conclude that uptake of these drugs in cells is primarily driven by the albumin-bound form. Consequently, it is suggested to estimate the uptake kinetic parameters with cell-based assays incubated in 100% human serum or to make a

  1. Glabridin induces glucose uptake via the AMP-activated protein kinase pathway in muscle cells.

    PubMed

    Sawada, Keisuke; Yamashita, Yoko; Zhang, Tianshun; Nakagawa, Kaku; Ashida, Hitoshi

    2014-08-05

    The present study demonstrates that glabridin, a prenylated isoflavone in licorice, stimulates glucose uptake through the adenosine monophosphate-activated protein kinase (AMPK) pathway in L6 myotubes. Treatment with glabridin for 4h induced glucose uptake in a dose-dependent manner accompanied by the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane. Glabridin needed at least 4h to increase glucose uptake, while it significantly decreased glycogen and increased lactic acid within 15 min. Pharmacological inhibition of AMPK by Compound C suppressed the glabridin-induced glucose uptake, whereas phosphoinositide 3-kinase and Akt inhibition by LY294002 and Akt1/2 inhibitor, respectively, did not. Furthermore, glabridin induced AMPK phosphorylation, and siRNA for AMPK completely abolished glabridin-induced glucose uptake. We confirmed that glabridin-rich licorice extract prevent glucose intolerance accompanied by the AMPK-dependent GLUT4 translocation in the plasma membrane of mice skeletal muscle. These results indicate that glabridin may possess a therapeutic effect on metabolic disorders, such as diabetes and hyperglycemia, by modulating glucose metabolism through AMPK in skeletal muscle cells.

  2. Relationship between uptake of mercury vapor by mushrooms and its catalase activity

    SciTech Connect

    Ogata, M.; Kenmotsu, K.; Hirota, N.; Naito, M.

    1981-12-01

    The uptake of mercury vapor by mushrooms (Shiitake) artifically grown on an oak tree and the uptake in vitro by catalase extracts prepared from mushroom Hay Bacillus and spinach are reported. Mushrooms were exposed to 1.4 mg/Hg/cu m for 11 days. Measurement of total mercury was as previously described (Ogata et al. 1978, 1979). Levels in mushrooms ranged from 0.4 +/- 0.1 ..mu..g/g at 0.5 days to 4.6 +/- 0.2 ..mu..g/g at 10.5 days and steady-state thereafter. In in vitro studies Hy uptake by mushroom catalase extract was estimated by the perborate method. Uptake was found to parallel catalase activity and was inhibited by potassium cyanide, sodium azide, and 3-amino-1,2,4-triazole. Similar results were obtained with Hay Bacillus and spinach catalase extracts. Results suggest that the level of mercury in the mushroom can be used as an indicator of mercury pollution in the environment. It is also suggested that catalase has an important role in uptake of mercury vapor in the plant. 2 tables (JMT)

  3. Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel.

    PubMed

    Akopova, O V; Nosar, V I; Mankovskaya, I N; Sagach, V F

    2008-10-01

    The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.

  4. Constituents from Cistus salvifolius (Cistaceae) activate peroxisome proliferator-activated receptor-γ but not -δ and stimulate glucose uptake by adipocytes.

    PubMed

    Kühn, Claudia; Arapogianni, Niki Eliza; Halabalaki, Maria; Hempel, Jana; Hunger, Nicole; Wober, Jannette; Skaltsounis, Alexios Leandros; Vollmer, Günter

    2011-03-01

    A number of medicinal/culinary herbs have been reported to improve glucose metabolism and to yield hypoglycemic effects in patients with diabetes. Since stimulation of insulin sensitivity appears to be a potential mechanism, peroxisome proliferator-activated receptor (PPAR) γ is a likely target molecule for small lipophilic compounds derived from endogenous metabolism and nutrition. Functionally, PPAR γ integrates the control of energy, lipid, and glucose homeostasis. In addition, PPAR δ activity is involved in energy expenditure. Therefore the aim of this study was to investigate whether PPAR γ and PPAR δ as well as the stimulation of glucose uptake is activated by botanical products. CISTUS SALVIFOLIUS (Cistaceae) has been identified as a candidate botanical in a preliminary screening of extracts from medicinal plants of Greek flora. In a bioguided approach, crude extracts, fractions and in the end purified compounds have been evaluated for PPAR γ and PPAR δ specific activities using cell-based transactivation assays. Glucose uptake was measured by nonradioactive 2-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) uptake. Concerning PPAR γ several extracts induced reporter gene activity, and clear dose-response patterns (0.1-100 µg/mL) could be established in the case of the cyclohexane and dichloromethane extracts. Isolation of individual compounds from the cyclohexane extract revealed that at least 6 out of 7 compounds isolated were active with TRANS-cinnamic acid showing a clear dose-response pattern. In contrast, they were found to be inactive on PPAR δ. The same compounds, however, were also active in stimulating glucose uptake into 3T3-L1 adipocytes. In summary, the bioguided fractionation of CISTUS SALVIFOLIUS yields PPAR γ stimulating metabolites with differing chemical natures. In conclusion, PPAR γ represents a candidate molecule for the mediation of improvement of glucose metabolism by botanical/nutritional products.

  5. Endoplasmic reticulum potassium-hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes.

    PubMed

    Kuum, Malle; Veksler, Vladimir; Liiv, Joanna; Ventura-Clapier, Renee; Kaasik, Allen

    2012-02-01

    Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.

  6. Cellular uptake mechanisms of functionalised multi-walled carbon nanotubes by 3D electron tomography imaging

    NASA Astrophysics Data System (ADS)

    Al-Jamal, Khuloud T.; Nerl, Hannah; Müller, Karin H.; Ali-Boucetta, Hanene; Li, Shouping; Haynes, Peter D.; Jinschek, Joerg R.; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas; Porter, Alexandra E.

    2011-06-01

    Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed that MWNT-NH3+ were internalised in both phagocytic and non-phagocytic cells by any one of three mechanisms: (a) individually via membrane wrapping; (b) individually by direct membrane translocation; and (c) in clusters within vesicular compartments. At early time points following intracellular translocation, we noticed accumulation of nanotube material within various intracellular compartments, while a long-term (14-day) study using primary human macrophages revealed that MWNT-NH3+ were able to escape vesicular (phagosome) entrapment by translocating directly into the cytoplasm.Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed

  7. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

    NASA Astrophysics Data System (ADS)

    Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

    2015-07-01

    Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

  8. Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in Pectobacterium spp.

    PubMed

    Grinter, Rhys; Milner, Joel; Walker, Daniel

    2012-01-01

    In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.

  9. Arsenate Uptake by Calcite: Macroscopic and Spectroscopic Characterization of Adsorption and Incorporation Mechanisms

    SciTech Connect

    Alexandratos,V.; Elzinga, E.; Reeder, R.

    2007-01-01

    Batch uptake experiments and X-ray element mapping and spectroscopic techniques were used to investigate As(V) (arsenate) uptake mechanisms by calcite, including adsorption and coprecipitation. Batch sorption experiments in calcite-equilibrated suspensions (pH 8.3; PCO{sub 2} = 10{sup -3.5} atm) reveal rapid initial sorption to calcite, with sorption rate gradually decreasing with time as available sorption sites decrease. An As(V)-calcite sorption isotherm determined after 24 h equilibration exhibits Langmuir-like behavior up to As concentrations of 300 {mu}M. Maximum distribution coefficient values (K{sub d}), derived from a best fit to a Langmuir model, are {approx}190 L kg{sup -1}. Calcite single crystals grown in the presence of As(V) show well-developed rhombohedral morphology with characteristic growth hillocks on (10{bar 1}4) surfaces at low As(V) concentrations ({<=}5 {mu}M), but habit modification is evident at As(V) concentrations {>=}30 {mu}M in the form of macrostep development preferentially on the - vicinal surfaces of growth hillocks. Micro-X-ray fluorescence element mapping of (10{bar 1}4) surfaces shows preferential incorporation of As in the - vicinal faces relative to + vicinals. EXAFS fit results for both adsorption and coprecipitation samples confirm that As occurs in the 5+ oxidation state in tetrahedral coordination with oxygen, i.e., as arsenate. For adsorption samples, As(V) forms inner-sphere surface complexes via corner-sharing with Ca octahedra. As(V) coprecipitated with calcite substitutes in carbonate sites but with As off-centered, as indicated by two Ca shells, and with likely disruption of local structure. The results indicate that As(V) interacts strongly with the calcite surface, similar to often-cited analog phosphate, and uptake can occur via both adsorption and coprecipitation reactions. Therefore, calcite may be effective for partial removal of dissolved arsenate from aquatic and soil systems.

  10. Cell uptake mechanisms of PAMAM G4-FITC dendrimer in human myometrial cells

    NASA Astrophysics Data System (ADS)

    Oddone, Natalia; Zambrana, Ana I.; Tassano, Marcos; Porcal, Williams; Cabral, Pablo; Benech, Juan C.

    2013-07-01

    The high incidence and severity of diseases which involve smooth muscle dysfunction dictates the need of continued search for novel therapeutic strategies to treat these conditions. Dendrimers are branched macromolecules with multiple end-groups that can be functionalized for applications which include drug delivery. There is no data regarding the cellular uptake mechanisms used by dendrimers in smooth muscle human myometrial cells (HMC). Polyamidoamine G4 dendrimers were conjugated with fluorescein isothiocyanate (FITC) and the resulting conjugate (G4-FITC) was characterized using high-performance liquid chromatography, nuclear magnetic resonance, and atomic force microscopy. G4-FITC showed to have no significant effect on the primary culture HMC viability up to 48 h. HMC incubated with G4-FITC were analyzed by laser confocal microscopy. Peri-nuclear fluorescence distribution was observed at 5 h of incubation or more (24, 36, and 48 h). At 24 h, G4-FITC partially co-localized with lysotracker. Uptake of G4-FITC by HMC was slightly inhibited by filipin (8.0 ± 3.9 %) and significantly inhibited by chlorpromazine (63.5 ± 3.7 %). In non-electroporated HMC, G4-FITC was never observed inside the cell nucleus. Interestingly, we detected G4-FITC inside the nuclear domain of some electroporated cells. Thus, electroporation changed intracellular G4-FITC localization. Isolated nuclei of HMC incubated with G4-FITC showed fluorescence signal inside the nuclear domain. The results suggest that in HMC, G4-FITC is taken up by clathrin-mediated endocytosis with endosomal and lysosomal localization at 24 h. The combination of electroporation and dendrimers could be an interesting technology to electrotransfer drugs into smooth muscle cells cytosol and nuclei.

  11. Investigating the Uptake Mechanisms of Hydrogen Peroxide to Single and Polycrystalline Ice with a Novel Flow Tube System

    NASA Astrophysics Data System (ADS)

    Hong, Angela; Ammann, Markus; Bartels-Rausch, Thorsten

    2016-04-01

    Air-ice chemical interactions are important for describing the distribution and subsequent chemical fate of trace atmospheric gases within ice and snow and determining the oxidative capacities of these environments. The nature of this interaction is governed by a compound's physicochemical properties as well as ice microstructure. Hydrogen peroxide (H2O2), a reservoir of HOx radicals in the atmosphere and an important chromophore in snow and ice, is a trace gas that demonstrates complex uptake behaviour to frozen aqueous media by the reversible, fast adsorption to the air-ice interface, aggregation, and lateral interactions, and a slower process, ostensibly via uptake into the bulk. However, the exact mechanism and kinetics for the slow uptake of H2O2 and the size of this reservoir is unknown. It is important to describe and quantify this loss term, over environmentally-relevant timescales, accommodation of H2O2 into the bulk may be the dominant process which controls the composition and chemistry of the snow and overlying atmosphere. We hypothesize that the slow uptake of H2O2 occurs by diffusion into the grain boundaries of ice. To provide mechanistic insight to the macroscopic phenomenon of atmospheric gas uptake to ice, and discern various mechanisms including adsorption to air-ice interface and accommodation into the bulk through uptake into grain boundaries, we design, machine, and validate a novel flow reactor system featuring a Drilled Ice Flow Tube (DIFT). Our flow reactor system is uniquely suited to testing these uptake mechanisms: by controlling the degree of grain boundaries present in the DIFT (ie. monocrystalline or polycrystalline), we can directly observe the effect of the ice microstructure on the adsorptive and bulk uptake of trace atmospheric gases over long timescales (eg. on the order of hours). Here, we describe method development of the DIFT and demonstrate using polarised microscopy imagery that our experimental set-up allows for the direct

  12. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

    PubMed Central

    Gibbs, Peter E. M.; Lerner-Marmarosh, Nicole; Poulin, Amelia; Farah, Elie; Maines, Mahin D.

    2014-01-01

    Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. 290KYCCSRK, which increased IRK Vmax without changing Km, stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C292→A mutant did not stimulate glucose uptake; K296→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988–1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C1056, C1138, or C1234, were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.—Gibbs, P. E. M., Lerner-Marmarosh, N., Poulin, A., Farah, E., Maines, M. D. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor. PMID:24568842

  13. Tetrodotoxin-insensitive Na+ channel activator palytoxin inhibits tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Teraoka, K.; Azuma, M.; Oka, M.; Hamano, S. )

    1991-07-01

    The effects of the tetrodotoxin-insensitive Na+ channel activator palytoxin on both the secretion of endogenous catecholamines and the formation of 14C-catecholamines from (14C)tyrosine were examined using cultured bovine adrenal chromaffin cells. Palytoxin was shown to cause the stimulation of catecholamine secretion in a concentration-dependent manner. However, this toxin caused the reduction rather than the stimulation of 14C-catecholamine formation at the same concentrations. Palytoxin failed to cause any alteration in the activity of tyrosine hydroxylase prepared from bovine adrenal medulla. Furthermore, the uptake of (14C)tyrosine into the cells was shown to be inhibited by this toxin under the conditions in which the suppression of 14C-catecholamine formation was observed, and this inhibitory action on tyrosine uptake was closely correlated with that on catecholamine formation. The inhibitory action of palytoxin on tyrosine uptake into the cells was observed to be noncompetitive, and this effect was not altered by the removal of Na+ from the incubation mixture. These results suggest that palytoxin may be able to inhibit the uptake of (14C)tyrosine into the cells, resulting in the suppression of 14C-catecholamine formation, probably through its direct action on the plasma membranes of bovine adrenal chromaffin cells.

  14. Gallium scintigraphy demonstration of an appendiceal mucocele: a proposed mechanism of uptake

    SciTech Connect

    Alpert, L.; Friedman, R.

    1981-08-01

    An appendiceal mucocele demonstrated intense early avidity for Ga-67, despite the lack of inflammatory cells to account for the uptake. It is proposed that the acid mucopolysaccharide component of the mucus within the lumen and lining cells accounted for the uptake of the gallium ion, in a similar manner to the uptake of its analogue, the ferric ion, as demonstrated by intense staining of mucus by the colloidal iron technique.

  15. Molecular mechanisms for the regulation of insulin-stimulated glucose uptake by small guanosine triphosphatases in skeletal muscle and adipocytes.

    PubMed

    Satoh, Takaya

    2014-10-16

    Insulin is a hormone that regulates the blood glucose level by stimulating various physiological responses in its target tissues. In skeletal muscle and adipose tissue, insulin promotes membrane trafficking of the glucose transporter GLUT4 from GLUT4 storage vesicles to the plasma membrane, thereby facilitating the uptake of glucose from the circulation. Detailed mechanisms underlying insulin-dependent intracellular signal transduction for glucose uptake remain largely unknown. In this article, I give an overview on the recently identified signaling network involving Rab, Ras, and Rho family small guanosine triphosphatases (GTPases) that regulates glucose uptake in insulin-responsive tissues. In particular, the regulatory mechanisms for these small GTPases and the cross-talk between protein kinase and small GTPase cascades are highlighted.

  16. Whisking mechanics and active sensing.

    PubMed

    Bush, Nicholas E; Solla, Sara A; Hartmann, Mitra Jz

    2016-10-01

    We describe recent advances in quantifying the three-dimensional (3D) geometry and mechanics of whisking. Careful delineation of relevant 3D reference frames reveals important geometric and mechanical distinctions between the localization problem ('where' is an object) and the feature extraction problem ('what' is an object). Head-centered and resting-whisker reference frames lend themselves to quantifying temporal and kinematic cues used for object localization. The whisking-centered reference frame lends itself to quantifying the contact mechanics likely associated with feature extraction. We offer the 'windowed sampling' hypothesis for active sensing: that rats can estimate an object's spatial features by integrating mechanical information across whiskers during brief (25-60ms) windows of 'haptic enclosure' with the whiskers, a motion that resembles a hand grasp.

  17. Juglone disrupts root plasma membrane H+-ATPase activity and impairs water uptake, root respiration, and growth in soybean (Glycine max) and corn (Zea mays).

    PubMed

    Hejl, Angela M; Koster, Karen L

    2004-02-01

    Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

  18. Mechanisms for independent and combined effects of calorie restriction and acute exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats.

    PubMed

    Sharma, Naveen; Wang, Haiyan; Arias, Edward B; Castorena, Carlos M; Cartee, Gregory D

    2015-04-01

    Either calorie restriction [CR; consuming 60-65% of ad libitum (AL) intake] or acute exercise can independently improve insulin sensitivity in old age, but their combined effects on muscle insulin signaling and glucose uptake have previously been unknown. Accordingly, we assessed the independent and combined effects of CR (beginning at 14 wk old) and acute exercise (3-4 h postexercise) on insulin signaling and glucose uptake in insulin-stimulated epitrochlearis muscles from 30-mo-old rats. Either CR alone or exercise alone vs. AL sedentary controls induced greater insulin-stimulated glucose uptake. Combined CR and exercise vs. either treatment alone caused an additional increase in insulin-stimulated glucose uptake. Either CR or exercise alone vs. AL sedentary controls increased Akt Ser(473) and Akt Thr(308) phosphorylation. Combined CR and exercise further elevated Akt phosphorylation on both sites. CR alone, but not exercise alone, vs. AL sedentary controls significantly increased Akt substrate of 160 kDa (AS160) Ser(588) and Thr(642) phosphorylation. Combined CR and exercise did not further enhance AS160 phosphorylation. Exercise alone, but not CR alone, modestly increased GLUT4 abundance. Combined CR and exercise did not further elevate GLUT4 content. These results suggest that CR or acute exercise independently increases insulin-stimulated glucose uptake via overlapping (greater Akt phosphorylation) and distinct (greater AS160 phosphorylation for CR, greater GLUT4 for exercise) mechanisms. Our working hypothesis is that greater insulin-stimulated glucose uptake in the combined CR and exercise group vs. CR or exercise alone relies on greater Akt activation, leading to greater phosphorylation of one or more Akt substrates other than AS160.

  19. ReishiMax, mushroom based dietary supplement, inhibits adipocyte differentiation, stimulates glucose uptake and activates AMPK

    PubMed Central

    2011-01-01

    Background Obesity is a health hazard which is closely associated with various complications including insulin resistance, hypertension, dyslipidemia, atherosclerosis, type 2 diabetes and cancer. In spite of numerous preclinical and clinical interventions, the prevalence of obesity and its related disorders are on the rise demanding an urgent need for exploring novel therapeutic agents that can regulate adipogenesis. In the present study, we evaluated whether a dietary supplement ReishiMax (RM), containing triterpenes and polysaccharides extracted from medicinal mushroom Ganoderma lucidum, affects adipocyte differentiation and glucose uptake in 3T3-L1 cells. Methods 3T3-L1 pre-adipocytes were differentiated into adipocytes and treated with RM (0-300 μg/ml). Adipocyte differentiation/lipid uptake was evaluated by oil red O staining and triglyceride and glycerol concentrations were determined. Gene expression was evaluated by semi-quantitative RT-PCR and Western blot analysis. Glucose uptake was determined with [3H]-glucose. Results RM inhibited adipocyte differentiation through the suppresion of expression of adipogenic transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element binding element protein-1c (SREBP-1c) and CCAAT/enhancer binding protein-α (C/EBP-α). RM also suppressed expression of enzymes and proteins responsible for lipid synthesis, transport and storage: fatty acid synthase (FAS), acyl-CoA synthetase-1 (ACS1), fatty acid binding protein-4 (FABP4), fatty acid transport protein-1 (FATP1) and perilipin. RM induced AMP-activated protein kinase (AMPK) and increased glucose uptake by adipocytes. Conclusion Our study suggests that RM can control adipocyte differentiation and glucose uptake. The health benefits of ReishiMax warrant further clinical studies. PMID:21929808

  20. Facilitating Mitochondrial Calcium Uptake Improves Activation-Induced Cerebral Blood Flow and Behavior after mTBI

    PubMed Central

    Murugan, Madhuvika; Santhakumar, Vijayalakshmi; Kannurpatti, Sridhar S.

    2016-01-01

    Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca2+ overload. However, in the surviving cellular population, mitochondrial Ca2+ influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca2+ uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca2+ uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca2+ uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol

  1. Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes.

    PubMed

    Li, Weihua; Liang, Xiaojing; Zeng, Zhipeng; Yu, Kaizhen; Zhan, Shaopeng; Su, Qiang; Yan, Yinzhi; Mansai, Huseen; Qiao, Weitong; Yang, Qi; Qi, Zhongquan; Huang, Zhengrong

    2016-10-01

    Simvastatin,a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, is clinically used in the prevention and treatment of cardiovascular diseases. Numerous studies demonstrate that statins increase the risk of new-onset diabetes in long-term therapy, but mechanisms underpinning this effect are still unclear. Here, we investigated whether simvastatin inhibited the glucose uptake activity and the underlying mechanisms in C2C12 myotubes. Our studies showed that simvastatin significantly inhibited glucose uptake activity and GLUT4 translocation, whereas the effect was reversible with mevalonolactone (ML), which acts as an intermediate of cholesterol synthesis pathway. Mechanistically, the inhibition of glucose uptake and GLUT4 translocation elicited by simvastatin were associated with the suppression of the insulin receptor (IR)/IR substrate (IRS)/Akt signaling cascade. Simvastatin suppressed the phosphorylation of IR, IRS-1 and Akt, and total expression of IR or IRS-1, but did not affect Akt. Furthermore, simvastatin decreased Rac1 GTP binding. In conclusion, our findings indicate that simvastatin suppresses glucose uptake activity and GLUT4 translocation via IR-dependent IRS-1/PI3K/Akt pathway. These results provide an important new insight into the mechanism of statins on insulin sensitivity which may be associated with new-onset diabetes.

  2. New mechanism of lenalidomide activity.

    PubMed

    Keevan, Jacob; Figg, William D

    2014-08-01

    Lenalidomide is an immunomodulatory agent (IMiD) that has activity in hematologic cancer (e.g., multiple myeloma). The immunomodulatory and apoptotic properties are readily apparent in therapy. However, the exact mechanism of action has been difficult to quantify until recently when it was shown that another IMiD, thalidomide, binds to an E3 ubiquitin ligase complex constituent, CRBN. The article by Kronke et al. demonstrates that, by binding to CRBN and altering its selectivity, lenalidomide potentiates the ubiquitination and proteolysis of 2 specific proteins, IKZF1 and IKZF3. An article in the same issue, by Lu et al., supports these observations. IKZF1 and IKZF3 are transcription factors that are necessary for multiple myeloma, and repression of these transcription factors is a likely mechanism for lenalidomide activity in this disease.

  3. Plant-driven removal of heavy metals from soil: uptake, translocation, tolerance mechanism, challenges, and future perspectives.

    PubMed

    Thakur, Sveta; Singh, Lakhveer; Wahid, Zularisam Ab; Siddiqui, Muhammad Faisal; Atnaw, Samson Mekbib; Din, Mohd Fadhil Md

    2016-04-01

    Increasing heavy metal (HM) concentrations in the soil have become a significant problem in the modern industrialized world due to several anthropogenic activities. Heavy metals (HMs) are non-biodegradable and have long biological half lives; thus, once entered in food chain, their concentrations keep on increasing through biomagnification. The increased concentrations of heavy metals ultimately pose threat on human life also. The one captivating solution for this problem is to use green plants for HM removal from soil and render it harmless and reusable. Although this green technology called phytoremediation has many advantages over conventional methods of HM removal from soils, there are also many challenges that need to be addressed before making this technique practically feasible and useful on a large scale. In this review, we discuss the mechanisms of HM uptake, transport, and plant tolerance mechanisms to cope with increased HM concentrations. This review article also comprehensively discusses the advantages, major challenges, and future perspectives of phytoremediation of heavy metals from the soil.

  4. Biological uptake of polychlorinated biphenyls by Macoma balthica from sediment amended with activated carbon

    USGS Publications Warehouse

    McLeod, Pamela B.; van den Heuvel-Greve, Martine J.; Luoma, S.N.; Luthy, R.G.

    2007-01-01

    This work characterizes the efficacy of activated carbon amendment in reducing polychlorinated biphenyl (PCB) bioavailability to clams (Macoma balthica) from field-contaminated sediment (Hunters Point Naval Shipyard, San Francisco Bay, CA, USA) Test methods were developed for the use of clams to investigate the effects of sediment amendment on biological uptake. Sediment was mixed with activated carbon for one month. Bioaccumulation tests (28 d) were employed to assess the relationships between carbon dose and carbon particle size on observed reductions in clam biological uptake of PCBs. Extraction and cleanup protocols were developed for the clam tissue. Efficacy of activated carbon treatment was found to increase with both increasing carbon dose and decreasing carbon particle size. Average reductions in bioaccumulation of 22, 64, and 84% relative to untreated Hunters Point sediment were observed for carbon amendments of 0.34, 1.7, and 3.4%, respectively. Average bioaccumulation reductions of 41, 73, and 89% were observed for amendments (dose = 1.7% dry wt) with carbon particles of 180 to 250, 75 to 180, and 25 to 75 ??m, respectively, in diameter, indicating kinetic phenomena in these tests. Additionally, a biodynamic model quantifying clam PCB uptake from water and sediment as well as loss through elimination provided a good fit of experimental data. Model predictions suggest that the sediment ingestion route contributed 80 to 95% of the PCB burdens in the clams. ?? 2007 SETAC.

  5. Methylglyoxal and carboxyethyllysine reduce glutamate uptake and S100B secretion in the hippocampus independently of RAGE activation.

    PubMed

    Hansen, Fernanda; Battú, Cíntia Eickhoff; Dutra, Márcio Ferreira; Galland, Fabiana; Lirio, Franciane; Broetto, Núbia; Nardin, Patrícia; Gonçalves, Carlos-Alberto

    2016-02-01

    Diabetes is a metabolic disease characterized by high fasting-glucose levels. Diabetic complications have been associated with hyperglycemia and high levels of reactive compounds, such as methylglyoxal (MG) and advanced glycation endproducts (AGEs) formation derived from glucose. Diabetic patients have a higher risk of developing neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. Herein, we examined the effect of high glucose, MG and carboxyethyllysine (CEL), a MG-derived AGE of lysine, on oxidative, metabolic and astrocyte-specific parameters in acute hippocampal slices, and investigated some of the mechanisms that could mediate these effects. Glucose, MG and CEL did not alter reactive oxygen species (ROS) formation, glucose uptake or glutamine synthetase activity. However, glutamate uptake and S100B secretion were decreased after MG and CEL exposure. RAGE activation and glycation reactions, examined by aminoguanidine and L-lysine co-incubation, did not mediate these changes. Acute MG and CEL exposure, but not glucose, were able to induce similar effects on hippocampal slices, suggesting that conditions of high glucose concentrations are primarily toxic by elevating the rates of these glycation compounds, such as MG, and by generation of protein cross-links. Alterations in the secretion of S100B and the glutamatergic activity mediated by MG and AGEs can contribute to the brain dysfunction observed in diabetic patients.

  6. Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in Leukaemic K562 Cells

    PubMed Central

    Manicardi, Alex; Fabbri, Enrica; Tedeschi, Tullia; Sforza, Stefano; Bianchi, Nicoletta; Brognara, Eleonora; Gambari, Roberto; Marchelli, Rosangela; Corradini, Roberto

    2012-01-01

    A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 m urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding. PMID:22639449

  7. External carbonic anhydrase in three Caribbean corals: quantification of activity and role in CO2 uptake

    NASA Astrophysics Data System (ADS)

    Tansik, Anna L.; Fitt, William K.; Hopkinson, Brian M.

    2015-09-01

    Scleractinian corals have complicated inorganic carbon ( C i) transport pathways to support both photosynthesis, by their symbiotic dinoflagellates, and calcification. The first step in C i acquisition, uptake into the coral, is critical as the diffusive boundary layer limits the supply of CO2 to the surface and HCO3 - uptake is energy intensive. An external carbonic anhydrase (eCA) on the oral surface of corals is thought to facilitate CO2 uptake by converting HCO3 - into CO2, helping to overcome the limitation imposed by the boundary layer. However, this enzyme has not yet been identified or detected in corals, nor has its activity been quantified. We have developed a method to quantify eCA activity using a reaction-diffusion model to analyze data on 18O removal from labeled C i. Applying this technique to three species of Caribbean corals ( Orbicella faveolata, Porites astreoides, and Siderastrea radians) showed that all species have eCA and that the potential rates of CO2 generation by eCA greatly exceed photosynthetic rates. This demonstrates that eCA activity is sufficient to support its hypothesized role in CO2 supply. Inhibition of eCA severely reduces net photosynthesis in all species (on average by 46 ± 27 %), implying that CO2 generated by eCA is a major carbon source for photosynthesis. Because of the high permeability of membranes to CO2, CO2 uptake is likely driven by a concentration gradient across the cytoplasmic membrane. The ubiquity of eCA in corals from diverse genera and environments suggests that it is fundamental for photosynthetic CO2 supply.

  8. Elevated CO2 plus chronic warming reduce nitrogen uptake and levels or activities of nitrogen-uptake and -assimilatory proteins in tomato roots.

    PubMed

    Jayawardena, Dileepa M; Heckathorn, Scott A; Bista, Deepesh R; Mishra, Sasmita; Boldt, Jennifer K; Krause, Charles R

    2017-03-01

    Atmospheric CO2 enrichment is expected to often benefit plant growth, despite causing global warming and nitrogen (N) dilution in plants. Most plants primarily procure N as inorganic nitrate (NO3(-) ) or ammonium (NH4(+) ), using membrane-localized transport proteins in roots, which are key targets for improving N use. Although interactive effects of elevated CO2 , chronic warming and N form on N relations are expected, these have not been studied. In this study, tomato (Solanum lycopersicum) plants were grown at two levels of CO2 (400 or 700 ppm) and two temperature regimes (30 or 37°C), with NO3(-) or NH4(+) as the N source. Elevated CO2 plus chronic warming severely inhibited plant growth, regardless of N form, while individually they had smaller effects on growth. Although %N in roots was similar among all treatments, elevated CO2 plus warming decreased (1) N-uptake rate by roots, (2) total protein concentration in roots, indicating an inhibition of N assimilation and (3) shoot %N, indicating a potential inhibition of N translocation from roots to shoots. Under elevated CO2 plus warming, reduced NO3(-) -uptake rate per g root was correlated with a decrease in the concentration of NO3(-) -uptake proteins per g root, reduced NH4(+) uptake was correlated with decreased activity of NH4(+) -uptake proteins and reduced N assimilation was correlated with decreased concentration of N-assimilatory proteins. These results indicate that elevated CO2 and chronic warming can act synergistically to decrease plant N uptake and assimilation; hence, future global warming may decrease both plant growth and food quality (%N).

  9. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    PubMed

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.

  10. Structure-activity relations of leucine derivatives reveal critical moieties for cellular uptake and activation of mTORC1-mediated signaling.

    PubMed

    Nagamori, Shushi; Wiriyasermkul, Pattama; Okuda, Suguru; Kojima, Naoto; Hari, Yoshiyuki; Kiyonaka, Shigeki; Mori, Yasuo; Tominaga, Hideyuki; Ohgaki, Ryuichi; Kanai, Yoshikatsu

    2016-04-01

    Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.

  11. Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake

    PubMed Central

    Grinter, Rhys; Josts, Inokentijs; Zeth, Kornelius; Roszak, Aleksander W; McCaughey, Laura C; Cogdell, Richard J; Milner, Joel J; Kelly, Sharon M; Byron, Olwyn; Walker, Daniel

    2014-01-01

    The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium. PMID:24865810

  12. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    PubMed

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; p<0.0001), suggesting that peroxisome proliferator-activated receptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients

  13. Phytofiltration of arsenic and cadmium by using an aquatic plant, Micranthemum umbrosum: phytotoxicity, uptake kinetics, and mechanism.

    PubMed

    Islam, Md Shariful; Saito, Takeshi; Kurasaki, Masaaki

    2015-02-01

    Arsenic (As) and cadmium (Cd) are noxious and carcinogenic pollutants that can be removed from water by using emerging, ecofriendly, phytofiltration technology that employs Micranthemum umbrosum. After culturing M. umbrosum for 7 days in a hydroponic experiment, accumulation of 1219±44.11 µg As g(-1) and 799.40±30.95 µg Cd g(-1) were observed in the leaves, from 1000 µg As L(-1) and 1000 µg Cd L(-1) of water, respectively. Plant and water samples were analyzed for assessing the As and Cd accumulations, translocations, phytotoxic effects, uptake mechanisms and kinetics, and for evaluating the potential of M. umbrosum in As and Cd phytofiltration. The uptake pattern was leaf>stem>root for both pollutants. The plant showed higher resistance to As than to that to Cd. Uptake of inorganic As species was much greater than that of organic As and was found at above the substrate concentration. However, Cd showed similar uptake pattern to that of inorganic As species, and the data was better fit to a non-linear than a linear model. Low molecular weight substances that have thiol group(s) may be responsible for the binding of As in plants whereas Cd showed a different mechanism to that of As. M. umbrosum showed good As phytofiltration capabilities without any phytotoxic effects, but it was found to be a moderate accumulator of Cd with some phytotoxic effect compare to some other previously studied plant.

  14. Mechanisms for greater insulin-stimulated glucose uptake in normal and insulin-resistant skeletal muscle after acute exercise.

    PubMed

    Cartee, Gregory D

    2015-12-15

    Enhanced skeletal muscle and whole body insulin sensitivity can persist for up to 24-48 h after one exercise session. This review focuses on potential mechanisms for greater postexercise and insulin-stimulated glucose uptake (ISGU) by muscle in individuals with normal or reduced insulin sensitivity. A model is proposed for the processes underlying this improvement; i.e., triggers initiate events that activate subsequent memory elements, which store information that is relayed to mediators, which translate memory into action by controlling an end effector that directly executes increased insulin-stimulated glucose transport. Several candidates are potential triggers or memory elements, but none have been conclusively verified. Regarding potential mediators in both normal and insulin-resistant individuals, elevated postexercise ISGU with a physiological insulin dose coincides with greater Akt substrate of 160 kDa (AS160) phosphorylation without improved proximal insulin signaling at steps from insulin receptor binding to Akt activity. Causality remains to be established between greater AS160 phosphorylation and improved ISGU. The end effector for normal individuals is increased GLUT4 translocation, but this remains untested for insulin-resistant individuals postexercise. Following exercise, insulin-resistant individuals can attain ISGU values similar to nonexercising healthy controls, but after a comparable exercise protocol performed by both groups, ISGU for the insulin-resistant group has been consistently reported to be below postexercise values for the healthy group. Further research is required to fully understand the mechanisms underlying the improved postexercise ISGU in individuals with normal or subnormal insulin sensitivity and to explain the disparity between these groups after similar exercise.

  15. An updated model for nitrate uptake modelling in plants. II. Assessment of active root involvement in nitrate uptake based on integrated root system age: measured versus modelled outputs

    PubMed Central

    Malagoli, Philippe; Le Deunff, Erwan

    2014-01-01

    Background and Aims An updated version of a mechanistic structural–functional model was developed to predict nitrogen (N) uptake throughout the growth cycle by a crop of winter oilseed rape, Brassica napus, grown under field conditions. Methods The functional component of the model derives from a revisited conceptual framework that combines the thermodynamic Flow–Force interpretation of nitrate uptake isotherms and environmental and in planta effects on nitrate influx. Estimation of the root biomass (structural component) is based upon a combination of root mapping along the soil depth profile in the field and a relationship between the specific root length and external nitrate concentration. The root biomass contributing actively to N uptake was determined by introduction of an integrated root system age that allows assignment of a root absorption capacity at a specific age of the root. Key Results Simulations were well matched to measured data of N taken up under field conditions for three levels of N fertilization. The model outputs indicated that the two topsoil layers (0–30 and 30–60 cm) contained 75–88 % of the total root length and biomass, and accounted for 90–95 % of N taken up at harvest. Conclusions This conceptual framework provides a model of nitrate uptake that is able to respond to external nitrate fluctuations at both functional and structural levels. PMID:24709791

  16. Submaximal oxygen uptake kinetics, functional mobility, and physical activity in older adults with heart failure and reduced ejection fraction

    PubMed Central

    Hummel, Scott L; Herald, John; Alpert, Craig; Gretebeck, Kimberlee A; Champoux, Wendy S; Dengel, Donald R; Vaitkevicius, Peter V; Alexander, Neil B

    2016-01-01

    Background Submaximal oxygen uptake measures are more feasible and may better predict clinical cardiac outcomes than maximal tests in older adults with heart failure (HF). We examined relationships between maximal oxygen uptake, submaximal oxygen kinetics, functional mobility, and physical activity in older adults with HF and reduced ejection fraction. Methods Older adults with HF and reduced ejection fraction (n = 25, age 75 ± 7 years) were compared to 25 healthy age- and gender-matched controls. Assessments included a maximal treadmill test for peak oxygen uptake (VO2peak), oxygen uptake kinetics at onset of and on recovery from a submaximal treadmill test, functional mobility testing [Get Up and Go (GUG), Comfortable Gait Speed (CGS), Unipedal Stance (US)], and self-reported physical activity (PA). Results Compared to controls, HF had worse performance on GUG, CGS, and US, greater delays in submaximal oxygen uptake kinetics, and lower PA. In controls, VO2peak was more strongly associated with functional mobility and PA than submaximal oxygen uptake kinetics. In HF patients, submaximal oxygen uptake kinetics were similarly associated with GUG and CGS as VO2peak, but weakly associated with PA. Conclusions Based on their mobility performance, older HF patients with reduced ejection fraction are at risk for adverse functional outcomes. In this population, submaximal oxygen uptake measures may be equivalent to VO2 peak in predicting functional mobility, and in addition to being more feasible, may provide better insight into how aerobic function relates to mobility in older adults with HF. PMID:27594875

  17. Simultaneous monitoring of electrical capacitance and water uptake activity of plant root system

    NASA Astrophysics Data System (ADS)

    Cseresnyés, Imre; Takács, Tünde; Füzy, Anna; Rajkai, Kálmán

    2014-10-01

    Pot experiments were designed to test the applicability of root electrical capacitance measurement for in situ monitoring of root water uptake activity by growing cucumber and bean cultivars in a growth chamber. Half of the plants were inoculated with Funneliformis mosseae arbuscular mycorrhizal fungi, while the other half served as non-infected controls. Root electrical capacitance and daily transpiration were monitored during the whole plant ontogeny. Phenology-dependent changes of daily transpiration (related to root water uptake) and root electrical capacitance proved to be similar as they showed upward trends from seedling emergence to the beginning of flowering stage, and thereafter decreased continuously during fruit setting. A few days after arbuscular mycorrhizal fungi-colonization, daily transpiration and root electrical capacitance of infected plants became significantly higher than those of non-infected counterparts, and the relative increment of the measured parameters was greater for the more highly mycorrhizal-dependent bean cultivar compared to that of cucumber. Arbuscular mycorrhizal fungi colonization caused 29 and 69% relative increment in shoot dry mass for cucumbers and beans, respectively. Mycorrhization resulted in 37% increase in root dry mass for beans, but no significant difference was observed for cucumbers. Results indicate the potential of root electrical capacitance measurements for monitoring the changes and differences of root water uptake rate.

  18. The influence of hydrologic connectivity on ecosystem metabolism and nitrate uptake in an active beaver meadow

    NASA Astrophysics Data System (ADS)

    Wegener, P.; Covino, T. P.; Wohl, E.; Kampf, S. K.; Lacy, S.

    2015-12-01

    Wetlands have been widely demonstrated to provide important watershed services, such as the sequestration of carbon (C) and removal of nitrate (NO3-) from through-flowing water. Hydrologic connectivity (degree of water and associated material exchange) between floodplain water bodies (e.g., side channels, ponds) and the main channel influence rates of C accumulation and NO3- uptake, and the degree to which wetlands contribute to enhanced water quality at the catchment scale. However, environmental engineers have largely ignored the role of hydrologic connectivity in providing essential ecosystem services, and constructed wetlands are commonly built using compacted clay and berms that result in less groundwater and surface water exchange than observed in natural wetlands. In a study of an active beaver meadow (multithreaded, riparian wetland) in Rocky Mountain National Park, CO, we show how shifts in hydrology (connectivity, residence times, flow paths) from late spring snowmelt (high connectivity) to autumn/winter baseflow (low connectivity) influence ecosystem metabolism metrics (e.g., gross primary production, ecosystem respiration, and net ecosystem productivity) and NO3- uptake rates. We use a combination of mixing analyses, tracer tests, and hydrometric methods to evaluate shifts in surface and subsurface hydrologic connections between floodplain water bodies from snowmelt to baseflow. In the main channel and three floodplain water bodies, we quantify metabolism metrics and NO3- uptake kinetics across shifting flow regimes. Results from our research indicate that NO3- uptake and metabolism dynamics respond to changing levels of hydrologic connectivity to the main channel, emphasizing the importance of incorporating connectivity in wetland mitigation practices that seek to enhance water quality at the catchment scale.

  19. Arsenic in hydrothermal apatite: Oxidation state, mechanism of uptake, and comparison between experiments and nature

    NASA Astrophysics Data System (ADS)

    Liu, Weihua; Mei, Yuan; Etschmann, Barbara; Brugger, Joël; Pearce, Mark; Ryan, Chris G.; Borg, Stacey; Wykes, Jeremey; Kappen, Peter; Paterson, David; Boesenberg, Ulrike; Garrevoet, Jan; Moorhead, Gareth; Falkenberg, Gerald

    2017-01-01

    expected to be the dominant As form in hydrothermal fluids. These results show that incorporation of As in apatite is a complicated process, and sensitive to the local fluid composition during crystallization, and that some of the complexity in As zoning in partially reacted apatite may be due to local fluctuations of As(V)/As(III) ratios in the fluid and to kinetic effects during the mineral replacement reaction. Our study shows for the first time that As(III) can be incorporated into the apatite structure, although not as efficiently as As(V). Uptake of As(III) is probably highly dependent on the reaction mechanism. As(III)O33- moieties replace phosphate groups, but cause a high strain on the lattice; as a result, As(III) is easily exchanged (or oxidized) for As(V) during hydrothermal recrystallization, and the fully reacted grains only record the preferred oxidation state (i.e., As(V)) from mixed-oxidation state solutions. Overall this study shows that the observed oxidation state of As in apatite may not reflect the original As(III)/As(V) ratio of the parent fluid, due to the complex nature of As(III) uptake and possible in situ oxidation during recrystallization.

  20. Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.

    PubMed

    Harb, O S; Venkataraman, C; Haack, B J; Gao, L Y; Kwaik, Y A

    1998-01-01

    Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease. Intracellular replication of L. pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y. Abu Kwaik, Appl. Environ. Microbiol. 62:2022-2028, 1996). Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga. Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoan hosts are, in part, different. First, uptake of L. pneumophila by H. vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A. polyphaga. Second, attachment of L. pneumophila to H. vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins. In contrast, only a slight dephosphorylation of a 170-kDa protein of A. polyphaga is detected upon infection. Third, synthesis of H. vermiformis proteins but not of A. polyphaga proteins is required for uptake of L. pneumophila. Fourth, we have identified L. pneumophila mutants that are severely defective in attachment to A. polyphaga but which exhibit minor reductions in attachment to H. vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa. The data indicate a remarkable adaptation of L. pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell.

  1. Water uptake and mechanical characteristics of wood fiber-polypropylene composites

    NASA Astrophysics Data System (ADS)

    Viksne, A.; Bledzki, A. K.; Rence, L.; Berzina, R.

    2006-01-01

    The influence of mixing process (in a two-roll mill, high-speed mixer, or twin-screw extruder) on the strength properties of polypropylene/wood fiber composites was studied. The best results were obtained for composites compounded in a twin-screw extruder. The water uptake and the influence of moisture on the flexural strength (σfl) and modulus (Efl) were studied by immersion of the composites in water at 20, 50, and 90°C. Most strongly the moisture affected the value of Efl, but the degree of water uptake and the change in σfl and Efl also depended on temperature and the presence of a modificator—maleated polypropylene (MAH). MAH improved the strength properties of the composites both in dry and wet states and also decreased the extent of water uptake and swelling in cyclic (soaking/drying) tests.

  2. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells.

    PubMed

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2013-04-12

    Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA-PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  3. Prostate-Specific Membrane Antigen PET/CT: Uptake in Lymph Nodes With Active Sarcoidosis.

    PubMed

    Dias, André Henrique; Holm Vendelbo, Mikkel; Bouchelouche, Kirsten

    2017-03-01

    We describe 2 cases of Ga-PSMA PET/CT in prostate cancer patients. Both cases demonstrated symmetrical bilateral involvement of mediastinal and hilar lymph nodes besides findings in relation with prostatic disease. In both cases, endobronchial ultrasound-guided biopsy showed that the involvement of the thoracic lymph nodes was caused by nonnecrotic granulomas compatible with sarcoidosis. The cases demonstrated that increased Ga-PSMA uptake can be seen in lymph nodes with active sarcoidosis, with images mimicking those well known from FDG PET/CT. Because of these findings, granulomatous disease has to be included in the differential diagnostic evaluation of patients with Ga-PSMA-positive lymph nodes.

  4. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    PubMed

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  5. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2013-04-12

    Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  6. Exercise and Type 2 Diabetes: Molecular Mechanisms Regulating Glucose Uptake in Skeletal Muscle

    ERIC Educational Resources Information Center

    Stanford, Kristin I.; Goodyear, Laurie J.

    2014-01-01

    Exercise is a well-established tool to prevent and combat type 2 diabetes. Exercise improves whole body metabolic health in people with type 2 diabetes, and adaptations to skeletal muscle are essential for this improvement. An acute bout of exercise increases skeletal muscle glucose uptake, while chronic exercise training improves mitochondrial…

  7. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  8. Astrocytic glutamate uptake is slow and does not limit neuronal NMDA receptor activation in the neonatal neocortex.

    PubMed

    Hanson, Elizabeth; Armbruster, Moritz; Cantu, David; Andresen, Lauren; Taylor, Amaro; Danbolt, Niels Christian; Dulla, Chris G

    2015-10-01

    Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post-natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: (1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; (2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; (3) slow glutamate uptake is accompanied by lower expression of both GLT-1 and GLAST; (4) glutamate uptake is less dependent on GLT-1 in neonatal neocortex than in neonatal hippocampus; and (5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling.

  9. Simultaneous ultrastructural visualization of acetylcholinesterase activity and tritiated norepinephrine uptake in renal nerves

    SciTech Connect

    Barajas, L.; Wang, P.

    1983-02-01

    In this investigation we have combined the methods of ultrastructural demonstration of acetylcholinesterase activity with electron microscopic autoradiography for the demonstration of norepinephrine uptake. The results show electron-dense deposits indicative of acetylcholinesterase activity associated with perivascular axons overlaid by concentrations of silver grains representing exogenous tritiated norepinephrine. Forty-five percent of the intervaricose regions and 19% of the varicosities overlaid by autoradiographic grains showed ''moderate'' amounts of cholinesterase staining. A greater proportion of autoradiographic grains was observed on the varicosities than in the intervaricose regions; however, the amount of acetylcholinesterase activity was greater in the intervaricose regions than in the varicosities. This investigation provides evidence for the presence of periaxonal acetylcholinesterase staining in adrenergic axons in the rat kidney.

  10. Effect of compost temperature on oxygen uptake rate, specific growth rate and enzymatic activity of microorganisms in dairy cattle manure.

    PubMed

    Miyatake, Fumihito; Iwabuchi, Kazunori

    2006-05-01

    Investigations were carried out to find out the relationship between temperature and microbial activity in dairy cattle manure composting using oxygen uptake rate, specific growth rate and enzymatic activities during autothermal and isothermal composting experiments. In autothermal composting, oxygen uptake rate and specific growth rate were found to be most intensive in order of 43 degrees C, 60 degrees C and 54 degrees C. Isothermal composting at 54 degrees C resulted highest levels of enzymatic activity and promoted the volatile solids reduction. Based on the maximum enzymatic activity, specific growth rate appeared to be more closely linked with microbial activity in compost than with oxygen uptake rate. The enhancement of specific growth rate, enzymatic activity and volatile solids reduction were induced at 54 degrees C in cattle manure composting.

  11. Physicochemical, morphological and cellular uptake properties of lutein nanodispersions prepared by using surfactants with different stabilizing mechanisms.

    PubMed

    Tan, Tai Boon; Chu, Wern Cui; Yussof, Nor Shariffa; Abas, Faridah; Mirhosseini, Hamed; Cheah, Yoke Kqueen; Nehdi, Imededdine Arbi; Tan, Chin Ping

    2016-04-01

    In this study, we prepared a series of lutein nanodispersions via the solvent displacement method, by using surfactants with different stabilizing mechanisms. The surfactants used include Tween 80 (steric stabilization), sodium dodecyl sulfate (SDS; electrostatic stabilization), sodium caseinate (electrosteric stabilization) and SDS-Tween 80 (electrostatic-steric stabilization). We then characterized the resulting lutein nanodispersions in terms of their particle size, particle size distribution, zeta potential, lutein content, flow behavior, apparent viscosity, transmittance, color, morphological properties and their effects on cell viability and cellular uptake. The type of surfactant used significantly (p < 0.05) affected the physical properties of the nanodispersions, but the chemical properties (lutein content) remained unaffected. Transmission electron microscopy (TEM) images obtained from this study demonstrated that the solvent displacement method was capable of producing lutein nanodispersions containing spherical particles with sizes ranging from 66.20-125.25 nm, depending on the type of surfactant used. SDS and SDS-Tween 80 surfactants negatively affected the viability of the HT-29 cells used in this study. Thus, for the cellular uptake determination, only Tween 80 and sodium caseinate surfactants were used. The cellular uptake of the lutein nanodispersion stabilized by sodium caseinate was higher than that which was stabilized by Tween 80. All things considered, the type of surfactant with different stabilizing mechanisms did produce lutein nanodispersions with different characteristics. These findings would aid in future selection of surfactants in order to produce nanodispersions with desirable properties.

  12. Effects of ammonium and nitrate on nutrient uptake and activity of nitrogen assimilating enzymes in western hemlock

    SciTech Connect

    Knoepp, J.D.; Turner, D.P.; Tingey, D.T.

    1993-01-01

    Western hemlock seedlings were grown in nutrient solutions with ammonium, nitrate or ammonium plus nitrate as nitrogen sources. The objectives were to examine (1) possible selectivity for ammonium or nitrate as an N source, (2) the maintenance of charge balance during ammonium and nitrate uptake, and (3) the activity of the nitrogen assimilating enzymes, nitrate reductase, glutamine synthetase, and glutamine dehydrogenase, in relation to the uptake of different nitrogen sources. The uptake studies revealed that western hemlock takes up ammonium faster than nitrate and that ammonium partially inhibits nitrate uptake. Nitrate reductase activity varied with nitrate availability in root tissue, but showed no response in needles, indicating that most nitrate is reduced in the roots. Results indicate that western hemlock may be adapted to sites where NH(4+) is the predominate N source.

  13. Mechanics of soft active materials

    NASA Astrophysics Data System (ADS)

    Zhao, Xuanhe

    Soft active materials, mostly elastomers and polymeric gels, are being developed to mimic a salient feature of life: movement in response to stimuli. For example, when an electric voltage is applied across a layer of a dielectric elastomer, the layer reduces in thickness and expands in area, giving a strain greater than 100%. As another example, in response to a small change of pH or temperature, a hydrogel may absorb a large amount of water and increase its volume over 100 times. The mechanics involved in these processes is important, interesting, and not well understood. This thesis studies large deformations and instabilities in dielectric elastomers and polymeric gels. The thesis first presents a nonlinear field theory for deformable dielectrics. The theory uses measurable quantities to define field variables. The definitions lead to decoupled field equations, and electromechanical coupling enters the theory through material laws. We use the theory to study electromechanical instability and coexistent states in dielectric elastomers. A computational method is also developed to analyze inhomogeneous deformations in complicated structures of dielectric elastomers. The second part of the thesis discusses large deformation and mass transportation in polymeric gels. A gel can undergo large deformation of two modes: local rearrangement and long-range migration. We assume that the local rearrangement is instantaneous, and model the long-range migration by assuming that the solvent molecules diffuse inside the gel. We further study inhomogeneous and anisotropic deformations and instabilities in gels constrained by rigid materials.

  14. Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures.

    PubMed

    Horemans, Nele; Potters, Geert; De Wilde, Leen; Caubergs, Roland J

    2003-09-01

    Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.

  15. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    NASA Astrophysics Data System (ADS)

    Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

    2015-07-01

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  16. Active cell mechanics: Measurement and theory.

    PubMed

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  17. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels.

    PubMed

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C P; Huisman, Jef

    2015-11-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2.

  18. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels

    PubMed Central

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C. P.

    2015-01-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2. PMID:26319871

  19. PAR1 activation induces rapid changes in glutamate uptake and astrocyte morphology

    PubMed Central

    Sweeney, Amanda M.; Fleming, Kelsey E.; McCauley, John P.; Rodriguez, Marvin F.; Martin, Elliot T.; Sousa, Alioscka A.; Leapman, Richard D.; Scimemi, Annalisa

    2017-01-01

    The G-protein coupled, protease-activated receptor 1 (PAR1) is a membrane protein expressed in astrocytes. Fine astrocytic processes are in tight contact with neurons and blood vessels and shape excitatory synaptic transmission due to their abundant expression of glutamate transporters. PAR1 is proteolytically-activated by bloodstream serine proteases also involved in the formation of blood clots. PAR1 activation has been suggested to play a key role in pathological states like thrombosis, hemostasis and inflammation. What remains unclear is whether PAR1 activation also regulates glutamate uptake in astrocytes and how this shapes excitatory synaptic transmission among neurons. Here we show that, in the mouse hippocampus, PAR1 activation induces a rapid structural re-organization of the neuropil surrounding glutamatergic synapses, which is associated with faster clearance of synaptically-released glutamate from the extracellular space. This effect can be recapitulated using realistic 3D Monte Carlo reaction-diffusion simulations, based on axial scanning transmission electron microscopy (STEM) tomography reconstructions of excitatory synapses. The faster glutamate clearance induced by PAR1 activation leads to short- and long-term changes in excitatory synaptic transmission. Together, these findings identify PAR1 as an important regulator of glutamatergic signaling in the hippocampus and a possible target molecule to limit brain damage during hemorrhagic stroke. PMID:28256580

  20. Evaluating the uptake of Canada's new physical activity and sedentary behavior guidelines on service organizations' websites.

    PubMed

    Gainforth, Heather L; Berry, Tanya; Faulkner, Guy; Rhodes, Ryan E; Spence, John C; Tremblay, Mark S; Latimer-Cheung, Amy E

    2013-06-01

    New evidence-based physical activity and sedentary behavior guidelines for Canadians were launched in 2011. As a consequence, service organizations that promote physical activity directly to the public needed to change their promotion materials to reflect the new guidelines. Little is known about the rate at which service organizations adopt and integrate new evidence-based guidelines and determinants of guideline adoption. In this natural observational study, we evaluated the rate of online adoption of the new guidelines among key service organizations that promote physical activity and examined participation in a booster webinar as a supplemental dissemination strategy. One hundred fifty nine service organization websites were coded by one of six raters prior to the release of the new guidelines as well as at 3, 6, and 9 months after the release. Online adoption of the guidelines increased during the coding period with 51 % of organizations posting the guidelines or related information on their websites. Organizations' engagement in a webinar was associated with their adoption of the guidelines. The release of new Canadian Physical Activity and Sedentary Behaviour Guidelines led to increased guideline adoption on service organizations' websites. However, adoption was not universal. In order for the uptake of the new guidelines to be successful, further efforts need to be taken to ensure that service organizations present physical activity guidelines on their websites. Comprehensive, active dissemination strategies tailored to address organizational barriers are needed to ensure online guideline adoption.

  1. Nitrate uptake improvement by modified activated carbons developed from two species of pine cones.

    PubMed

    Nunell, G V; Fernandez, M E; Bonelli, P R; Cukierman, A L

    2015-02-15

    Activated carbons from two species of pine cones (Pinus canariensis and Cupressus sempervirens) were prepared by phosphoric acid activation and tested for the removal of nitrate ions from aqueous solution. To investigate the feasibility of improving their nitrate adsorption capacity, two different post-treatments—a thermal treatment and a treatment with saturated urea solution—were also applied to the prepared activated carbons. Comparison of the treated and untreated activated carbons showed that both post-treatments improved the nitrate adsorption performance more than twice. The maximum adsorption capacity, as evaluated from determination of the adsorption isotherms for the P. canariensis based carbons, and their proper representation by the Langmuir model, demonstrated that the post-treatment with the urea solution led to activated carbons with increased nitrate removal effectiveness, even superior to other reported results. Enhancements in their adsorption capacity could be mainly ascribed to higher contents of nitrogen and basic functional groups, whereas porous structure of the activated carbons did not seem to play a key role in the nitrate uptake.

  2. The interrelationship between muscle oxygenation, muscle activation, and pulmonary oxygen uptake to incremental ramp exercise: influence of aerobic fitness.

    PubMed

    Boone, Jan; Barstow, Thomas J; Celie, Bert; Prieur, Fabrice; Bourgois, Jan

    2016-01-01

    We investigated whether muscle and ventilatory responses to incremental ramp exercise would be influenced by aerobic fitness status by means of a cross-sectional study with a large subject population. Sixty-four male students (age: 21.2 ± 3.2 years) with a heterogeneous peak oxygen uptake (51.9 ± 6.3 mL·min(-1)·kg(-1), range 39.7-66.2 mL·min(-1)·kg(-1)) performed an incremental ramp cycle test (20-35 W·min(-1)) to exhaustion. Breath-by-breath gas exchange was recorded, and muscle activation and oxygenation were measured with surface electromyography and near-infrared spectroscopy, respectively. The integrated electromyography (iEMG), mean power frequency (MPF), deoxygenated [hemoglobin and myoglobin] (deoxy[Hb+Mb]), and total[Hb+Mb] responses were set out as functions of work rate and fitted with a double linear function. The respiratory compensation point (RCP) was compared and correlated with the breakpoints (BPs) (as percentage of peak oxygen uptake) in muscle activation and oxygenation. The BP in total[Hb+Mb] (83.2% ± 3.0% peak oxygen uptake) preceded (P < 0.001) the BP in iEMG (86.7% ± 4.0% peak oxygen uptake) and MPF (86.3% ± 4.1% peak oxygen uptake), which in turn preceded (P < 0.01) the BP in deoxy[Hb+Mb] (88.2% ± 4.5% peak oxygen uptake) and RCP (87.4% ± 4.5% peak oxygen uptake). Furthermore, the peak oxygen uptake was significantly (P < 0.001) positively correlated to the BPs and RCP, indicating that the BPs in total[Hb+Mb] (r = 0.66; P < 0.001), deoxy[Hb+Mb] (r = 0.76; P < 0.001), iEMG (r = 0.61; P < 0.001), MPF (r = 0.63; P < 0.001), and RCP (r = 0.75; P < 0.001) occurred at a higher percentage of peak oxygen uptake in subjects with a higher peak oxygen uptake. In this study a close relationship between muscle oxygenation, activation, and pulmonary oxygen uptake was found, occurring in a cascade of events. In subjects with a higher aerobic fitness level this cascade occurred at a higher relative intensity.

  3. Correlation between mechanical stress by finite element analysis and 18F-fluoride PET uptake in hip osteoarthritis patients.

    PubMed

    Hirata, Yasuhide; Inaba, Yutaka; Kobayashi, Naomi; Ike, Hiroyuki; Yukizawa, Yohei; Fujimaki, Hiroshi; Tezuka, Taro; Tateishi, Ukihide; Inoue, Tomio; Saito, Tomoyuki

    2015-01-01

    18F-fluoride positron emission tomography (18F-fluoride PET) is a functional imaging modality used primarily to detect increased bone metabolism. Increased 18F-fluoride PET uptake suggests an association between increased bone metabolism and load stress at the subchondral level. This study therefore examined the relationship between equivalent stress distribution calculated by finite element analysis and 18F-fluoride PET uptake in patients with hip osteoarthritis. The study examined 34 hips of 17 patients who presented to our clinic with hip pain, and were diagnosed with osteoarthritis or pre-osteoarthritis. The hips with trauma, infection, or bone metastasis of cancer were excluded. Three-dimensional models of each hip were created from computed tomography data to calculate the maximum equivalent stress by finite element analysis, which was compared with the maximum standardized uptake value (SUVmax) examined by 18F-fluoride PET. The SUVmax and equivalent stress were correlated (Spearman's rank correlation coefficient ρ=0.752), and higher equivalent stress values were noted in higher SUVmax patients. The correlation between SUVmax and maximum equivalent stress in osteoarthritic hips suggests the possibility that 18F-fluoride PET detect increased bone metabolism at sites of stress concentration. This study demonstrates the correlation between mechanical stress and bone remodeling acceleration in hip osteoarthritis.

  4. Cadmium induced oxalic acid secretion and its role in metal uptake and detoxification mechanisms in Phanerochaete chrysosporium.

    PubMed

    Xu, Piao; Leng, Yang; Zeng, Guangming; Huang, Danlian; Lai, Cui; Zhao, Meihua; Wei, Zhen; Li, Ningjie; Huang, Chao; Zhang, Chen; Li, Fangling; Cheng, Min

    2015-01-01

    This study examines the role of oxalic acid in the uptake of Cd and participation in detoxification process in Phanerochaete chrysosporium. Cd-induced oxalic acid secretion was observed with growth inhibition and enzyme inactivation (LiP and MnP) of P. chrysosporium. The peak value of oxalic acid concentration was 16.6 mM at initial Cd concentration of 100 mg L(-1). During the short-term uptake experiments, the uptake of Cd was enhanced and accelerated in the presence of oxalic acid and resulted in alleviated growth and enzyme inhibition ratios. The formation of a metal-oxalate complex therefore may provide a detoxification mechanism via effect on metal bioavailability, whereby many fungi can survive and grow in environments containing high concentrations of toxic metals. The present findings will advance the understanding of fungal resistance to metal stress, which could show promise for a more useful application of microbial technology in the treatment of metal-polluted waste.

  5. Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake

    PubMed Central

    Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

    2015-01-01

    Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver. PMID:26083030

  6. Both immanently high active iron contents and increased root ferrous uptake in response to low iron stress contribute to the iron deficiency tolerance in Malus xiaojinensis.

    PubMed

    Zha, Qian; Wang, Yi; Zhang, Xin-Zhong; Han, Zhen-Hai

    2014-01-01

    To better understand the mechanism of low-iron stress tolerance in Malus xiaojinensis, the differences in physiological parameters and gene expression between an iron deficiency-sensitive species, Malus baccata, and an iron deficiency-tolerant species, M. xiaojinensis were investigated under low-iron (4 μM Fe) conditions. Under iron sufficient conditions, the expressions of iron uptake- and transport-related genes, i.e. FIT1, IRT1, CS1, FRD3 and NRMAP1, and the immanent leaf and root active iron contents were higher in M. xiaojinensis than those in M. baccata. However, on the first three days of low iron stress, the rhizospheric pH decreased and the root ferric chelate reductase (FCR) activity and the expression of ferrous uptake- and iron transport-related genes in the roots increased significantly only in M. xiaojinensis. Leaf chlorosis occurred on the 3rd and the 9th day after low-iron treatment in M. baccata and M. xiaojinensis, respectively. The expression of iron relocalization-related genes, such as NAS1, FRD3 and NRMAP3, increased after the 5th or 6th day of low iron stress in leaves of M. xiaojinensis, whereas the expression of NAS1, FRD3 and NRMAP3 in the leaves of M. baccata increased immediately after the onset of low iron treatment. Conclusively, the relative high active iron contents caused by the immanently active root ferrous uptake and the increased root ferrous uptake in response to low iron stress were the dominant mechanisms for the tolerance to iron deficiency in M. xiaojinensis.

  7. Spatial distribution of microorganisms and measurements of oxygen uptake rate and ammonia uptake rate activity in a drinking water biofilter.

    PubMed

    Madoni, P; Davoli, D; Fontani, N; Cucchi, A; Rossi, F

    2001-04-01

    The biofilm characteristics (population dynamics and biofilm composition) in a biological filter for the removal of iron, manganese and ammonium were studied in a drinking water treatment plant. The objective was to examine the spatial distribution and biological composition of active biomass that grows in a biological filter and to verify the effect of the backwashing on the quantity of fixed biomass and on the density and activity of the biological population. Heterotrophic microorganisms activity was higher in the upper layer of the filter. Nitrifying microorganisms colonized the biofilter in a stratified manner and their activity was higher in the second layer of the filter. A total of 14 species of ciliated protozoa and 7 species of filamentous microorganisms were found in the biofilters. Ciliates were concentrated in the filterbed layer in which the heterotrophic activity was higher. The grazing activity of ciliates on heterotrophic bacteria reduced the competition pressure on nitrifying microorganisms, supporting their growth and thus raising the ammonium removal efficiency. In general, filamentous microorganisms appeared to be indifferent to operating changes in the plant such as backwashing and filtering cycles. Crenothrix was the prevalent filamentous microorganism in terms of both frequency and abundance; it was found prevalently in the first layer where the oxidisation of iron and manganese occurred.

  8. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food.

  9. Exocytosis mechanisms underlying insulin release and glucose uptake: conserved roles for Munc18c and syntaxin 4.

    PubMed

    Jewell, Jenna L; Oh, Eunjin; Thurmond, Debbie C

    2010-03-01

    Type 2 diabetes has been coined "a two-hit disease," as it involves specific defects of glucose-stimulated insulin secretion from the pancreatic beta cells in addition to defects in peripheral tissue insulin action required for glucose uptake. Both of these processes, insulin secretion and glucose uptake, are mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein core complexes composed of syntaxin, SNAP-23/25, and VAMP proteins. The SNARE core complex is regulated by the Sec1/Munc18 (SM) family of proteins, which selectively bind to their cognate syntaxin isoforms with high affinity. The process of insulin secretion uses multiple Munc18-syntaxin isoform pairs, whereas insulin action in the peripheral tissues appears to use only the Munc18c-syntaxin 4 pair. Importantly, recent reports have linked obesity and Type 2 diabetes in humans with changes in protein levels and single nucleotide polymorphisms (SNPs) of Munc18 and syntaxin isoforms relevant to these exocytotic processes, although the molecular mechanisms underlying the observed phenotypes remain incomplete (5, 104, 144). Given the conservation of these proteins in two seemingly disparate processes and the need to design and implement novel and more effective clinical interventions, it will be vitally important to delineate the mechanisms governing these conserved SNARE-mediated exocytosis events. Thus, we provide here an up-to-date historical review of advancements in defining the roles and molecular mechanisms of Munc18-syntaxin complexes in the pathophysiology of Type 2 diabetes.

  10. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    PubMed

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  11. Brown adipose tissue (Na+-K+)-ATPase activity and substrate uptake during the breeding cycle of rats.

    PubMed

    Zamora, F; Arola, L

    1989-05-01

    Brown adipose tissue (Na+-K+)-ATPase activity, in vitro glucose and 2-aminoisobutyric acid uptake, as well as mitochondrial GDP-binding and succinate dehydrogenase activity were determined in order to study the relationship between these parameters and the thermogenic status. Analysis were carried out on control animal, pregnant rats, dams and pups during lactation, GDP-binding, (Na+-K+)-ATPase and glucose uptake were found to be decreased in brown adipose tissue from pregnant rats and dams, and increased in pups, 2-aminoisobutyric acid uptake was only increased in pups, but no changes were observed in the other experimental groups tested. GDP-binding and (Na+-K+)-ATPase activity showed a parallelism which suggests that the enzyme is a good index of thermogenic status of the animal.

  12. Studies on the mechanism of pyrophosphate-mediated uptake of iron from transferrin by isolated rat-liver mitochondria.

    PubMed

    Konopka, K; Romslo, I

    1981-07-01

    1. Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1--1.5 nmol mg protein-1 h-1, or approximately 60% of the amount of iron mobilized from transferrin. 2. The uptake declines if respiration is inhibited, substrate is deleted, or the experiments are run under anaerobic conditions. Substrate depletion and respiratory inhibitors are less inhibitory under anaerobic conditions. 3. More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bathophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration-inhibited mitochondria under aerobic conditions is not available for heme synthesis. 4. With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate. 5. The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous iron thus formed is available for heme synthesis.

  13. Noscapine chemosensitization enhances docetaxel anticancer activity and nanocarrier uptake in triple negative breast cancer

    PubMed Central

    Doddapaneni, Ravi; Patel, Ketan; Chowdhury, Nusrat; Singh, Mandip

    2016-01-01

    Chemosensitization and enhanced delivery to solid tumor are widely explored strategies to augment the anticancer efficacy of existing chemotherapeutics agents. The aim of current research was to investigate the role of low dose Noscapine (Nos) in potentiating docetaxel cytotoxicity and enhancing tumor penetration of nanocarriers. The objectives are; (1) To evaluate the chemo-sensitizing effect of Nos in combination with docetaxel (DTX), and to elucidate the possible mechanism (2) To investigate the effect of low dose Nos on tumor stroma and enhancing nanocarrier uptake in triple negative breast cancer (TNBC) bearing nude mice. Cytotoxicity and flow cytometry analysis of DTX in Nos (4 µM) pre-treated MDA-MB-231 cells showed 3.0-fold increase in cell killing and 30% increase in number of late apoptotic cells, respectively. Stress transducer p38 phosphorylation was significantly upregulated with Nos exposure. DTX showed remarkable downregulation in expression of bcl-2, survivin and pAKT in Nos pre-treated MDA-MB-231 cells. Nos pre-sensitization significantly (p < 0.02) enhanced the anti-migration effect of DTX. In vivo studies in orthotopic TNBC tumor bearing mice showed marked reduction in tumor collagen-I levels and significantly (p < 0.03) higher intra-tumoral uptake of coumarin-6 loaded PEGylated liposomes (7-fold) in Nos treated group. Chemo-sensitization and anti-fibrotic effect of Nos could be a promising approach to increase anticancer efficacy of DTX which can be used for other nanomedicinal products. PMID:27177833

  14. Iodomethylnorcholesterol uptake in an aldosteronoma shown by dexamethasone-suppression scintigraphy: Relationship to adenoma size and functional activity

    SciTech Connect

    Nomura, K.; Kusakabe, K.; Maki, M.; Ito, Y.; Aiba, M.; Demura, H. )

    1990-10-01

    Dexamethasone-suppression (DS) adrenal scintigraphy localizes an aldosteronoma, but with false-negative results, i.e. 2 of 19 cases in our study. Our aim was to clarify the clinical meaningfulness of this test. Adrenal iodomethyl-norcholesterol (NP-59) uptake on the adenoma side correlated with the estimated adenoma volume (n = 15, r = 0.843, P less than 0.001). Accordingly, the uptake ratio on the adenoma side to that on the opposite side depended on the adenoma volume (r = 0.683, P less than 0.01). This explains the false-negative results (uptake ratio less than 2) in two cases with small adenomas. The NP-59 uptake correlated weakly with the plasma aldosterone level (r = 0.516, P less than 0.05). This result indicates the low correlation between NP-59 uptake and the ability to secrete aldosterone. NP-59 accumulation in the surgically removed gland was analyzed by autoradiography in six cases where DS scintigraphy was done just before surgery. The density was higher in the adenoma cells than in the adjacent cortical cells in five cases, but the difference was rather small, i.e., within a 2-fold difference in four cases. In one case, almost the same density was observed in both types of cells. Thus, the laterality of NP-59 uptake primarily depends on the adenoma volume although NP-59 uptake somewhat reflects the adenoma's ability to secrete aldosterone or the adenoma cell's activity in accumulating NP-59. Care must be taken in interpreting the findings from DS scintigraphy where the adenoma is small or adrenal uptake is low.

  15. Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

    2016-03-01

    Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

  16. Influence of anionic surface-active agents on the uptake of heavy metals by water hyacinth (Eichhornia crassipes)

    SciTech Connect

    Muramoto, S.; Oki, Y.

    1984-10-01

    In a previous paper, the ability of water hyacinth to remove toxic heavy metals, cadmium, lead, and mercury, from a metal-containing solution was reported. However, information on the effects of surface-active agents on the metal uptake from waste water by water hyacinth is insufficient. Surface-active agents including anionic detergents have been found in lake, ponds, and rivers polluted by waste from industry and municipal sewage treatment plants. The present study examines the uptake of cadmium or nickel in the presence of the anionic detergent sodium dedecyl sulfate.

  17. Multifunctional organic–inorganic hybrid nanoparticles and nanosheets based on chitosan derivative and layered double hydroxide: cellular uptake mechanism and application for topical ocular drug delivery

    PubMed Central

    Chi, Huibo; Gu, Yan; Xu, Tingting; Cao, Feng

    2017-01-01

    To study the cellular uptake mechanism of multifunctional organic–inorganic hybrid nanoparticles and nanosheets, new chitosan–glutathione–valine–valine-layered double hydroxide (CG-VV-LDH) nanosheets with active targeting to peptide transporter-1 (PepT-1) were prepared, characterized and further compared with CG-VV-LDH nanoparticles. Both organic–inorganic hybrid nanoparticles and nanosheets showed a sustained release in vitro and prolonged precorneal retention time in vivo, but CG-VV-LDH nanoparticles showed superior permeability in the isolated cornea of rabbits than CG-VV-LDH nanosheets. Furthermore, results of cellular uptake on human corneal epithelial primary cells (HCEpiC) and retinal pigment epithelial (ARPE-19) cells indicated that both clathrin-mediated endocytosis and active transport of PepT-1 are involved in the internalization of CG-VV-LDH nanoparticles and CG-VV-LDH nanosheets. In summary, the CG-VV-LDH nanoparticle may be a promising carrier as a topical ocular drug delivery system for the treatment of ocular diseases of mid-posterior segments, while the CG-VV-LDH nanosheet may be suitable for the treatment of ocular surface diseases. PMID:28280329

  18. Atypical Dopamine Uptake Inhibitors that Provide Clues About Cocaine's Mechanism at the Dopamine Transporter

    NASA Astrophysics Data System (ADS)

    Hauck Newman, Amy; Katz, Jonathan L.

    The dopamine transporter (DAT) has been a primary target for cocaine abuse/addiction medication discovery. However predicted addiction liability and limited clinical evaluation has provided a formidable challenge for development of these agents for human use. The unique and atypical pharmacological profile of the benztropine (BZT) class of dopamine uptake inhibitors, in preclinical models of cocaine effects and abuse, has encouraged further development of these agents. Moreover, in vivo studies have challenged the original DAT hypothesis and demonstrated that DAT occupancy and subsequent increases in dopamine produced by BZT analogues are significantly delayed and long lasting, as compared to cocaine. These important and distinctive elements are critical to the lack of abuse liability among BZT analogues, and improve their potential for development as treatments for cocaine abuse and possibly other neuropsychiatric disorders.

  19. Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition.

    PubMed

    Salameh, Therese S; Bullock, Kristin M; Hujoel, Isabel A; Niehoff, Michael L; Wolden-Hanson, Tami; Kim, Junghyun; Morley, John E; Farr, Susan A; Banks, William A

    2015-01-01

    Intranasal insulin has shown efficacy in patients with Alzheimer's disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain.

  20. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    PubMed

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-09

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

  1. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps

    PubMed Central

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A. S.; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-01-01

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K+ uptake system in the Venus flytrap. In search of K+ transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K+-transporter genes into Xenopus oocytes, however, both putative K+ transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K+ transporter 1 (AKT1), we coexpressed the putative K+ transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K+ uptake. DmKT1 was found to be a K+-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around −120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K+, reducing its concentration from millimolar levels down to trace levels. PMID:25997445

  2. Experimental evaluation of starch utilization mechanism by activated sludge.

    PubMed

    Karahan, Ozlem; Martins, António; Orhon, Derin; van Loosdrecht, Mark C M

    2006-04-05

    The study aimed to explore the conversion processes of hydrolysable substrates by activated sludge. Experimental data were collected from a sequencing batch reactor (SBR) and from batch tests using activated sludge acclimated to native potato starch (NPS). Parallel batch tests were run with NPS (particulate), soluble starch (SolS), maltose, and glucose for comparative evaluation. The fate of organic carbon in the reactor was followed directly by measuring substrate, poly-glucose, and oxygen uptake rate. Results indicated that adsorption was the dominant mechanism for starch removal with subsequent enzymatic hydrolysis inside the flocs. The role of bulk liquid enzyme activity was minimal. Starch was observed to hydrolyze to maltose rather than glucose. The behavior of NPS and SolS was quite similar to maltose in terms of poly-glucose formation and oxygen uptake. Since the simplest hydrolysis product was maltose, the biomass was not acclimated to glucose and thus, glucose exhibited a significantly different removal and storage pattern. The study also showed that differentiation of readily biodegradable and slowly biodegradable COD should better be based on the kinetics of their utilization rather than simple physical characterization.

  3. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.

  4. Molecular Mechanisms of Anthracycline Activity

    NASA Astrophysics Data System (ADS)

    Beretta, Giovanni Luca; Zunino, Franco

    On the basis of evidence that anthracyclines are DNA intercalating agents and DNA is the primary target, a large number of analogs and related intercalators have been developed. However, doxorubicin and closely related anthracyclines still remain among the most effective antitumor agents. Multiple mechanisms have been proposed to explain their efficacy. They include inhibition of DNA-dependent functions, free radical formation, and membrane interactions. The primary mechanism of action is now ascribed to drug interference with the function of DNA topoisomerase II. The stabilization of the topoisomerase-mediated cleavable complex results in a specific type of DNA damage (i.e., double-strand protein-associated DNA breaks). The drug-stabilized cleavable complex is a potentially reversible molecular event and its persistence, as a consequence of strong DNA binding, may be recognized as an apoptotic stimulus. Indirect evidence supports the notion that the bioreductive processes of the quinone moiety generating the semiquinone radical with concomitant production of reactive oxygen species may contribute to the drug effects. The cellular defense mechanisms and response to genotoxic/cytotoxic stress appear to be critical determinants of the tumor sensitivity to anthracyclines.

  5. Active Uptake of Amino Acids by Leaves of an Epiphytic Vascular Plant, Tillandsia paucifolia (Bromeliaceae) 1

    PubMed Central

    Nyman, Leslie Paul; Davis, James P.; O'Dell, Stephen J.; Arditti, Joseph; Stephens, Grover C.; Benzing, David H.

    1987-01-01

    Specialized epidermal trichomes on the leaves of the epiphyte, Tillandsia paucifolia (Bromeliaceae) accumulate amino acids from solution. Simultaneous net uptake of 17 amino acids was determined using high performance liquid chromatography. Uptake occurs against concentration gradients at least as high as 104. Images Fig. 2 Fig. 3 PMID:16665307

  6. Single exposure to cocaine impairs aspartate uptake in the pre-frontal cortex via dopamine D1-receptor dependent mechanisms.

    PubMed

    Sathler, Matheus Figueiredo; Stutz, Bernardo; Martins, Robertta Silva; Dos Santos Pereira, Maurício; Pecinalli, Ney Roner; Santos, Luis E; Taveira-da-Silva, Rosilane; Lowe, Jennifer; de Freitas, Isis Grigorio; de Melo Reis, Ricardo Augusto; Manhães, Alex C; Kubrusly, Regina C C

    2016-08-04

    Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.

  7. Exploring equity in uptake of the NHS Health Check and a nested physical activity intervention trial

    PubMed Central

    Attwood, S.; Morton, K.; Sutton, S.

    2016-01-01

    Background Socio-demographic factors characterizing disadvantage may influence uptake of preventative health interventions such as the NHS Health Check and research trials informing their content. Methods A cross-sectional study examining socio-demographic characteristics of participants and non-participants to the NHS Health Check and a nested trial of very brief physical activity interventions within this context. Age, gender, Index of Multiple Deprivation (IMD) and ethnicity were extracted from patient records of four General Practices (GP) in England. Results In multivariate analyses controlling for GP surgery, the odds of participation in the Health Check were higher for older patients (OR 1.05, 95% CI 1.04–1.07) and lower from areas of greater deprivation (IMD Quintiles 4 versus 1, OR 0.37, 95% CI 0.18–0.76, 5 versus 1 OR 0.42, 95% CI 0.20–0.88). Older patients were more likely to participate in the physical activity trial (OR 1.04, 95% CI 1.02–1.06). Conclusions Younger patients and those living in areas of greater deprivation may be at risk of non-participation in the NHS Health Check, while younger age also predicted non-participation in a nested research trial. The role that GP-surgery-specific factors play in influencing participation across different socio-demographic groups requires further exploration. PMID:26036701

  8. Oxygen uptake and energy expenditure for children during rock climbing activity.

    PubMed

    Watts, Phillip Baxter; Ostrowski, Megan L

    2014-02-01

    The purpose of this study was to measure oxygen uptake and energy expenditure in children during rock climbing activity. 29 children (age = 10.9 ± 1.7 yr) participated in the study. A commercially available rock climbing structure with ample features for submaximal effort climbing provided continuous terrain. Participants were instructed to climb at a comfortable pace. Following an initial 5-min rest, each child climbed one sustained 5-min bout followed by 5-min sitting recovery for a total of 10 min (SUS). This was immediately followed by five 1-min climbing + 1-min recovery intervals for a second total of 10 min (INT). Expired air was analyzed continuously. Energy expenditure (EE) was determined via the Weir method for 10-s intervals throughout the full protocol. The total energy expenditure in kilocalories during the 10-min SUS period was 34.3 ± 11.3 kcal. Energy expenditure during the 10-min INT period averaged 39.3 ± 13.1 kcal and was significantly higher than during SUS (p < .05). The mean total EE for SUS + INT was 73.7 ± 24.2 kcal. EE was correlated with body mass; r = .86. The rock climbing tasks employed in this study produced EE levels similar to what have been reported in children for stair climbing, sports/games activities, and easy jogging.

  9. The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells.

    PubMed

    Koppolu, Bhanuprasanth; Zaharoff, David A

    2013-03-01

    Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. In this study, the influence of chitosan encapsulation on antigen uptake, activation and presentation by antigen presenting cells (APCs) is explored. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and ovalbumin (OVA) were used as model protein antigens and encapsulated in chitosan particles via precipitation-coacervation at loading efficiencies >89%. Formulation conditions were manipulated to create antigen-encapsulated chitosan particles (AgCPs) with discrete nominal sizes (300 nm, 1 μm, and 3 μm). Uptake of AgCPs by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs.

  10. Water uptake mechanism and germination of Erythrina velutina seeds treated with atmospheric plasma.

    PubMed

    Alves Junior, Clodomiro; de Oliveira Vitoriano, Jussier; da Silva, Dinnara Layza Souza; de Lima Farias, Mikelly; de Lima Dantas, Nadjamara Bandeira

    2016-09-27

    The effect of plasma applied to mulungu (Erythrina velutina) seeds was studied to verify its influence on the germination, water absorption, wettability and structure of the seeds. The plasma jet used in this study was produced by dielectric barrier discharge (DBD) in a helium gas flow of 0.03 L/s at a distance of 13 mm for 60 s. The plasma treatment significantly affected the seed germination rate, which was approximately 5% higher than that of the untreated group. Micropyle and hilum contributed a greater proportion to uptake. When sealed in the hilar or micropyle regions the amount of water absorbed into the seed decreased approximately 75% compared to the unsealed seed. This difference suggests that these two regions together act cooperatively in the water absorption. However, when plasma treated seed was blocked in the micropyle region, water absorption was higher higher than in seeds blocked hilum. This difference suggests that the plasma treatment changed the wettability of the hilum more effectively than it changed the micropyle. These results indicate that plasma can significantly change the hydrophilicity, water absorption and percentage of seed germination in E. velutina.

  11. Water uptake mechanism and germination of Erythrina velutina seeds treated with atmospheric plasma

    NASA Astrophysics Data System (ADS)

    Alves Junior, Clodomiro; de Oliveira Vitoriano, Jussier; da Silva, Dinnara Layza Souza; de Lima Farias, Mikelly; de Lima Dantas, Nadjamara Bandeira

    2016-09-01

    The effect of plasma applied to mulungu (Erythrina velutina) seeds was studied to verify its influence on the germination, water absorption, wettability and structure of the seeds. The plasma jet used in this study was produced by dielectric barrier discharge (DBD) in a helium gas flow of 0.03 L/s at a distance of 13 mm for 60 s. The plasma treatment significantly affected the seed germination rate, which was approximately 5% higher than that of the untreated group. Micropyle and hilum contributed a greater proportion to uptake. When sealed in the hilar or micropyle regions the amount of water absorbed into the seed decreased approximately 75% compared to the unsealed seed. This difference suggests that these two regions together act cooperatively in the water absorption. However, when plasma treated seed was blocked in the micropyle region, water absorption was higher higher than in seeds blocked hilum. This difference suggests that the plasma treatment changed the wettability of the hilum more effectively than it changed the micropyle. These results indicate that plasma can significantly change the hydrophilicity, water absorption and percentage of seed germination in E. velutina.

  12. Water uptake mechanism and germination of Erythrina velutina seeds treated with atmospheric plasma

    PubMed Central

    Alves Junior, Clodomiro; de Oliveira Vitoriano, Jussier; da Silva, Dinnara Layza Souza; de Lima Farias, Mikelly; de Lima Dantas, Nadjamara Bandeira

    2016-01-01

    The effect of plasma applied to mulungu (Erythrina velutina) seeds was studied to verify its influence on the germination, water absorption, wettability and structure of the seeds. The plasma jet used in this study was produced by dielectric barrier discharge (DBD) in a helium gas flow of 0.03 L/s at a distance of 13 mm for 60 s. The plasma treatment significantly affected the seed germination rate, which was approximately 5% higher than that of the untreated group. Micropyle and hilum contributed a greater proportion to uptake. When sealed in the hilar or micropyle regions the amount of water absorbed into the seed decreased approximately 75% compared to the unsealed seed. This difference suggests that these two regions together act cooperatively in the water absorption. However, when plasma treated seed was blocked in the micropyle region, water absorption was higher higher than in seeds blocked hilum. This difference suggests that the plasma treatment changed the wettability of the hilum more effectively than it changed the micropyle. These results indicate that plasma can significantly change the hydrophilicity, water absorption and percentage of seed germination in E. velutina. PMID:27670654

  13. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake

    PubMed Central

    Salter, Rebecca C.; Foka, Pelagia; Davies, Thomas S.; Gallagher, Hayley; Michael, Daryn R.; Ashlin, Tim G.; Ramji, Dipak P.

    2016-01-01

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis. PMID:27687241

  14. The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake.

    PubMed

    Salter, Rebecca C; Foka, Pelagia; Davies, Thomas S; Gallagher, Hayley; Michael, Daryn R; Ashlin, Tim G; Ramji, Dipak P

    2016-09-30

    The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis.

  15. Effects of Body Mass Index on Task-Related Oxygen Uptake and Dyspnea during Activities of Daily Life in COPD

    PubMed Central

    Vaes, Anouk W.; Franssen, Frits M. E.; Meijer, Kenneth; Cuijpers, Martijn W. J.; Wouters, Emiel F. M.; Rutten, Erica P. A.; Spruit, Martijn A.

    2012-01-01

    Background Patients with COPD use a higher proportion of their peak aerobic capacity during the performance of domestic activities of daily life (ADLs) compared to healthy peers, accompanied by a higher degree of task-related symptoms. To date, the influence of body mass index (BMI) on the task-related metabolic demands remains unknown in patients with COPD. Therefore, the aim of our study was to determine the effects of BMI on metabolic load during the performance of 5 consecutive domestic ADLs in patients with COPD. Methodology Ninety-four COPD patients and 20 healhty peers performed 5 consecutive, self-paced domestic ADLs putting on socks, shoes and vest; folding 8 towels; putting away groceries; washing up 4 dishes, cups and saucers; and sweeping the floor for 4 min. Task-related oxygen uptake and ventilation were assessed using a mobile oxycon, while Borg scores were used to assess task-related dyspnea and fatigue. Principal Findings 1. Relative task-related oxygen uptake after the performance of domestic ADLs was increased in patients with COPD compared to healthy elderly, whereas absolute oxygen uptake is similar between groups; 2. Relative oxygen uptake and oxygen uptake per kilogram fat-free mass were comparable between BMI groups; and 3. Borg symptom scores for dyspnea en fatigue were comparable between BMI groups. Conclusion Patients with COPD in different BMI groups perform self-paced domestic ADLs at the same relative metabolic load, accompanied by comparable Borg symptom scores for dyspnea and fatigue. PMID:22815922

  16. Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

    PubMed Central

    Liu, Jian; Copland, David A.; Horie, Shintaro; Wu, Wei-Kang; Chen, Mei; Xu, Yunhe; Paul Morgan, B.; Mack, Matthias; Xu, Heping; Nicholson, Lindsay B.; Dick, Andrew D.

    2013-01-01

    Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV) as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE) and Bruch’s membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b+ cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF + Arg-1+ myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF + Arg-1+ phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain. PMID:23977372

  17. The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake.

    PubMed

    Fischinger, Stephanie A; Schulze, Joachim

    2010-09-01

    Replacement of N(2) by argon in the air around nodules directs nitrogenase electron flow in its total onto H(+) resulting in increased nodule H(2) evolution (total nitrogenase activity (TNA)). However, argon application induces a so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with decreased nodule oxygen permeability. Consequently, TNA measurements tend to underestimate total nitrogenase activity. It is unclear whether the decline in oxygen diffusion into nodules induces the Ar-ID, or whether a decline in nitrogenase activity is followed by lower nodule O(2) uptake. The objective of the present work was to examine the time sequence of the decline in nodule H(2) evolution and O(2) uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O(2), was tested through comparative measurement of (15)N(2) uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of (15)N(2) uptake. A five min application of Ar/O(2) was without effect on the subsequent H(2) evolution in ambient air. A parallel experiment on control plants revealed that a decrease in nodule oxygen uptake began several minutes after the onset of the decline in H(2) evolution. We conclude that the primary effect of the replacement of N(2) by argon differs from oxygen diffusion control. A gas exchange system allowing an immediate taking of TNA yields reliable results and does not disturb nodule activity. Gas exchange measurements provide a powerful tool for studying nodule physiology and should be combined with material from molecular studies.

  18. Forms of selenium affect its transport, uptake and glutathione peroxidase activity in the Caco-2 cell model.

    PubMed

    Wang, Yanbo; Fu, Linglin

    2012-10-01

    The experiment was designed to investigate the effect of selenium (Se) chemical forms (sodium selenite, selenium nanoparticle [nano-Se] and selenomethionine) on the transport, uptake and glutathione peroxidase (GSH-Px) activity in the Caco-2 cell model. The transport and uptake of different forms of Se (0.1 μmol l(-1)) across the Caco-2 cell monolayer were carried out in two directions (apical [AP] to basolateral [BL] and BL to AP) for 2 h, respectively, and the apparent permeability coefficient (P(app)), transport efficiency and uptake efficiency were all calculated. In the present study, the transport and uptake of three forms of Se were time-dependent both in AP to BL and BL to AP directions. By the end of 2 h, the transport efficiencies of selenomethionine and nano-Se were higher than that of sodium selenite (P<0.05). The highest uptake efficiency (P<0.05) was observed in cells treated with nano-Se and significant difference (P<0.05) was also observed between the cells incubated with sodium selenite and selenomethionine. As for the P(app), sodium selenite (P<0.05) had the lowest values compared with that of selenomethionine and nano-Se, in both AP-BL and BL-AP. However, no significant differences were observed in GSH-Px activities. These results indicated that the efficiency of Se in the Caco-2 cells varied with its chemical forms, which might be associated with the differences in Se transport and uptake.

  19. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells.

    PubMed

    Krolopp, James E; Thornton, Shantaé M; Abbott, Marcia J

    2016-01-01

    Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

  20. IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

    PubMed Central

    Krolopp, James E.; Thornton, Shantaé M.; Abbott, Marcia J.

    2016-01-01

    Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism. PMID:28066259

  1. Biodynamic modeling of PCB uptake by Macoma balthica and Corbicula fluminea from sediment amended with activated carbon

    USGS Publications Warehouse

    McLeod, Pamela B.; Luoma, S.N.; Luthy, R.G.

    2008-01-01

    Activated carbon amendment was assessed in the laboratory as a remediation strategy for freshwater sediment contaminated with polychlorinated biphenyls (PCBs) from the Grasse River (near Massena, NY). Three end points were evaluated: aqueous equilibrium PCB concentration, uptake into semipermeable membrane devices (SPMDs), and 28-day bioaccumulation in the clam Corbicula fluminea. PCB uptake by water, SPMDs, and clams followed similar trends, with reductions increasing as a function of carbon dose. Average percent reductions in clam tissue PCBs were 67, 86, and 95% for activated carbon doses of 0.7, 1.3, and 2.5% dry wt, respectively. A biodynamic model that incorporates sediment geochemistry and dietary and aqueous uptake routes was found to agree well with observed uptake by C. fluminea in our laboratory test systems. Results from this study were compared to 28-day bioaccumulation experiments involving PCB-contaminated sediment from Hunters Point Naval Shipyard (San Francisco Bay, CA) and the clam Macoma balthica. Due to differences in feeding strategy, M. balthica deposit-feeds whereas C. fluminea filter-feeds, the relative importance of the aqueous uptake route is predicted to be much higher for C. fluminea than for M. balthica. Whereas M. balthica takes up approximately 90% of its body burden through sediment ingestion, C. fluminea only accumulates approximately 45% via this route. In both cases, results strongly suggest that it is the mass transfer of PCBs from native sediment to added carbon particles, not merely reductions in aqueous PCB concentrations, that effectively reduces PCB bioavailability and uptake by sediment-dwelling organisms. ?? 2008 American Chemical Society.

  2. Biodynamic modeling of PCB uptake by Macoma balthica and Corbicula fluminea from sediment amended with activated carbon.

    PubMed

    McLeod, Pamela B; Luoma, Samuel N; Luthy, Richard G

    2008-01-15

    Activated carbon amendment was assessed in the laboratory as a remediation strategy for freshwater sediment contaminated with polychlorinated biphenyls (PCBs) from the Grasse River (near Massena, NY). Three end points were evaluated: aqueous equilibrium PCB concentration, uptake into semipermeable membrane devices (SPMDs), and 28-day bioaccumulation in the clam Corbicula fluminea. PCB uptake by water, SPMDs, and clams followed similar trends, with reductions increasing as a function of carbon dose. Average percent reductions in clam tissue PCBs were 67, 86, and 95% for activated carbon doses of 0.7, 1.3, and 2.5% dry wt, respectively. A biodynamic model that incorporates sediment geochemistry and dietary and aqueous uptake routes was found to agree well with observed uptake by C. fluminea in our laboratory test systems. Results from this study were compared to 28-day bioaccumulation experiments involving PCB-contaminated sediment from Hunters Point Naval Shipyard (San Francisco Bay, CA) and the clam Macoma balthica. Due to differences in feeding strategy, M. balthica deposit-feeds whereas C. fluminea filter-feeds, the relative importance of the aqueous uptake route is predicted to be much higher for C. fluminea than for M. balthica. Whereas M. balthica takes up approximately 90% of its body burden through sediment ingestion, C. fluminea only accumulates approximately 45% via this route. In both cases, results strongly suggest that it is the mass transfer of PCBs from native sediment to added carbon particles, not merely reductions in aqueous PCB concentrations, that effectively reduces PCB bioavailability and uptake by sediment-dwelling organisms.

  3. Stimulation of AMP-activated protein kinase and enhancement of basal glucose uptake in muscle cells by quercetin and quercetin glycosides, active principles of the antidiabetic medicinal plant Vaccinium vitis-idaea.

    PubMed

    Eid, Hoda M; Martineau, Louis C; Saleem, Ammar; Muhammad, Asim; Vallerand, Diane; Benhaddou-Andaloussi, Ali; Nistor, Lidia; Afshar, Arvind; Arnason, John T; Haddad, Pierre S

    2010-07-01

    Several medicinal plants that stimulate glucose uptake in skeletal muscle cells were identified from among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. This study aimed to elucidate the mechanism of action of one of these products, the berries of Vaccinium vitis idaea, as well as to isolate and identify its active constituents using a classical bioassay-guided fractionation approach. Western immunoblot analysis in C2C12 muscle cells revealed that the ethanol extract of the berries stimulated the insulin-independent AMP-activated protein kinase (AMPK) pathway. The extract mildly inhibited ADP-stimulated oxygen consumption in isolated mitochondria, an effect consistent with metabolic stress and the ensuing stimulation of AMPK. This mechanism is highly analogous to that of Metformin. Fractionation guided by glucose uptake activity resulted in the isolation of ten compounds. The two most active, quercetin-3-O-glycosides, enhanced glucose uptake by 38-59% (50 muM; 18 h treatment) in the absence of insulin. Quercetin aglycone, a minor constituent, stimulated uptake by 37%. The quercetin glycosides and the aglycone stimulated the AMPK pathway at concentrations of 25-100 muM, but only the aglycone inhibited ATP synthase in isolated mitochondria (by 34 and 79% at 25 and 100 muM, respectively). This discrepancy suggests that the activity of the glycosides may require hydrolysis to the aglycone form. These findings indicate that quercetin and quercetin 3-O-glycosides are responsible for the antidiabetic activity of V. vitis crude berry extract mediated by AMPK. These common plant products may thus have potential applications for the prevention and treatment of insulin resistance and other metabolic diseases.

  4. Hypoglycemic Effect of Opuntia ficus-indica var. saboten Is Due to Enhanced Peripheral Glucose Uptake through Activation of AMPK/p38 MAPK Pathway.

    PubMed

    Leem, Kang-Hyun; Kim, Myung-Gyou; Hahm, Young-Tae; Kim, Hye Kyung

    2016-12-09

    Opuntia ficus-indica var. saboten (OFS) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, detailed mechanisms underlying hypoglycemic effects remain unclear. In this study, the mechanism underlying the hypoglycemic activity of OFS was evaluated using in vitro and in vivo systems. OFS treatment inhibited α-glucosidase activity and intestinal glucose absorption assessed by Na⁺-dependent glucose uptake using brush border membrane vesicles. AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle, and p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. In the present study, OFS dose-dependently increased glucose uptake in L6 muscle cells. The AMPK and p38 MAPK phosphorylations were stimulated by OFS, and inhibitors of AMPK (compound C) and p38 MAPK (SB203580) abolished the effects of OFS. Furthermore, OFS increased glucose transporter 4 (GLUT4) translocation to the plasma membrane. OFS administration (1 g/kg and 2 g/kg body weight) in db/db mice dose-dependently ameliorated hyperglycemia, hyperinsulinemia, and glucose tolerance. Insulin resistance assessed by homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index were also dose-dependently improved with OFS treatment. OFS administration improved pancreatic function through increased β-cell mass in db/db mice. These findings suggest that OFS acts by inhibiting glucose absorption from the intestine and enhancing glucose uptake from insulin-sensitive muscle cells through the AMPK/p38 MAPK signaling pathway.

  5. Hypoglycemic Effect of Opuntia ficus-indica var. saboten Is Due to Enhanced Peripheral Glucose Uptake through Activation of AMPK/p38 MAPK Pathway

    PubMed Central

    Leem, Kang-Hyun; Kim, Myung-Gyou; Hahm, Young-Tae; Kim, Hye Kyung

    2016-01-01

    Opuntia ficus-indica var. saboten (OFS) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, detailed mechanisms underlying hypoglycemic effects remain unclear. In this study, the mechanism underlying the hypoglycemic activity of OFS was evaluated using in vitro and in vivo systems. OFS treatment inhibited α-glucosidase activity and intestinal glucose absorption assessed by Na+-dependent glucose uptake using brush border membrane vesicles. AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle, and p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. In the present study, OFS dose-dependently increased glucose uptake in L6 muscle cells. The AMPK and p38 MAPK phosphorylations were stimulated by OFS, and inhibitors of AMPK (compound C) and p38 MAPK (SB203580) abolished the effects of OFS. Furthermore, OFS increased glucose transporter 4 (GLUT4) translocation to the plasma membrane. OFS administration (1 g/kg and 2 g/kg body weight) in db/db mice dose-dependently ameliorated hyperglycemia, hyperinsulinemia, and glucose tolerance. Insulin resistance assessed by homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index were also dose-dependently improved with OFS treatment. OFS administration improved pancreatic function through increased β-cell mass in db/db mice. These findings suggest that OFS acts by inhibiting glucose absorption from the intestine and enhancing glucose uptake from insulin-sensitive muscle cells through the AMPK/p38 MAPK signaling pathway. PMID:27941667

  6. Role of the scavenger receptor in the uptake of methylamine-activated alpha 2-macroglobulin by rat liver.

    PubMed Central

    van Dijk, M C; Boers, W; Linthorst, C; van Berkel, T J

    1992-01-01

    Alpha 2-Macroglobulin (alpha 2M) requires activation by small nucleophiles (e.g. methylamine; giving alpha 2M-Me) or proteolytic enzymes (e.g. trypsin; giving alpha 2M-Tr) in order to be rapidly removed from the circulation by the liver. Separation of rat liver cells into parenchymal, endothelial and Kupffer cells at 10 min after injection indicates that liver uptake of alpha 2M-Me is shared between parenchymal and endothelial cells, with relative contributions of 51.3% and 48.3% respectively of total liver-associated radioactivity. In contrast, alpha 2M-Tr is almost exclusively taken up by the parenchymal cells (90.1% of liver-associated radioactivity). A preinjection of 5 mg of poly(inosinic acid) decreased liver uptake of alpha 2M-Me to 39.9% of the control value, while it had no effect on liver uptake of alpha 2M-Tr. It appears that poly(inosinic acid) specifically reduces the uptake of alpha 2M-Me in vivo by endothelial cells, leaving uptake by parenchymal cells unaffected. In vitro studies with isolated liver cells indicate that the association of alpha 2M-Me with endothelial cells is 21-fold higher per mg of cell protein than with parenchymal cells. The capacity of endothelial cells to degrade alpha 2M-Me appears to be 46 times higher than that of parenchymal cells. Competition studies show that poly(inosinic acid) or acetylated low-density lipoprotein effectively competes with the association of alpha 2M-Me with endothelial and Kupffer cells, but association with parenchymal cells is unaffected. It is suggested that activation of alpha 2M by methylamine induces a charge distribution on the protein which triggers specific uptake by the scavenger receptor on endothelial cells. It is concluded that the uptake of alpha 2M-Me by the scavenger receptor might function as an additional system for the uptake of activated alpha 2M. Images Fig. 11. PMID:1280102

  7. Remarkable Improvement in the Mechanical Properties and CO2 Uptake of MOFs Brought About by Covalent Linking to Graphene.

    PubMed

    Kumar, Ram; Raut, Devaraj; Ramamurty, Upadrasta; Rao, C N R

    2016-06-27

    Metal-organic frameworks (MOFs) are exceptional as gas adsorbents but their mechanical properties are poor. We present a successful strategy to improve the mechanical properties along with gas adsorption characteristics, wherein graphene (Gr) is covalently bonded with M/DOBDC (M=Mg(2+) , Ni(2+) , or Co(2+) , DOBDC=2,5-dioxido-1,4-benzene dicarboxylate) MOFs. The surface area of the graphene-MOF composites increases up to 200-300 m(2)  g(-1) whereas the CO2 uptake increases by ca. 3-5 wt % at 0.15 atm and by 6-10 wt % at 1 atm. What is significant is that the composites exhibit improved mechanical properties. In the case of Mg/DOBDC, a three-fold increase in both the elastic modulus and hardness with 5 wt % graphene reinforcement is observed. Improvement in both the mechanical properties and gas adsorption characteristics of porous MOFs on linking them to graphene is a novel observation and suggests a new avenue for the design and synthesis of porous materials.

  8. Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

    PubMed

    Noyer, C M; Immenschuh, S; Liem, H H; Muller-Eberhard, U; Wolkoff, A W

    1998-07-01

    Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

  9. Estrogen effects on thyroid iodide uptake and thyroperoxidase activity in normal and ovariectomized rats.

    PubMed

    Lima, Lívia P; Barros, Inês A; Lisbôa, Patrícia C; Araújo, Renata L; Silva, Alba C M; Rosenthal, Doris; Ferreira, Andrea C F; Carvalho, Denise P

    2006-08-01

    Sex steroids interfere with the pituitary-thyroid axis function, although the reports have been controversial and no conclusive data is available. Some previous reports indicate that estradiol might also regulate thyroid function through a direct action on the thyrocytes. In this report, we examined the effects of low and high doses of estradiol administered to control and ovariectomized adult female rats and to pre-pubertal females. We demonstrate that estradiol administration to both intact adult and pre-pubertal females causes a significant increase in the relative thyroid weight. Serum T3 is significantly decreased in ovariectomized rats, and is normalized by estrogen replacement. Neither doses of estrogen produced a significant change in serum TSH and total T4 in ovariectomized, adult intact and pre-pubertal rats. The highest, supraphysiological, estradiol dose produced a significant increase in thyroid iodide uptake in ovariectomized and in pre-pubertal rats, but not in control adult females. Thyroperoxidase activity was significantly higher in intact adult rats treated with both estradiol doses and in ovariectomized rats treated with the highest estradiol dose. Since serum TSH levels were not significantly changed, we suggest a direct action of estradiol on the thyroid gland, which depends on the age and on the previous gonad status of the animal.

  10. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

    SciTech Connect

    Peterson, K.M.; Alderete, J.F.

    1984-08-01

    Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

  11. Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response.

    PubMed

    dos Santos, Nathália Villa; Matias, Andreza Cândido; Higa, Guilherme Shigueto Vilar; Kihara, Alexandre Hiroaki; Cerchiaro, Giselle

    2015-01-01

    The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion.

  12. Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity.

    PubMed

    Fang, Xian-Kang; Gao, Jie; Zhu, Dan-Ni

    2008-03-12

    Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist. When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

  13. AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway.

    PubMed

    Chen, Bo; Li, Jin; Zhu, Haibo

    2016-10-01

    The differentiation of macrophages into lipid-laden foam cells is a hallmark in early-stage atherosclerosis. The developmental role of adenosine monophosphate-activated protein kinase (AMPK) in a transformation of foam cells, especially in macrophage cholesterol uptake that remains undetermined. Here we demonstrate that AMPK activation in response to IMM-H007 or AICAR resulted in a decrease in macrophage cholesterol uptake and thus inhibited foam cell formation in macrophages mediated by oxidized low-density lipoprotein (oxLDL). This functional change was caused by a downregulation of mRNA and protein expression of LOX-1 but not other scavenger receptors, including scavenger receptor-A (SR-A), CD36 and scavenger receptor-BI (SR-BI). The expression of LOX-1 was regulated by AMPK activation induced decreased phosphorylation of nuclear transcription factor NF-κB, since siRNA interference or dominant negative AMPK overexpression significantly promotes Ser536 dephosphorylation of NF-κB p65 and thus increases LOX-1 expression. Moreover, pharmacological AMPK activation was shown to promote protein phosphatase 2A (PP2A) activity and the specific PP2A inhibitor, okadaic acid, could prevent the effects of IMM-H007 or AICAR on NF-κB and LOX-1. In vivo, pharmacological AMPK activation reduced the lesion size of atherosclerosis and the expression of LOX-1 in aortas in apolipoprotein E-deficient mice. Our current findings suggest a novel mechanism of LOX-1 regulation by AMPK to attenuate macrophage oxLDL uptake and atherosclerosis.

  14. Uptake Mechanisms of Eu(III) on Hydroxyapatite: A Potential Permeable Reactive Barrier Backfill Material for Trapping Trivalent Minor Actinides.

    PubMed

    Xu, Lin; Zheng, Tao; Yang, Shitong; Zhang, Linjuan; Wang, Jianqiang; Liu, Wei; Chen, Lanhua; Diwu, Juan; Chai, Zhifang; Wang, Shuao

    2016-04-05

    The permeable reactive barrier (PRB) technique has attracted an increasing level of attention for the in situ remediation of contaminated groundwater. In this study, the macroscopic uptake behaviors and microscopic speciation of Eu(III) on hydroxyapatite (HAP) were investigated by a combination of theoretical modeling, batch experiments, powder X-ray diffraction (PXRD) fitting, and X-ray absorption spectroscopy (XAS). The underlying removal mechanisms were identified to further assess the application potential of HAP as an effective PRB backfill material. The macroscopic analysis revealed that nearly all dissolved Eu(III) in solution was removed at pH 6.5 within an extremely short reaction time of 5 min. In addition, the thermodynamic calculations, desorption experiments, and PXRD and XAS analyses definitely confirmed the formation of the EuPO4·H2O(s) phase during the process of uptake of dissolved Eu(III) by HAP via the dissolution-precipitation mechanism. A detailed comparison of the present experimental findings and related HAP-metal systems suggests that the relative contribution of precipitation to the total Eu(III) removal increases as the P:Eu ratio decreases. The dosage of HAP-based PRB for the remediation of groundwater polluted by Eu(III) and analogous trivalent actinides [e.g., Am(III) and Cm(III)] should be strictly controlled depending on the dissolved Eu(III) concentration to obtain an optimal P:M (M represents Eu, Am, or Cm) ratio and treatment efficiency.

  15. The Yin: An adverse health perspective of nanoceria: uptake, distribution, accumulation, and mechanisms of its toxicity

    PubMed Central

    Yokel, Robert A.; Hussain, Salik; Garantziotis, Stavros; Demokritou, Philip; Castranova, Vincent; Cassee, Flemming R.

    2014-01-01

    This critical review evolved from a SNO Special Workshop on Nanoceria panel presentation addressing the toxicological risks of nanoceria: accumulation, target organs, and issues of clearance; how exposure dose/concentration, exposure route, and experimental preparation/model influence the different reported effects of nanoceria; and how can safer by design concepts be applied to nanoceria? It focuses on the most relevant routes of human nanoceria exposure and uptake, disposition, persistence, and resultant adverse effects. The pulmonary, oral, dermal, and topical ocular exposure routes are addressed as well as the intravenous route, as the latter provides a reference for the pharmacokinetic fate of nanoceria once introduced into blood. Nanoceria reaching the blood is primarily distributed to mononuclear phagocytic system organs. Available data suggest nanoceria’s distribution is not greatly affected by dose, shape, or dosing schedule. Significant attention has been paid to the inhalation exposure route. Nanoceria distribution from the lung to the rest of the body is less than 1% of the deposited dose, and from the gastrointestinal tract even less. Intracellular nanoceria and organ burdens persist for at least months, suggesting very slow clearance rates. The acute toxicity of nanoceria is very low. However, large/accumulated doses produce granuloma in the lung and liver, and fibrosis in the lung. Toxicity, including genotoxicity, increases with exposure time; the effects disappear slowly, possibly due to nanoceria’s biopersistence. Nanoceria may exert toxicity through oxidative stress. Adverse effects seen at sites distal to exposure may be due to nanoceria translocation or released biomolecules. An example is elevated oxidative stress indicators in the brain, in the absence of appreciable brain nanoceria. Nanoceria may change its nature in biological environments and cause changes in biological molecules. Increased toxicity has been related to greater surface

  16. Ecophysiological mechanisms characterising fen and bog species: focus on variations in nitrogen uptake traits under different soil-water pH.

    PubMed

    Nakamura, Takatoshi; Nakamura, Motoka

    2012-04-01

    Although the productivity and nitrogen (N)-use traits of mire plants differ dramatically between fens and bogs, soil N richness does not necessarily differ, whereas the soil-water pH is distinctly lower in bogs than in fens. The ecophysiological mechanisms underlying these relations are unclear. To assess the relative availability of N forms in relation to soil-water pH, we focused on the net N uptake rate per unit root weight (NNUR), glutamine synthetase activity and nitrate reductase activity, and performed reciprocal transplant experiments with the seedlings of fen (Carex lyngbyei) and bog (C. middendorffii) sedge species in intact habitat sites. The soil-water pH was clearly lower at the bog site, but the NH(4) (+), NO(3) (-) or dissolved organic-N concentrations did not differ between the fen and bog sites. The activity of both enzymes for inorganic-N assimilation did not differ among the sites and species. However, the fen species grown at bog sites showed a drastic decrease in the NNUR, suggesting a suppression of organic-N uptake. The bog species showed no NNUR difference between the sites. These results indicate that inorganic-N availability does not differ between the two habitats, but organic-N availability is lowered in a low-pH bog, particularly in the case of fen species. Therefore, the relative availability of N forms shows species-specific variations that depend on the differences in the soil-water pH of root zone, even at similar N richness, which would play a key role in plant distribution strategies in relation to the fen-bog gradient.

  17. Direct evidence of iron uptake by the Gram-positive siderophore-shuttle mechanism without iron reduction.

    PubMed

    Fukushima, Tatsuya; Allred, Benjamin E; Raymond, Kenneth N

    2014-09-19

    Iron is an essential element for all organisms, and microorganisms produce small molecule iron-chelators, siderophores, to efficiently acquire Fe(III). Gram-positive bacteria possess lipoprotein siderophore-binding proteins (SBPs) on the membrane. Some of the SBPs bind both apo-siderophores (iron-free) and Fe-siderophore (iron-chelated) and only import Fe-siderophores. When the SBP initially binds an apo-siderophore, the SBP uses the Gram-positive siderophore-shuttle mechanism (the SBPs exchange Fe(III) from a Fe-siderophore to the apo-siderophore bound to the protein) and/or displacement mechanism (the apo-siderophore bound to the SBP is released and a Fe-siderophore is then bound to the protein) to import the Fe-siderophore. Previously, we reported that the Bacillus cereus SBP, YxeB, exchanges Fe(III) from a ferrioxamine B (FO) to a desferrioxamine B (DFO) bound to YxeB using the siderophore-shuttle mechanism although the iron exchange was indirectly elucidated. Synthetic Cr-DFO (inert metal FO analog) and Ga-DFO (nonreducible FO analog) are bound to YxeB and imported via YxeB and the corresponding permeases and ATPase. YxeB exchanges Fe(III) from FO and Ga(III) from Ga-DFO to DFO bound to the protein, indicating that the metal-exchange occurs without metal reduction. YxeB also binds DFO derivatives including acetylated DFO (apo-siderophore) and acetylated FO (AcFO, Fe-siderophore). The iron from AcFO is transferred to DFO when bound to YxeB, giving direct evidence of iron exchange. Moreover, YxeB also uses the displacement mechanism when ferrichrome (Fch) is added to the DFO:YxeB complex. Uptake by the displacement mechanism is a minor pathway compared to the shuttle mechanism.

  18. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  19. Mechanism of potassium ion uptake by the Na+/K+-ATPase

    PubMed Central

    Castillo, Juan P.; Rui, Huan; Basilio, Daniel; Das, Avisek; Roux, Benoît; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

    2015-01-01

    The Na+/K+-ATPase restores sodium (Na+) and potassium (K+) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na+ ions are released, followed by the binding and occlusion of two K+ ions. While the mechanisms of Na+ release have been well characterized by the study of transient Na+ currents, smaller and faster transient currents mediated by external K+ have been more difficult to study. Here we show that external K+ ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K+ gating different from that of Na+ occlusion. PMID:26205423

  20. Mechanism of potassium ion uptake by the Na(+)/K(+)-ATPase.

    PubMed

    Castillo, Juan P; Rui, Huan; Basilio, Daniel; Das, Avisek; Roux, Benoît; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

    2015-07-24

    The Na(+)/K(+)-ATPase restores sodium (Na(+)) and potassium (K(+)) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na(+) ions are released, followed by the binding and occlusion of two K(+) ions. While the mechanisms of Na(+) release have been well characterized by the study of transient Na(+) currents, smaller and faster transient currents mediated by external K(+) have been more difficult to study. Here we show that external K(+) ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K(+) gating different from that of Na(+) occlusion.

  1. Mechanism of potassium ion uptake by the Na+/K+-ATPase

    NASA Astrophysics Data System (ADS)

    Castillo, Juan P.; Rui, Huan; Basilio, Daniel; Das, Avisek; Roux, Benoît; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

    2015-07-01

    The Na+/K+-ATPase restores sodium (Na+) and potassium (K+) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na+ ions are released, followed by the binding and occlusion of two K+ ions. While the mechanisms of Na+ release have been well characterized by the study of transient Na+ currents, smaller and faster transient currents mediated by external K+ have been more difficult to study. Here we show that external K+ ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K+ gating different from that of Na+ occlusion.

  2. Inactive lipoprotein lipase (LPL) alone increases selective cholesterol ester uptake in vivo, whereas in the presence of active LPL it also increases triglyceride hydrolysis and whole particle lipoprotein uptake.

    PubMed

    Merkel, Martin; Heeren, Jörg; Dudeck, Wiebke; Rinninger, Franz; Radner, Herbert; Breslow, Jan L; Goldberg, Ira J; Zechner, Rudolf; Greten, Heiner

    2002-03-01

    We have previously shown that transgenic expression of catalytically inactive lipoprotein lipase (LPL) in muscle (Mck-N-LPL) enhances triglyceride hydrolysis as well as whole particle lipoprotein and selective cholesterol ester uptake. In the current study, we have examined whether these functions can be performed by inactive LPL alone or require the presence of active LPL expressed in the same tissue. To study inactive LPL in the presence of active LPL in the same tissue, the Mck-N-LPL transgene was bred onto the heterozygous LPL-deficient (LPL1) background. At 18 h of age, Mck-N-LPL reduced triglycerides by 35% and markedly increased muscle lipid droplets. In adult mice, it reduced triglycerides by 40% and increased lipoprotein particle uptake into muscle by 60% and cholesterol ester uptake by 110%. To study inactive LPL alone, the Mck-N-LPL transgene was bred onto the LPL-deficient (LPL0) background. These mice die at approximately 24 h of age. At 18 h of age, in the absence of active LPL, inactive LPL expression did not diminish triglycerides nor did it result in the accumulation of muscle lipid droplets. To study inactive LPL in the absence of active LPL in the same tissue in adult animals, the Mck-N-LPL transgene was bred onto mice that only expressed active LPL in the heart (LPL0/He-LPL). In this case, Mck-N-LPL did not reduce triglycerides or increase the uptake of lipoprotein particles but did increase muscle uptake of chylomicron and very low density lipoprotein cholesterol ester by 40%. Thus, in the presence of active LPL in the same tissue, inactive LPL augments triglyceride hydrolysis and increases whole particle triglyceride-rich lipoprotein and selective cholesterol ester uptake. In the absence of active LPL in the same tissue, inactive LPL only mediates selective cholesterol ester uptake.

  3. The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis.

    PubMed

    Chan, Olivia; Burke, J Daniel; Gao, Darrin F; Fish, Eleanor N

    2012-08-24

    Recruitment of effector T cells to sites of infection or inflammation is essential for an effective adaptive immune response. The chemokine CCL5 (RANTES) activates its cognate receptor, CCR5, to initiate cellular functions, including chemotaxis. In earlier studies, we reported that CCL5-induced CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Specifically, CCL5-mediated mTOR activation contributes to T cell chemotaxis by initiating the synthesis of chemotaxis-related proteins. Up-regulation of chemotaxis-related proteins may prime T cells for efficient migration. It is now clear that mTOR is also a central regulator of nutrient sensing and glycolysis. Herein we describe a role for CCL5-mediated glucose uptake and ATP accumulation to meet the energy demands of chemotaxis in activated T cells. We provide evidence that CCL5 is able to induce glucose uptake in an mTOR-dependent manner. CCL5 treatment of ex vivo activated human CD3(+) T cells also induced the activation of the nutrient-sensing kinase AMPK and downstream substrates ACC-1, PFKFB-2, and GSK-3β. Using 2-deoxy-d-glucose, an inhibitor of glucose uptake, and compound C, an inhibitor of AMPK, experimental data are presented that demonstrate that CCL5-mediated T cell chemotaxis is dependent on glucose, as these inhibitors inhibit CCL5-mediated chemotaxis in a dose-dependent manner. Altogether, these findings suggest that both glycolysis and AMPK signaling are required for efficient T cell migration in response to CCL5. These studies extend the role of CCL5 mediated CCR5 signaling beyond lymphocyte chemotaxis and demonstrate a role for chemokines in promoting glucose uptake and ATP production to match energy demands of migration.

  4. Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

    PubMed

    Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

    2014-01-15

    Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

  5. Mechanisms of nickel uptake, and hyperaccumulation by plants and implications to soil remediation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil contamination by heavy metals like Ni was originally restricted to metalliferous soils but in recent years it has become a general problem due to the increasingly frequent anthropogenic activities. Because of the characteristics of cost-effectiveness, environmental friendliness, and fewer side...

  6. Siderophore-mediated mechanism of gallium uptake demonstrated in the microorganism Ustilago sphaerogena

    SciTech Connect

    Emery, T.; Hoffer, P.B.

    1980-10-01

    The radioactive gallium analog of ferrichrome, Ga-67 deferriferrichrome, has been prepared and compared with ferrichrome in the specific siderophore-transport system of Ustilago sphaerogena. The gallium analog is taken up by the cells in an active transport process indistinguishable from that of ferrichrome. The findings may explain how Ga(III) mimics Fe(III) in clinical use.

  7. Empowerment beliefs and intention to uptake cervical cancer screening: three psychosocial mediating mechanisms.

    PubMed

    Luszczynska, Aleksandra; Durawa, Alicja B; Scholz, Urte; Knoll, Nina

    2012-01-01

    Three studies tested if the associations between women's empowerment beliefs and intentions to attend cervical cancer screening could be explained by mediating psychological mechanisms: control-related beliefs, well being-related beliefs, and beliefs and evaluations referring to social functioning. Data were collected from January to March 2011 in the rural and urban areas across regions of Poland. Study 1 (N = 386) indicated that women with strong empowerment harbored stronger self-efficacy and beliefs that screening participation would make them feel in control of their own health and body. These two types of cognitions were, in turn, associated with stronger cervical cancer screening intentions. Results of Study 2 (N = 527) confirmed three significant well being-related mediators in the relationship between empowerment beliefs and cervical cancer screening: perceived benefits of screening related to well being, appearance satisfaction, discomfort- and shame-related barriers for screening. Finally, Study 3 (N = 424) showed that empowerment enabled receiving higher social support for cervical cancer screening, promoted perceiving fewer barriers for cervical cancer screening-related communication and more social benefits of engaging in cervical cancer screening. Support for cervical cancer screening, social barriers, and benefits were, in turn, related to screening intentions. Across the studies similar shares of intention variance were explained, and thus the hypothesized mediating mechanisms may have similar explanatory power.

  8. Potential fluid mechanic pathways of platelet activation.

    PubMed

    Shadden, Shawn C; Hendabadi, Sahar

    2013-06-01

    Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.

  9. Uptake of Free Choline by Isolated Perfused Rat Liver

    NASA Astrophysics Data System (ADS)

    Zeisel, Steven H.; Story, David L.; Wurtman, Richard J.; Brunengraber, Henri

    1980-08-01

    The uptake of free choline by isolated perfused rat liver was characterized. A saturable uptake mechanism [Ka=0.17± 0.07 mM (SD); Vmax=0.84± 0.16\\ μ mol/min × g dry weight] and a nonsaturable mechanism (through which uptake is proportional to choline concentration in the perfusate) were identified. Most of the choline transported into hepatocytes was converted to betaine, phosphorylcholine, or lecithin. Free choline also accumulated within the intracellular space, suggesting that choline oxidase activity does not always limit choline's uptake by the liver.

  10. Modulation of methylmercury uptake by methionine: Prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism

    PubMed Central

    Roos, Daniel Henrique; Puntel, Robson Luiz; Farina, Marcelo; Aschner, Michael; Bohrer, Denise; Rocha, João Batista T.; de Vargas Barbosa, Nilda B.

    2016-01-01

    Methylmercury (MeHg) is an ubiquitous environmental pollutant which is transported into the mammalian cells when present as the methylmercury-cysteine conjugate (MeHg–Cys). With special emphasis on hepatic cells, due to their particular propensity to accumulate an appreciable amount of Hg after exposure to MeHg, this study was performed to evaluate the effects of methionine (Met) on Hg uptake, reactive species (RS) formation, oxygen consumption and mitochondrial function/cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg–Cys complex. The liver slices were pre-treated with Met (250 μM) 15 min before being exposed to MeHg (25 μM) or MeHg–Cys (25 μM each) for 30 min at 37 °C. The treatment with MeHg caused a significant increase in the Hg concentration in both liver slices and mitochondria isolated from liver slices. Moreover, the Hg uptake was higher in the group exposed to the MeHg–Cys complex. In the DCF (dichlorofluorescein) assay, the exposure to MeHg and MeHg–Cys produced a significant increase in DFC reactive species (DFC-RS) formation only in the mitochondria isolated from liver slices. As observed with Hg uptake, DFC-RS levels were significantly higher in the mitochondria treated with the MeHg–Cys complex compared to MeHg alone. MeHg exposure also caused a marked decrease in the oxygen consumption of liver slices when compared to the control group, and this effect was more pronounced in the liver slices treated with the MeHg–Cys complex. Similarly, the loss of mitochondrial activity/cell viability was greater in liver slices exposed to the MeHg–Cys complex when compared to slices treated only with MeHg. In all studied parameters, Met pre-treatment was effective in preventing the MeHg-and/or MeHg–Cys-induced toxicity in both liver slices and mitochondria. Part of the protection afforded by Met against MeHg may be related to a direct interaction with MeHg or to the

  11. Modulation of methylmercury uptake by methionine: Prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism

    SciTech Connect

    Roos, Daniel Henrique; Puntel, Robson Luiz; Farina, Marcelo; Aschner, Michael; Bohrer, Denise; Rocha, Joao Batista T.; Vargas Barbosa, Nilda B. de

    2011-04-01

    Methylmercury (MeHg) is an ubiquitous environmental pollutant which is transported into the mammalian cells when present as the methylmercury-cysteine conjugate (MeHg-Cys). With special emphasis on hepatic cells, due to their particular propensity to accumulate an appreciable amount of Hg after exposure to MeHg, this study was performed to evaluate the effects of methionine (Met) on Hg uptake, reactive species (RS) formation, oxygen consumption and mitochondrial function/cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg-Cys complex. The liver slices were pre-treated with Met (250 {mu}M) 15 min before being exposed to MeHg (25 {mu}M) or MeHg-Cys (25 {mu}M each) for 30 min at 37 {sup o}C. The treatment with MeHg caused a significant increase in the Hg concentration in both liver slices and mitochondria isolated from liver slices. Moreover, the Hg uptake was higher in the group exposed to the MeHg-Cys complex. In the DCF (dichlorofluorescein) assay, the exposure to MeHg and MeHg-Cys produced a significant increase in DFC reactive species (DFC-RS) formation only in the mitochondria isolated from liver slices. As observed with Hg uptake, DFC-RS levels were significantly higher in the mitochondria treated with the MeHg-Cys complex compared to MeHg alone. MeHg exposure also caused a marked decrease in the oxygen consumption of liver slices when compared to the control group, and this effect was more pronounced in the liver slices treated with the MeHg-Cys complex. Similarly, the loss of mitochondrial activity/cell viability was greater in liver slices exposed to the MeHg-Cys complex when compared to slices treated only with MeHg. In all studied parameters, Met pre-treatment was effective in preventing the MeHg- and/or MeHg-Cys-induced toxicity in both liver slices and mitochondria. Part of the protection afforded by Met against MeHg may be related to a direct interaction with MeHg or to the competition

  12. The Use of a Mercury Biosensor to Evaluate the Bioavailability of Mercury-Thiol Complexes and Mechanisms of Mercury Uptake in Bacteria.

    PubMed

    Ndu, Udonna; Barkay, Tamar; Mason, Robert P; Traore Schartup, Amina; Al-Farawati, Radwan; Liu, Jie; Reinfelder, John R

    2015-01-01

    As mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)2) and Hg-glutathione (Hg(GSH)2) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)2 was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.

  13. Modelling orange tree root water uptake active area by minimally invasive ERT data and transpiration measurements

    NASA Astrophysics Data System (ADS)

    Vanella, Daniela; Boaga, Jacopo; Perri, Maria Teresa; Consoli, Simona; Cassiani, Giorgio

    2015-04-01

    The comprehension of the hydrological processes involving plant root dynamics is crucial for implementing water saving measures in agriculture. This is particular urgent in areas, like those Mediterranean, characterized by scarce water availability. The study of root water dynamics should not be separated from a more general analysis of the mass and energy fluxes transferred in the soil-plant-atmosphere continuum. In our study, in order to carry this inclusive approach, minimal invasive 3D time-lapse electrical resistivity tomography (ERT) for soil moisture estimation was combined with plant transpiration fluxes directly measured with Sap Flow (SF) techniques and Eddy Covariance methods, and volumetric soil moisture measurements by TDR probes. The main objective of this inclusive approach was to accurately define root-zone water dynamics and individuate the root-area effectively active for water and nutrient uptake process. The monitoring was carried out in Eastern Sicily (south Italy) in summers 2013 and 2014, within an experimental orange orchard farm. During the first year of experiment (October 2013), ERT measurements were carried out around the pertinent volume of one fully irrigated tree, characterized by a vegetation ground cover of 70%; in the second year (June 2014), ERT monitoring was conducted considering a cutting plant, thus to evaluate soil water dynamics without the significant plant transpiration contribution. In order to explore the hydrological dynamics of the root zone volume surrounded by the monitored tree, the resistivity data acquired during the ERT monitoring were converted into soil moisture content distribution by a laboratory calibration based on the soil electrical properties as a function of moisture content and pore water electrical conductivity. By using ERT data in conjunction with the agro-meteorological information (i.e. irrigation rates, rainfall, evapotranspiration by Eddy Covariance, transpiration by Sap Flow and soil moisture

  14. Biophysical mechanisms of trichloroethene uptake and loss in baldcypress growing in shallow contaminated groundwater

    USGS Publications Warehouse

    Nietch, C.T.; Morris, J.T.; Vroblesky, D.A.

    1999-01-01

    Wetland vegetation may be useful in the remediation of shallow contaminated aquifers. Mesocosm experiments were conducted to describe the regulatory mechanisms affecting trichloroethene (TCE) removal rates from groundwater by flood-adapted wetland trees at a contaminated site. TCE flux through baldcypress [Taxodium distichum (L) Rich] seedlings grown in glass- carboys decreased from day to night and from August to December. The diel fluctuation coincided with changes in leaf-level physiology, as the daytime flux was significantly correlated with net photosynthesis but not with respiration at night. A decrease in seedling water use from summer to winter explained the large seasonal difference in TCE flux. A simple model that simulates gas-phase diffusion through aerenchyma tested the importance of diffusion of TCE vapor from roots to the stem. The modeled diffusive flux was within 64% of the observed value during the winter but could only explain 8% of the summer flux. Seedling water use was a good estimator of flux during the summer. Hence, evapotranspiration (ET) in the summer may serve as a good predictor for the potential of TCE removal by baldcypress trees, while diffusive flux may better approximate potential contaminant loss in the winter.Wetland vegetation may be useful in the remediation of shallow contaminated aquifers. Mesocosm experiments were conducted to describe the regulatory mechanisms affecting trichloroethene (TCE) removal rates from groundwater by flood-adapted wetland trees at a contaminated site. TCE flux through baldcypress [Taxodium distichum (L) Rich] seedlings grown in glass-carboys decreased from day to night and from August to December. The diel fluctuation coincided with changes in leaf-level physiology, as the daytime flux was significantly correlated with net photosynthesis but not with respiration at night. A decrease in seedling water use from summer to winter explained the large seasonal difference in TCE flux. A simple model that

  15. Chemical carcinogenesis in feral fish: uptake, activation, and detoxication of organic xenobiotics.

    PubMed Central

    Varanasi, U; Stein, J E; Nishimoto, M; Reichert, W L; Collier, T K

    1987-01-01

    The high prevalence of liver neoplasms in English sole (Parophrys vetulus) and substantially lower prevalence of neoplasms in a closely related species, starry flounder (Platichthys stellatus) captured from industrialized waterways, provide a unique opportunity to compare biochemical processes involved in chemical carcinogenesis in feral fish species. Because levels of aromatic hydrocarbons (AHs) in urban sediments are correlated with prevalences of liver neoplasms in English sole, we have initiated detailed studies to evaluate the effects of endogenous and exogenous factors on uptake, activation and detoxication of carcinogenic AHs, such as benzo[a]pyrene (BaP), using spectroscopic, chromatographic, and radiometric techniques. The results obtained thus far show that sole readily takes up AHs associated with sediment from urban areas and that the presence of other xenobiotics, such as PCBs, in sediment increases tissue concentrations of BaP metabolites. Extensive metabolism of BaP occurred whether sole was exposed to this AH via sediment, per os, or intraperitoneally. Substantial modification of hepatic DNA occurred and persisted for a period of 2-4 weeks after a single exposure to BaP. The level of covalent binding of BaP intermediates to hepatic DNA was 10-fold higher in juvenile than adult sole and 90-fold higher in juvenile sole than in Sprague-Dawley rat, a species which is resistant to BaP-induced hepatocarcinogenesis. The level of chemical modification of hepatic DNA in juvenile flounder was 2-4 fold lower than that for juvenile sole and concentration of BaP 7,8-diol glucuronide in bile of sole was significantly higher than that in flounder bile, although the rate of formation of BaP 7,8-diol by hepatic microsomes was comparable for both species. Moreover, liver microsomes from both species, in the presence of exogenous DNA, metabolized BaP into essentially a single adduct, identified as (+)anti-7,8-diol-9,10-epoxy-7,8,9,10-tetrahydroBaP-dG. These results

  16. Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats

    PubMed Central

    Wang, Haiyan; Arias, Edward B.

    2016-01-01

    Skeletal muscle insulin resistance is associated with many common age-related diseases, but moderate calorie restriction (CR) can substantially elevate glucose uptake by insulin-stimulated skeletal muscle from both young and old rats. The current study evaluated the isolated epitrochlearis muscle from ∼24.5-mo-old rats that were either fed ad libitum (AL) or subjected to CR (consuming ∼65% of ad libitum, AL, intake beginning at ∼22.5 mo old). Some muscles were also incubated with MK-2206, a potent and selective Akt inhibitor. The most important results were that in isolated muscles, CR vs. AL resulted in 1) greater insulin-stimulated glucose uptake 2) that was accompanied by significantly increased insulin-mediated activation of Akt2, as indicated by greater phosphorylation on both Thr309 and Ser474 along with greater Akt2 activity, 3) concomitant with enhanced phosphorylation of several Akt substrates, including an Akt substrate of 160 kDa on Thr642 and Ser588, filamin C on Ser2213 and proline-rich Akt substrate of 40 kDa on Thr246, but not TBC1D1 on Thr596; and 4) each of the CR effects was eliminated by MK-2206. These data provide compelling new evidence linking greater Akt2 activation to the CR-induced elevation of insulin-stimulated glucose uptake by muscle from old animals. PMID:26739650

  17. The Design, Synthesis and Structure-Activity Relationship of Mixed Serotonin, Norepinephrine and Dopamine Uptake Inhibitors

    NASA Astrophysics Data System (ADS)

    Chen, Zhengming; Yang, Ji; Skolnick, Phil

    The evolution of antidepressants over the past four decades has involved the replacement of drugs with a multiplicity of effects (e.g., TCAs) by those with selective actions (i.e., SSRIs). This strategy was employed to reduce the adverse effects of TCAs, largely by eliminating interactions with certain neurotransmitters or receptors. Although these more selective compounds may be better tolerated by patients, selective drugs, specifically SSRIs, are not superior to older drugs in treating depressed patients as measured by response and remission rates. It may be an advantage to increase synaptic levels of both serotonin and norepinephrine, as in the case of dual uptake inhibitors like duloxetine and venlafaxine. An important recent development has been the emergence of the triple-uptake inhibitors (TUIs/SNDRIs), which inhibit the uptake of the three neurotransmitters most closely linked to depression: serotonin, norepinephrine, and dopamine. Preclinical studies and clinical trials indicate that a drug inhibiting the reuptake of all three of these neurotransmitters could produce more rapid onset of action and greater efficacy than traditional antidepressants. This review will detail the medicinal chemistry involved in the design, synthesis and discovery of mixed serotonin, norepinephrine and dopamine transporter uptake inhibitors.

  18. Effect of anesthetics on microglial activation and nanoparticle uptake: Implications for drug delivery in traumatic brain injury.

    PubMed

    Kannan, Gokul; Kambhampati, Siva P; Kudchadkar, Sapna R

    2017-03-21

    Traumatic brain injury (TBI) is a serious public health problem, often with devastating consequences for patients and their families. Affordable and timely therapies can have a substantial impact on outcomes in severe TBI. Despite the common use of sedatives and anesthetics in the acute phase of TBI management, their effect on glial cells is not well understood. We investigated the effect of a commonly used sedative, pentobarbital, on glial cells and their uptake of nanoparticles. First, we studied how pentobarbital affects BV2 mouse microglial cells in culture. The cell morphology was imaged by confocal microscopy and analyzed. Our results suggest that microglia change to a more swollen, 'activated' shape with pentobarbital (cell area increased by approximately 20%, p<0.001). Such glial activation may have negative implications for the ability of the injured brain to clear edema. Second, we investigated how pentobarbital treatment affected nanoparticle uptake. BV-2 mouse microglial cells in the presence and absence of pentobarbital were treated with fluorescently-labeled, hydroxyl-functionalized poly(amidoamine) dendrimer nanoparticles (Dendrimer-Cy5). We demonstrated that the presence of pentobarbital increased the dendrimer nanoparticle uptake significantly (~2-fold both 2 and 6h following treatment). This semi-quantitative fluorescence assessment was broadly consistent among confocal image analysis, flow cytometry, and fluorescence quantification of cell-extracted dendrimer-Cy5. Although anesthetics appear to activate microglia, the increased uptake of dendrimer nanoparticles in their presence can be exploited to deliver drug-loaded nanoparticles directly to microglia after TBI. These drugs could restore glial and glymphatic function, enabling efficient drainage of waste and fluid from the brain and effectively improving recovery after TBI. A key future direction is to validate these findings in TBI models.

  19. Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

    2015-07-01

    4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes.

  20. [Activation and inhibitory mechanisms of blood platelets].

    PubMed

    Suzuki-Inoue, Katsue

    2014-07-01

    Exposure of platelets to subendothelial matrices initiates physiological hemostasis and pathological thrombosis. Under high shear stress, von Willebrand factor bridges newly exposed collagen to glycoprotein (GP) Ib on platelets. This initial tethering facilitates association between the collagen receptor GPVI and collagen, which generates tyrosine kinase-dependent activation signals, followed by release of secondary mediators and integrin activation. Activated integrin can bind to their ligands including fibrinogen. The released secondary mediators, ADP and thromboxane A2, activate integrin of flowing platelets, which enables formation of platelet thrombi by binding of activated flowing platelets and adhered platelets to collagen via binding between activated aIIbbeta3 integrin and fibrinogen. Platelets also have inhibitory mechanisms, which help to prevent unwanted platelet activation in vivo.

  1. Spontaneous mechanical activity in depolarized frog ventricle

    PubMed Central

    1976-01-01

    Spontaneous mechanical activity can be produced in depolarized frog ventricle by bathing the tissue in a solution with low Na, Iow Ca, and high K+. The contractions can be inhibited by depleting the tissue of Ca first, but they are relatively insensitive to changes in either extracellular [Ca++] or [Ca++]/[Na+]2. They are terminated very rapidly by raising [Na+] to 40 mM. Local anesthetics enhance the spontaneous activity in proportion to the concentration of their free base form. These contractions occur relatively rhythmically for several hours. Since the preparation is multicellular, this suggests a mechanism for intercellular communication without change in membrane potential. PMID:822122

  2. Ionic status, calcium uptake, and Ca2+-ATPase activity during early development in the purple sea urchin (Strongylocentrotus purpuratus).

    PubMed

    Tellis, Margaret S; Lauer, Mariana M; Nadella, Sunita; Bianchini, Adalto; Wood, Chris M

    2013-10-01

    Ionic status during early development was investigated in the purple sea urchin. Whole body cation concentrations (Ca(2+), Na(+), K(+), Mg(2+)), unidirectional Ca(2+) uptake rates measured with (45)Ca(2+), Ca(2+)-ATPase activity, and growth were examined at 12h intervals over the first 96h of development. Whole body Ca(2+) concentration was low initially but increased steadily by >15-fold through to the pluteus stage. Whole body Mg(2+), K(+) and Na(+) levels exhibited diverse patterns, but all increased at 72-96h. Ca(2+) uptake rates were low during initial cell cleavages at 12h but increased greatly at blastulation (24h) and then again at gastrulation (48h), declining thereafter in the pluteus stage, but increasing slightly at 96h. Ca(2+)-ATPase activity was initially low but increased at blastulation through gastrulation (24-48h) but declined thereafter in the pluteus stage. Embryonic weights did not change over most of development, but were significantly higher at 96h. Overall, the gastrulation stage displayed the most pronounced changes, as Ca(2+) uptake and accumulation and Ca(2+)-ATPase levels were the highest at this stage, likely involved in mineralization of the spicule. Biomarkers of Ca(2+) metabolism may be good endpoints for potential future toxicity studies.

  3. Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations

    SciTech Connect

    Lomax, T.L.; Mehlhorn, R.J.; Briggs, W.R.

    1985-10-01

    Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake.

  4. Active principle of swine prostate extract: I. Isolation of active principle activating prostatic acid phosphatase and its effect on testosterone uptake of the prostate in castrated rats.

    PubMed

    Yoshida, Y; Mori, H; Inami, K; Koda, A

    1991-07-01

    There have been several reports concerning the therapeutic effect of an extract from animal prostates on benign prostatic hypertrophy. Previously, we reported that the swine prostate extract (PE) had the activity to enhance human prostatic acid phosphatase (PAPase) activity in vitro, and to increase the muscular tonicity of the urinary bladder by directly acting upon vesical muscles, suggesting that PE have an activity to elevate the intravesical voiding pressure in vivo. In the present study, it was attempted to isolate such an active principle of PE as activates human prostatic acid phosphatase (PAPase). The finally purified PE (PPE) was assessed as to some physico-chemical and pharmacological properties. 1) PPE was found to be a peptide with a molecular weight of about 8,800, composed largely of neutral amino acids (approximately 70%) and few of aromatic amino acids. 2) PPE activated PAPase in a dose-dependent fashion, resulting in an increase of the enzyme activity approximately twice in a dose of 2 X 10(-5) g/ml of PPE. Furthermore, PPE recovered PAPase activity dose-dependently from the 50% inhibition by 2 X 10(-3) M L-tartaric acid. 3) In castrated rats, the 3H-testosterone uptake of the prostate was significantly suppressed by the oral administration of PPE. PPE might be one of active principles of PE for the therapeutic effect on prostatic hypertrophy.

  5. Isolation and characterization of Pichia heedii mutants defective in xylose uptake

    SciTech Connect

    Does, A.L.; Bisson, L.F. )

    1990-11-01

    To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.

  6. Oncogenic activation of the PI3K/Akt pathway promotes cellular glucose uptake by downregulating the expression of thioredoxin-interacting protein.

    PubMed

    Hong, Shin Yee; Yu, Fa-Xing; Luo, Yan; Hagen, Thilo

    2016-05-01

    Oncogenic activation of the PI3K/Akt pathway is known to play an important role to promote glucose metabolism in cancer cells. However, the molecular mechanism through which the PI3K/Akt signalling pathway promotes glucose utilisation in cancer cells is still not well understood. It has recently been shown that the oncogenic activation of the PI3K/Akt/mTOR signalling in lung adenocarcinoma is important in promoting the localisation of glucose transporter 1 (GLUT1) at the plasma membrane. We thus hypothesised that the effect of constitutive activation of the PI3K/AKT signalling on glucose metabolism is mediated by thioredoxin interacting protein (TXNIP), a known regulator of the GLUT1 plasma membrane localisation. Consistent with previous studies, inhibition of the PI3K/Akt pathway decreased cellular glucose uptake. Furthermore, inhibition of PI3K/Akt signalling in non-small cell lung cancer (NSCLC) cell lines using clinically used tyrosine kinase inhibitors (TKIs) resulted in a decrease in GLUT1 membrane localisation. We also observed that inhibition of the PI3K/Akt pathway in various cell lines, including NSCLC cells, resulted in an increase in TXNIP expression. Importantly, knockdown of TXNIP using siRNA in the NSCLC cells promoted GLUT1 to be localised at the plasma membrane and reversed the effect of PI3K/Akt inhibitors. Together, our results suggest that the oncogenic activation of PI3K/Akt signalling promotes cellular glucose uptake, at least in part, through the regulation of TXNIP expression. This mechanism may contribute to the Warburg effect in cancer cells.

  7. Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo.

    PubMed

    Kemnade, Jan Ole; Seethammagari, Mamatha; Collinson-Pautz, Mathew; Kaur, Hardeep; Spencer, David M; McCormick, Alison A

    2014-07-16

    Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.

  8. The aspect ratio effect of drug nanocrystals on cellular internalization efficiency, uptake mechanisms, and in vitro and in vivo anticancer efficiencies

    NASA Astrophysics Data System (ADS)

    Tian, Baishun; Zhang, Xiujuan; Yu, Caitong; Zhou, Mengjiao; Zhang, Xiaohong

    2015-02-01

    In this paper, we investigated the aspect ratio (AR) effect of anticancer drug nanocrystals (NCs) on their cellular internalization efficiency, uptake mechanisms, biodistributions as well as in vitro and in vivo antitumor efficiencies. Both confocal imaging and flow cytometry show that shorter NCs with AR = 1.3 have a much faster cellular uptake rate and a much higher anticancer efficacy than longer NCs. All NCs with different ARs were found to enter the cells via an energy-dependent clathrin-mediated pathway. In vivo experiments indicate that NCs with higher ARs have a shorter half-life and are more easily captured by the liver, while the corresponding tumor uptake decreased. We also observed that NCs with the smallest AR have the highest therapeutic efficacy with appreciably less weight loss. These results would assist in the future design of drug NCs and may lead to the development of new drug nanostructures for biomedical applications.In this paper, we investigated the aspect ratio (AR) effect of anticancer drug nanocrystals (NCs) on their cellular internalization efficiency, uptake mechanisms, biodistributions as well as in vitro and in vivo antitumor efficiencies. Both confocal imaging and flow cytometry show that shorter NCs with AR = 1.3 have a much faster cellular uptake rate and a much higher anticancer efficacy than longer NCs. All NCs with different ARs were found to enter the cells via an energy-dependent clathrin-mediated pathway. In vivo experiments indicate that NCs with higher ARs have a shorter half-life and are more easily captured by the liver, while the corresponding tumor uptake decreased. We also observed that NCs with the smallest AR have the highest therapeutic efficacy with appreciably less weight loss. These results would assist in the future design of drug NCs and may lead to the development of new drug nanostructures for biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06743f

  9. Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity.

    PubMed

    Gill, R T; Cha, H J; Jain, A; Rao, G; Bentley, W E

    1998-07-20

    The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

  10. Molecular mechanisms regulating NLRP3 inflammasome activation

    PubMed Central

    Jo, Eun-Kyeong; Kim, Jin Kyung; Shin, Dong-Min; Sasakawa, Chihiro

    2016-01-01

    Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome. PMID:26549800

  11. Duality of G protein-coupled mechanisms for beta-adrenergic activation of NKCC activity in skeletal muscle.

    PubMed

    Gosmanov, Aidar R; Wong, Jennifer A; Thomason, Donald B

    2002-10-01

    Skeletal muscle Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity provides a potential mechanism for regulated K(+) uptake. beta-Adrenergic receptor (beta-AR) activation stimulates skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We examined potential G protein-coupled pathways for beta-AR-stimulated NKCC activity. Inhibition of G(s)-coupled PKA blocked isoproterenol-stimulated NKCC activity in both the slow-twitch soleus muscle and the fast-twitch plantaris muscle. However, the PKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP (8-BrcAMP) were not sufficient to activate NKCC in the plantaris and partially stimulated NKCC activity in the soleus. Isoproterenol-stimulated NKCC activity in the soleus was abolished by pretreatment with pertussis toxin (PTX), indicating a G(i)-coupled mechanism. PTX did not affect the 8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded the isoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus, consistent with NKCC's MAPK dependency. Inhibition of isoproterenol-stimulated ERK activity by PTX treatment was associated with an increase in Akt activation and phosphorylation of Raf-1 on the inhibitory residue Ser(259). These results demonstrate a novel, muscle phenotype-dependent mechanism for beta-AR-mediated NKCC activation that involves both G(s) and G(i) protein-coupled mechanisms.

  12. Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

    PubMed

    Choi, Kyeong-Mi; Kim, Wonkyun; Hong, Jin Tae; Yoo, Hwan-Soo

    2017-02-01

    Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

  13. 1α,25-dihydroxyvitamin D(3) mechanism of action: modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats.

    PubMed

    Zanatta, Leila; Goulart, Paola Bez; Gonçalves, Renata; Pierozan, Paula; Winkelmann-Duarte, Elisa C; Woehl, Viviane Mara; Pessoa-Pureur, Regina; Silva, Fátima Regina Mena Barreto; Zamoner, Ariane

    2012-10-01

    The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D(3) (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10 day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca(2+) uptake and potassium and chloride channels. Also, the Na(+)/K(+)-ATPase inactivation by ouabain mimicked the 1,25D action on (45)Ca(2+) uptake. The Na(+)/K(+)-ATPase inhibition observed herein might lead to intracellular Na(+) accumulation with subsequent L-VDCC opening and consequently increased (45)Ca(2+) (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin-dependent protein kinase (PKCaMII), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase p38 (p38(MAPK)). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.

  14. Effect of Solanum surattense on mitochondrial enzymes in diabetic rats and in vitro glucose uptake activity in L6 myotubes

    PubMed Central

    Sridevi, Muruhan; Kalaiarasi, Pannerselvam; Pugalendi, Kodukkur Viswanathan

    2015-01-01

    Background: S. surattense is widely used in Siddha medicine for various ailments. Objective: The aim was to evaluate the impact of alcoholic leaf-extract of S. surattense on mitochondrial enzymes in streptozotocin (STZ) induced diabetic rats and to study the in vitro muscle glucose uptake activity on L6 myotubes. Materials and Methods: The male albino Wistar rats were randomly divided into five groups of six animals each. Diabetes was induced by intraperitoneal injection of STZ (40 mg/kg body weight). After being confirmed the diabetic rats were treated with alcoholic leaf-extract of S. surattense (100 mg/kg body weight) for 45 days. The biochemical estimations (liver mitochondrial enzymes, antioxidants, thiobarbituric acid reactive substances [TBARS]) and histopathological studies were performed. Further, the in vitro muscle glucose uptake activity in L6 myotubes and messenger RNA (mRNA) expression of glucose transporter-4 (GLUT-4) was performed. Results: In diabetic rats, the activities of liver mitochondrial enzymes were found to be significantly lowered. The mitochondrial TBARS level increased, whereas the activities/level of enzymatic and non-enzymatic antioxidants decreased in diabetic rats. Administration of S. surattense to diabetic rats significantly reversed the above parameters toward normalcy. Furthermore in diabetic rats, the histopathological studies showed growth of adipose tissue and shrinkage of islets in the pancreas, liver showed fatty change with mild inflammation of portal triad, and kidney showed messangial capillary proliferation of glomeruli and fatty infiltration of tubules. Treatment with S. surattense brought back these changes to near normalcy. The extract was analyzed for in vitro muscle glucose uptake activity in L6 myotubes and mRNA expression of GLUT-4 by semi-quantitative reverse transcriptase-polymerase chain reaction. One nano gram per millilitre of S. surattense leaf-extract gave 115% glucose uptake on L6 myotubes. It also showed

  15. Decreases in Maximal Oxygen Uptake Following Long-duration Spaceflight: Role of Convective and Diffusive O2 Transport Mechanisms.

    PubMed

    Ade, Carl J; Broxterman, Ryan M; Moore, Alan; Barstow, Thomas J

    2017-02-02

    We have previously predicted that the decrease in maximal oxygen uptake (VO2max) that accompanies time in microgravity reflects decrements in both convective and diffusive O2 transport to the mitochondria of the contracting myocytes. The aim of this investigation was therefore to quantify the relative changes in convective O2 transport (QO2) and O2 diffusing capacity (DO2) following long duration spaceflight. In 9 astronauts, resting hemoglobin concentration ([Hb]), VO2max, maximal cardiac output (QTmax), and differences in arterial and venous O2 contents (CaO2-CvO2) were obtained retrospectively for International Space Station Increments 19 through 33 (April 2009-November 2012). QO2 and DO2 were calculated from these variables via integration of Fick's Principle of Mass Conservation and Fick's Law of Diffusion. VO2max significantly decreased from pre- to post-flight (-53.9 ± 45.5%, P =0.008). The significant decrease in Q ̇_Tmax (-7.8±9.1%, P =0.05), despite an unchanged [Hb] resulted in a significantly decreased QO2 (-11.4±10.5%, P = 0.02). DO2 significantly decreased from pre- to post-flight by -27.5±24.5% (P =0.04), as did the peak CaO2-CvO2 (-9.2±7.5%, P =0.007). Using linear regression analysis, changes in VO2max were significantly correlated with changes in DO2 (R2=0.47; P = 0.04). These data suggest that space flight decreases both convective and diffusive O2 transport. These results have practical implications for future long-duration space missions and highlight the need to resolve the specific mechanisms underlying these spaceflight-induced changes along the O2 transport pathway.

  16. Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition.

    PubMed

    Goss, Greg; Gilmour, Kathleen; Hawkings, Guy; Brumbach, Jonathan H; Huynh, Maily; Galvez, Fernando

    2011-07-01

    The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.

  17. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    SciTech Connect

    Moreira, Liliana; Araújo, Isabel; Costa, Tito; Correia-Branco, Ana; Faria, Ana; Martel, Fátima; Keating, Elisa

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  18. Mechanobiocatalysis: Modulating Enzymatic Activity with Mechanical Force

    DTIC Science & Technology

    2015-09-28

    displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers resulted in...intrinsic activities displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers ...of eYFP-containing polymer composites via the application of mechanical force, as well as showing that the photophysical properties displayed by

  19. In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

    PubMed

    Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

    2006-06-01

    The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

  20. Topological mechanics: from metamaterials to active matter

    NASA Astrophysics Data System (ADS)

    Vitelli, Vincenzo

    2015-03-01

    Mechanical metamaterials are artificial structures with unusual properties, such as negative Poisson ratio, bistability or tunable acoustic response, which originate in the geometry of their unit cell. At the heart of such unusual behavior is often a mechanism: a motion that does not significantly stretch or compress the links between constituent elements. When activated by motors or external fields, these soft motions become the building blocks of robots and smart materials. In this talk, we discuss topological mechanisms that possess two key properties: (i) their existence cannot be traced to a local imbalance between degrees of freedom and constraints (ii) they are robust against a wide range of structural deformations or changes in material parameters. The continuum elasticity of these mechanical structures is captured by non-linear field theories with a topological boundary term similar to topological insulators and quantum Hall systems. We present several applications of these concepts to the design and experimental realization of 2D and 3D topological structures based on linkages, origami, buckling meta-materials and lastly active media that break time-reversal symmetry.

  1. Mechanically activated artificial cell by using microfluidics

    PubMed Central

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  2. Mechanically activated artificial cell by using microfluidics

    NASA Astrophysics Data System (ADS)

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-09-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

  3. Methylphenidate Decreases ATP Levels and Impairs Glutamate Uptake and Na(+),K(+)-ATPase Activity in Juvenile Rat Hippocampus.

    PubMed

    Schmitz, Felipe; Pierozan, Paula; Rodrigues, André F; Biasibetti, Helena; Grings, Mateus; Zanotto, Bruna; Coelho, Daniella M; Vargas, Carmen R; Leipnitz, Guilhian; Wyse, Angela T S

    2016-11-14

    The study of the long-term neurological consequences of early exposure with methylphenidate (MPH) is very important since this psychostimulant has been widely misused by children and adolescents who do not meet full diagnostic criteria for ADHD. The aim of this study was to examine the effect of early chronic exposure with MPH on amino acids profile, glutamatergic and Na(+),K(+)-ATPase homeostasis, as well as redox and energy status in the hippocampus of juvenile rats. Wistar male rats received intraperitoneal injections of MPH (2.0 mg/kg) or saline solution (controls), once a day, from the 15th to the 45th day of age. Results showed that MPH altered amino acid profile in the hippocampus, decreasing glutamine levels. Glutamate uptake and Na(+),K(+)-ATPase activity were decreased after chronic MPH exposure in the hippocampus of rats. No changes were observed in the immunocontents of glutamate transporters (GLAST and GLT-1), and catalytic subunits of Na(+),K(+)-ATPase (α1, α2, and α3), as well as redox status. Moreover, MPH provoked a decrease in ATP levels in the hippocampus of chronically exposed rats, while citrate synthase, succinate dehydrogenase, respiratory chain complexes activities (II, II-III, and IV), as well as mitochondrial mass and mitochondrial membrane potential were not altered. Taken together, our results suggest that chronic MPH exposure at early age impairs glutamate uptake and Na(+),K(+)-ATPase activity probably by decreasing in ATP levels observed in rat hippocampus.

  4. Use of reference chemicals to determine passive uptake rates of common indoor air VOCs by collocation deployment of active and passive samplers.

    PubMed

    Xian, Qiming; Feng, Yong-Lai; Chan, Cecilia C; Zhu, Jiping

    2011-09-01

    Passive samplers have become more popular in their application in the measurement of airborne chemicals. For volatile organic compounds, the rate of a chemical's diffusivity is a determining factor in the quantity of the chemical being collected for a given passive sampler. While uptake rate of a chemical in the passive sampler can be determined either by collocation deployment of both active and passive samplers or use of controlled facilities such as environmental chambers, a new approach without a need for accurate active flow rate in the collocation experiment was demonstrated in this study. This approach uses chemicals of known uptake rates as references to calculate the actual flow rate of the active sampling in the collocation experiment. The active sampling rate in turn can be used in the determination of the uptake rates of all other chemicals present in the passive samplers. The advantage of such approach is the elimination of the errors in actual active sampling rate associated with low flow employed in the collocation experiment. Using this approach, passive uptake rates of more than 80 volatile organic compounds commonly present in indoor air were determined. These experimentally determined uptake rates correlate well with air diffusivity of the chemicals, indicating the regression equation describing such correlation might be useful in predicting the uptake rates of other volatile organic chemicals in indoor air based on their air diffusivity.

  5. Influence of biologically-active substances on {sup 137}Cs and heavy metals uptake by Barley plant

    SciTech Connect

    Kruglov, Stanislav; Filipas, Alexander

    2007-07-01

    Available in abstract form only. Full text of publication follows: When solving the problem of contaminated agricultural lands rehabilitation, most of attention is concentrated on the effective means which allow the obtaining of ecologically safe production. The minimization of radionuclides and heavy metals (HM) content in farm products on the basis of their migration characteristics in agro-landscapes and with the regard for different factors influencing contaminants behavior in the soil-plant system is of great significance. Our investigation has shown that the effect of biologically active substances (BAS) using for seeds treatment on {sup 137}Cs transfer to barley grown on Cd contaminated soil was dependent on their properties and dosage, characteristics of soil contamination and biological peculiarities of plants, including stage of plants development. Seeds treatment by plant growth regulator Zircon resulted in a significant increase in {sup 137}Cs activity in harvest (40- 50%), increase in K concentration and significant reduction in Ca concentration. Increased Cd content in soil reduced {sup 137}Cs transfer to barley plants by 30-60% (p<0,05) and Zircon application further reduced its concentration. Ambiol and El also reduced {sup 137}Cs uptake by roots and Cd and Pb phyto-toxicity. The experimental data do not make it possible to link the BAS effect on inhibition of {sup 137}Cs absorption by plants directly with their influence on HM phyto-toxicity. The dependence of Concentration Ratio of {sup 137}Cs on the Ambiol and El dose was not proportional and the most significant decrease in the radionuclide uptake by plants was reported with the use of dose showing the most pronounced stimulating effect on the barley growth and development. The pre-sowing seed treatment with Ambiol increased Pb absorption by 35-50% and, on the contrary, decreased Cd uptake by plants by 30-40%. (authors)

  6. The use of a mercury biosensor to evaluate the bioavailability of mercury-thiol complexes and mechanisms of mercury uptake in bacteria

    SciTech Connect

    Ndu, Udonna; Barkay, Tamar; Mason, Robert P.; Schartup, Amina Traore; Al-Farawati, Radwan; Liu, Jie; Reinfelder, John R.; Chang, Yung -Fu

    2015-09-15

    We discuss as mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)2) and Hg-glutathione (Hg(GSH)2) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)2 was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.

  7. The use of a mercury biosensor to evaluate the bioavailability of mercury-thiol complexes and mechanisms of mercury uptake in bacteria

    DOE PAGES

    Ndu, Udonna; Barkay, Tamar; Mason, Robert P.; ...

    2015-09-15

    We discuss as mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversionmore » of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)2) and Hg-glutathione (Hg(GSH)2) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)2 was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.« less

  8. Mechanism of FGF receptor dimerization and activation

    NASA Astrophysics Data System (ADS)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  9. Electrophysiological and amperometric evidence that modafinil blocks the dopamine uptake transporter to induce behavioral activation.

    PubMed

    Federici, M; Latagliata, E C; Rizzo, F R; Ledonne, A; Gu, H H; Romigi, A; Nisticò, R; Puglisi-Allegra, S; Mercuri, N B

    2013-11-12

    Although the wake-promoting drug modafinil has been shown to bind quite exclusively to the dopamine transporter (DAT), its action in the brain has been thought to be partially independent from the facilitation of the dopaminergic signals. Here we used electrophysiological and amperometric techniques to investigate the effects of modafinil on the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and on the synaptic overflow of dopamine in the dorsal striatum from the sliced tissue of wild-type and cocaine-insensitive genetically modified mice (DAT-CI). Moreover, we examined the consequences of modafinil administration on the locomotor behavior of wild-type and DAT-CI mice. In in vitro experiments, modafinil inhibited the spontaneous firing discharge of the dopaminergic neurons. More consistently, it potentiated firing inhibition and the membrane responses caused by exogenously applied dopamine on these cells. Furthermore, it augmented the stimulus-evoked outflow of DA in the striatum. Noteworthy, modafinil caused locomotor activation in wild-type mice. On the other hand, neither the electrophysiological nor the behavioral effects of modafinil were detected in DAT-CI animals. These results demonstrate that modafinil potentiates brain dopaminergic signals via DAT inhibition by acting at the same binding site of cocaine. Therefore, this mechanism of action explains most of the pharmacological properties of this compound in the clinical setting.

  10. Impact of chronic subthalamic high-frequency stimulation on metabolic basal ganglia activity: a 2-deoxyglucose uptake and cytochrome oxidase mRNA study in a macaque model of Parkinson's disease.

    PubMed

    Meissner, Wassilios; Guigoni, Celine; Cirilli, Laetitia; Garret, Maurice; Bioulac, Bernard H; Gross, Christian E; Bezard, Erwan; Benazzouz, Abdelhamid

    2007-03-01

    The mechanisms of action of high-frequency stimulation (HFS) of the subthalamic nucleus (STN) remain only partially understood. Hitherto, experimental studies have suggested that STN-HFS reduces the activity of STN neurons. However, some recent reports have challenged this view, showing that STN-HFS might also increase the activity of globus pallidus internalis (GPi) neurons that are under strong excitatory drive of the STN. In addition, most results emanate from studies applying acute STN-HFS, while parkinsonian patients receive chronic stimulation. Thus, the present study was designed to assess the effect of chronic (10 days) STN-HFS in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primate. For this purpose, 2-deoxyglucose (2-DG) uptake, a measure of global synaptic activity, was assessed in the basal ganglia and the motor thalamus after chronic unilateral STN-HFS. Cytochrome oxidase subunit 1 (COI) mRNA expression, a marker of efferent metabolic activity, was additionally assessed in the globus pallidus. Chronic STN-HFS (i) reversed abnormally decreased 2-DG uptake in the STN of parkinsonian nonhuman primates, (ii) reversed abnormally increased 2-DG accumulation in the GPi while COI mRNA expression was increased, suggesting global activation of GPi neurons, and (iii) reversed abnormally increased 2-DG uptake in the ventrolateral motor thalamus nucleus. The simultaneous decrease in 2-DG uptake and increase in COI mRNA expression are difficult to reconcile with the current model of basal ganglia function and suggest that the mechanisms by which STN-HFS exerts its clinical benefits are more complex than a simple reversal of abnormal activity in the STN and its targets.

  11. Magnetic field effect on growth, arsenic uptake, and total amylolytic activity on mesquite (Prosopis juliflora x P. velutina) seeds

    NASA Astrophysics Data System (ADS)

    Flores-Tavizón, Edith; Mokgalaka-Matlala, Ntebogeng S.; Elizalde Galindo, José T.; Castillo-Michelle, Hiram; Peralta-Videa, Jose R.; Gardea-Torresdey, Jorge L.

    2012-04-01

    Magnetic field is closely related to the cell metabolism of plants [N. A. Belyavskaya, Adv. Space Res. 34, 1566 (2004)]. In order to see the effect of magnetic field on the plant growth, arsenic uptake, and total amylolytic activity of mesquite (Prosopis juliflora x P. velutina) seeds, ten sets of 80 seeds were selected to be oriented with the long axis parallel or randomly oriented to an external magnetic field. The external magnetic field magnitude was 1 T, and the exposition time t = 30 min. Then, the seeds were stored for three days in a plastic bag and then sown on paper towels in a modified Hoagland's nutrient solution. After three days of germination in the dark and three days in light, seedlings were grown hydroponically in modified Hoagland's nutrient solution (high PO42-) containing 0, 10, or 20 ppm of arsenic as As (III) and (V). The results show that the germination ratios, growth, elongation, arsenic uptake, and total amylolytic activity of the long axis oriented mesquite seeds were much higher than those of the randomly oriented seeds. Also, these two sets of seeds showed higher properties than the ones that were not exposed to external magnetic field.

  12. Effect of leukotriene C4 on electromechanical activity and Ca/sup 2 +/ uptake in taenia coli

    SciTech Connect

    Zschauer, A.; Matthews, E.K.; Richardson, B.P.

    1988-10-01

    The actions of leukotriene C4 (LTC4) on electromechanical activity and /sup 45/Ca/sup 2 +/ uptake in guinea pig taenia coli were investigated. The contractile action of LTC4 was abolished by the removal of extracellular Ca/sup 2 +/. LTC4 concentrations eliciting a maximal contraction in normal medium produced no response in preparations depolarized with KCl. In single sucrose gap studies, LTC4 increased both the frequency of electrical spiking and tension. These effects were blocked by the dihydropyridine Ca/sup 2 +/-channel antagonist PY 108-068 and by the leukotriene receptor antagonist FPL 55712. In double sucrose gap experiments, LTC4 caused a small depolarization without measurable change in membrane conductivity; increased spontaneous electrical activity was again accompanied by an increase in tension. LTC4 caused a detectable increase in /sup 45/Ca/sup 2 +/ uptake only at extracellular Ca/sup 2 +/ concentrations less than 1 mM, and this was again inhibited by PY 108-068 or FPL 55712. It is concluded that the contractile effects of LTC4 in guinea pig taenia coli occur as a consequence of its ability to open voltage-sensitive Ca/sup 2 +/ channels, an effect that may occur independently of membrane depolarization.

  13. Activities of the Institute for Mechanical Engineering

    NASA Astrophysics Data System (ADS)

    The Institute of Mechanical Engineering (IME) is part of Canada's National Research Council. Its mission is to undertake, support, promote, and disseminate research and development in the mechanical engineering aspects of three vital sectors of the Canadian economy: transportation, resource industries, and manufacturing. The IME achieves its mission by performing research and development in its own facilities; by developing, providing, and transferring expertise and knowledge; by making its research facilities available to collaborators and clients; and by participating in international liaison and collaborative research activities. Six research programs are conducted in the IME: Advanced Manufacturing Technology; Coastal Zone Engineering; Cold Regions Engineering; Combustion and Fluids Engineering; Ground Transportation Technology; and Machinery and Engine Technology. The rationale and major research thrusts of each program are described, and specific achievements in 1991-92 are reviewed. Lists of technical reports and papers presented by IME personnel are also included.

  14. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  15. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  16. Contraction stimulates muscle glucose uptake independent of atypical PKC.

    PubMed

    Yu, Haiyan; Fujii, Nobuharu L; Toyoda, Taro; An, Ding; Farese, Robert V; Leitges, Michael; Hirshman, Michael F; Mul, Joram D; Goodyear, Laurie J

    2015-11-01

    Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC-λ/ζ) have been shown to be necessary for insulin-, AICAR-, and metformin-stimulated glucose uptake in skeletal muscle, but not for treadmill exercise-stimulated muscle glucose uptake. To investigate if PKC-λ/ζ activity is required for contraction-stimulated muscle glucose uptake, we used mice with tibialis anterior muscle-specific overexpression of an empty vector (WT), wild-type PKC-ζ (PKC-ζ(WT)), or an enzymatically inactive T410A-PKC-ζ mutant (PKC-ζ(T410A)). We also studied skeletal muscle-specific PKC-λ knockout (MλKO) mice. Basal glucose uptake was similar between WT, PKC-ζ(WT), and PKC-ζ(T410A) tibialis anterior muscles. In contrast, in situ contraction-stimulated glucose uptake was increased in PKC-ζ(T410A) tibialis anterior muscles compared to WT or PKC-ζ(WT) tibialis anterior muscles. Furthermore, in vitro contraction-stimulated glucose uptake was greater in soleus muscles of MλKO mice than WT controls. Thus, loss of PKC-λ/ζ activity increases contraction-stimulated muscle glucose uptake. These data clearly demonstrate that PKC-λζ activity is not necessary for contraction-stimulated glucose uptake.

  17. Cellular Uptake and Ultrastructural Localization Underlie the Pro-apoptotic Activity of a Hydrocarbon-stapled BIM BH3 Peptide.

    PubMed

    Edwards, Amanda L; Wachter, Franziska; Lammert, Margaret; Huhn, Annissa J; Luccarelli, James; Bird, Gregory H; Walensky, Loren D

    2015-09-18

    Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.

  18. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  19. Mechanism and active variety of allelochemicals

    USGS Publications Warehouse

    Peng, S.-L.; Wen, J.; Guo, Q.-F.

    2004-01-01

    This article summarizes allelochemicals' active variety, its potential causes and function mechanisms. Allelochemicals' activity varies with temperature, photoperiod, water and soils during natural processes, with its initial concentration, compound structure and mixed degree during functional processes, with plant accessions, tissues and maturity within-species, and with research techniques and operation processes. The prospective developmental aspects of allelopathy studies in the future are discussed. Future research should focus on: (1) to identify and purify allelochemicals more effectively, especially for agriculture, (2) the functions of allelopathy at the molecular structure level, (3) using allelopathy to explain plant species interactions, (4) allelopathy as a driving force of succession, and (5) the significance of allelopathy in the evolutionary processes.

  20. Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation.

    PubMed

    Alam, Anwar; Puri, Niti; Saxena, Rajiv K

    2016-09-01

    The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1(+) cells as well as the activated subset of NK cells (NK1.1(+)CD69(+) cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1(+) phosphatidylserine(+)) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.

  1. The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training

    PubMed Central

    Vigelsø, Andreas; Andersen, Nynne B; Dela, Flemming

    2014-01-01

    Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and changes in CS activity is often assumed. However, this relationship and absolute values of CS and maximal oxygen uptake (V.O2max) has never been assessed across different studies. A systematic PubMed search on literature published from 1983 to 2013 was performed. The search profile included: citrate, synthase, human, skeletal, muscle, training, not electrical stimulation, not in-vitro, not rats. Studies that reported changes in CS activity and V.O2max were included. Different training types and subject populations were analyzed independently to assess correlation between relative changes in V.O2max and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P < 0.001) between the relative change in V.O2max and the relative change in CS activity. All reported absolute values of CS and V.O2max did not correlate (r =- 0.07, n = 148, P = 0.4). Training induced changes in whole body oxidative capacity is matched by changes in muscle CS activity in a nearly 1:1 relationship. Absolute values of CS across different studies cannot be compared unless a standardized analytical method is used by all laboratories. PMID:25057335

  2. Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat

    PubMed Central

    Allen, George C.; Grimm, Daniel T.; Elkan, Gerald H.

    1991-01-01

    Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 μM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 μM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 μM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min−1 mg of protein−1 and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and β-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h−1 resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C2H4h−1 mg (dry weight)−1, and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C2H4 h−1 mg (dry weight)−1. However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in “washout,” proving that ORS571 can grow on N2 and that there was not a nitrogen source in the medium that could substitute. PMID:16348585

  3. Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens.

    PubMed

    Aquino-Miranda, Guillermo; Escamilla-Sánchez, Juan; González-Pantoja, Raúl; Bueno-Nava, Antonio; Arias-Montaño, José-Antonio

    2016-07-01

    We studied the effect of activating histamine H3 receptors (H3Rs) on rat nucleus accumbens (rNAcc) dopaminergic transmission by analyzing [(3)H]-dopamine uptake by synaptosomes, and dopamine synthesis and depolarization-evoked [(3)H]-dopamine release in slices. The uptake of [(3)H]-dopamine by rNAcc synaptosomes was not affected by the H3R agonist RAMH (10(-10)-10(-6) M). In rNAcc slices perfusion with RAMH (1 μM) had no significant effect on [(3)H]-dopamine release evoked by depolarization with 30 mM K(+) (91.4 ± 4.5% of controls). The blockade of dopamine D2 autoreceptors with sulpiride (1 μM) enhanced K(+)-evoked [(3)H]-dopamine release (168.8 ± 15.5% of controls), but under this condition RAMH (1 μM) also failed to affect [(3)H]-dopamine release. Dopamine synthesis was evaluated in rNAcc slices incubated with the l-dihydroxyphenylalanine (DOPA) decarboxylase inhibitor NSD-1015 (1 mM). Forskolin-induced DOPA accumulation (220.1 ± 10.4% of controls) was significantly reduced by RAMH (41.1 ± 6.5% and 43.5 ± 9.1% inhibition at 100 nM and 1 μM, respectively), and this effect was prevented by the H3R antagonist ciproxifan (10 μM). DOPA accumulation induced by preventing cAMP degradation with IBMX (iso-butyl-methylxantine, 1 mM) or by activating receptors for the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) with PACAP-27 (1 μM) was reduced (IBMX) or prevented (PACAP-27) by RAMH (100 nM). In contrast, DOPA accumulation induced by 8-Bromo-cAMP (1 mM) was not affected by RAMH (100 nM). These results indicate that in rNAcc H3Rs do not modulate dopamine uptake or release, but regulate dopamine synthesis by inhibiting cAMP formation and thus PKA activation. This article is part of the Special Issue entitled 'Histamine Receptors'.

  4. Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry 1

    PubMed Central

    Tjepkema, John D.; Cartica, Robert J.

    1982-01-01

    Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O2 partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O2 microelectrode measurements which showed that the O2 partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O2 uptake as a function of temperature and partial pressure of O2 were consistent with diffusion limitation of O2 uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O2 regulation in legume and Parasponia nodules is very similar and differs from O2 regulation in actionorhizal nodules. Images PMID:16662284

  5. Measurement of gas/water uptake coefficients for trace gases active in the marine environment

    SciTech Connect

    Davidovits, P. . Dept. of Chemistry); Worsnop, D.W.; Zahniser, M.S.; Kolb, C.E. . Center for Chemical and Environmental Physics)

    1992-02-01

    Ocean produced reduced sulfur compounds including dimethylsulfide (DMS), hydrogen sulfide (H{sub 2}S), carbon disulfide (CS{sub 2}), methyl mercaptan (CH{sub 3}CH) and carbonyl sulfide (OCS) deliver a sulfur burden to the atmosphere which is roughly equal to sulfur oxides produced by fossil fuel combustion. These species and their oxidation products dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO{sub 2}) and methane sulfonic acid (MSA) dominate aerosol and CCN production in clean marine air. Furthermore, oxidation of reduced sulfur species will be strongly influenced by NO{sub x}/O{sub 3} chemistry in marine atmospheres. The multiphase chemical processes for these species must be understood in order to study the evolving role of combustion produced sulfur oxides over the oceans. We have measured the chemical and physical parameters affecting the uptake of reduced sulfur compounds, their oxidation products, ozone, and nitrogen oxides by the ocean's surface, and marine clouds, fogs, and aerosols. These parameters include: gas/surface mass accommodation coefficients; physical and chemically modified (effective) Henry's law constants; and surface and liquid phase reaction constants. These parameters are critical to understanding both the interaction of gaseous trace species with cloud and fog droplets and the deposition of trace gaseous species to dew covered, fresh water and marine surfaces.

  6. Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes

    PubMed Central

    Saeed, Mohammad F.; Kolokoltsov, Andrey A.; Albrecht, Thomas; Davey, Robert A.

    2010-01-01

    Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as

  7. Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca²+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake.

    PubMed

    Dal-Cim, T; Martins, W C; Santos, A R S; Tasca, C I

    2011-06-02

    Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as modulation of glutamate transmission and neuronal protection against excitotoxic damage. GD are spontaneously released to the extracellular space from cultured astrocytes and during oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway involvement in the mechanisms related to the neuroprotective role of guanosine in rat hippocampal slices subjected to OGD. The addition of guanosine (100 μM) to hippocampal slices subjected to 15 min of OGD and followed by 2 h of re-oxygenation is neuroprotective. The presence of K+ channel blockers, glibenclamide (20 μM) or apamin (300 nM), revealed that neuroprotective effect of guanosine was not dependent on ATP-sensitive K+ channels or small conductance Ca²+-activated K+ channels. The presence of charybdotoxin (100 nM), a large conductance Ca²+-activated K+ channel (BK) blocker, inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect was inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 μM) in the re-oxygenation period. Guanosine promoted an increase in Akt protein phosphorylation. However, the presence of charybdotoxin blocked such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the increased expression of phospho-Akt protein.

  8. Uptake of Optional Activities Leads to Improved Performance in a Biomedical Sciences Class

    ERIC Educational Resources Information Center

    Verkade, Heather; Lim, Saw Hoon

    2015-01-01

    Optional (non-assessed) learning activities are a learning tool that may help students achieve their desired grade, or help students with lower levels of previous experience in the topic. This study examines the implementation of, and outcomes from, two optional activities, one online and one paper-based. The activities complemented the lectures…

  9. Mechanism of Highly Synchronized Bilateral Hippocampal Activity

    PubMed Central

    Wang, Y.; Toprani, S.; Tang, Y.; Vrabec, T.; Durand, D.M.

    2014-01-01

    In vivo studies of epileptiform discharges in the hippocampi of rodents have shown that bilateral seizure activity can sometimes be synchronized with very small delays (< 2 ms). This observed small time delay of epileptiform activity between the left and right CA3 regions is unexpected given the physiological propagation time across the hemispheres (> 6 ms). The goal of this study is to determine the mechanisms of this tight synchronization with in-vitro electrophysiology techniques and computer simulations. The hypothesis of a common source was first eliminated by using an in-vitro preparation containing both hippocampi with a functional ventral hippocampal commissure (VHC) and no other tissue. Next, the hypothesis that a noisy baseline could mask the underlying synchronous activity between the two hemispheres was ruled out by low noise in-vivo recordings and computer simulation of the noisy environment. Then we built a novel bilateral CA3 model to test the hypothesis that the phenomenon of very small left-to-right propagation delay of seizure activity is a product of epileptic cell network dynamics. We found that the commissural tract connectivity could decrease the delay between seizure events recorded from two sides while the activity propagated longitudinally along the CA3 layer thereby yielding delays much smaller than the propagation time between the two sides. The modeling results indicate that both recurrent and feedforward inhibition were required for shortening the bilateral propagation delay and depended critically on the length of the commissural fiber tract as well as the number of cells involved in seizure generation. These combined modeling/experimental studies indicate that it is possible to explain near perfect synchronization between the two hemispheres by taking into account the structure of the hippocampal network. PMID:24262205

  10. Mechanism of base activation of persulfate.

    PubMed

    Furman, Olha S; Teel, Amy L; Watts, Richard J

    2010-08-15

    Base is the most commonly used activator of persulfate for the treatment of contaminated groundwater by in situ chemical oxidation (ISCO). A mechanism for the base activation of persulfate is proposed involving the base-catalyzed hydrolysis of persulfate to hydroperoxide anion and sulfate followed by the reduction of another persulfate molecule by hydroperoxide. Reduction by hydroperoxide decomposes persulfate into sulfate radical and sulfate anion, and hydroperoxide is oxidized to superoxide. The base-catalyzed hydrolysis of persulfate was supported by kinetic analyses of persulfate decomposition at various base:persulfate molar ratios and an increased rate of persulfate decomposition in D(2)O vs H(2)O. Stoichiometric analyses confirmed that hydroperoxide reacts with persulfate in a 1:1 molar ratio. Addition of hydroperoxide to basic persulfate systems resulted in rapid decomposition of the hydroperoxide and persulfate and decomposition of the superoxide probe hexachloroethane. The presence of superoxide was confirmed with scavenging by Cu(II). Electron spin resonance spectroscopy confirmed the generation of sulfate radical, hydroxyl radical, and superoxide. The results of this research are consistent with the widespread reactivity reported for base-activated persulfate when it is used for ISCO.

  11. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells

    PubMed Central

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells. PMID:27536106

  12. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

    PubMed

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

  13. Screening of medicinal plants for PPPAR-alpha and PPAR-gamma activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPAR-alpha/gamma activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A report...

  14. Diphenyl diselenide elicits antidepressant-like activity in rats exposed to monosodium glutamate: A contribution of serotonin uptake and Na(+), K(+)-ATPase activity.

    PubMed

    Quines, Caroline B; Rosa, Suzan G; Velasquez, Daniela; Da Rocha, Juliana T; Neto, José S S; Nogueira, Cristina W

    2016-03-15

    Depression is a disorder with symptoms manifested at the psychological, behavioral and physiological levels. Monosodium glutamate (MSG) is the most widely used additive in the food industry; however, some adverse effects induced by this additive have been demonstrated in experimental animals and humans, including functional and behavioral alterations. The aim of this study was to investigate the possible antidepressant-like effect of diphenyl diselenide (PhSe)2, an organoselenium compound with pharmacological properties already documented, in the depressive-like behavior induced by MSG in rats. Male and female newborn Wistar rats were divided in control and MSG groups, which received, respectively, a daily subcutaneous injection of saline (0.9%) or MSG (4g/kg/day) from the 1st to 5th postnatal day. At 60th day of life, animals received (PhSe)2 (10mg/kg, intragastrically) 25min before spontaneous locomotor and forced swimming tests (FST). The cerebral cortices of rats were removed to determine [(3)H] serotonin (5-HT) uptake and Na(+), K(+)-ATPase activity. A single administration of (PhSe)2 was effective against locomotor hyperactivity caused by MSG in rats. (PhSe)2 treatment protected against the increase in the immobility time and a decrease in the latency for the first episode of immobility in the FST induced by MSG. Furthermore, (PhSe)2 reduced the [(3)H] 5-HT uptake and restored Na(+), K(+)-ATPase activity altered by MSG. In the present study a single administration of (PhSe)2 elicited an antidepressant-like effect and decrease the synaptosomal [(3)H] 5-HT uptake and an increase in the Na(+), K(+)-ATPase activity in MSG-treated rats.

  15. Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules

    SciTech Connect

    Monroe, J.D. ); LaRue, T.A. )

    1989-10-01

    The interaction between carbon substrates and O{sub 2} and their effects on nitrogenase activity (C{sub 2}H{sub 2}) were examined in detached nodules of pea (Pisum sativum L. cv Sparkle). The internal O{sub 2} concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O{sub 2} for 2 hours resulted in a 2- to 10-fold increase in internal O{sub 2}, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O{sub 2} was lowered. Nitrogenase activity was stimulated by succinate but only at high external O{sub 2}. Oxygen uptake increased linearly with external O{sub 2} but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O{sub 2} concentration within detached nodules.

  16. Uptake and toxicity of polycyclic aromatic hydrocarbons in terrestrial springtails--studying bioconcentration kinetics and linking toxicity to chemical activity.

    PubMed

    Schmidt, Stine Nørgaard; Smith, Kilian Eric Christopher; Holmstrup, Martin; Mayer, Philipp

    2013-02-01

    Passive dosing applies a polymer loaded with test compound(s) to establish and maintain constant exposure in laboratory experiments. Passive dosing with the silicone poly(dimethylsiloxane) was used to control exposure of the terrestrial springtail Folsomia candida to six polycyclic aromatic hydrocarbons (PAHs) in bioconcentration and toxicity experiments. Folsomia candida could move freely on the PAH-loaded silicone, resulting in exposure via air and direct contact. The bioconcentration kinetics indicated efficient uptake of naphthalene, anthracene, and pyrene through air and (near) equilibrium partitioning of these PAHs to lipids and possibly the waxy layer of the springtail cuticle. Toxicities of naphthalene, phenanthrene, and pyrene were related to chemical activity, which quantifies the energetic level and drives spontaneous processes including diffusive biouptake. Chemical activity-response relationships yielded effective lethal chemical activities (La50s) well within the expected range for baseline toxicity (0.01-0.1). Effective lethal body burdens for naphthalene and pyrene exceeded the expected range of 2 to 8 mmol kg(-1) fresh weight, which again indicated the waxy layer to be a sorbing phase. Finally, chemical activities were converted into equilibrium partitioning concentrations in lipids yielding effective lethal concentrations for naphthalene and phenanthrene in good correspondence with the lethal membrane burden for baseline toxicity (40-160 mmol kg(-1) lipid). Passive dosing was a practical approach for tightly controlling PAH exposure, which in turn provided new experimental possibilities and findings.

  17. [Experiments on the mechanism of action of vascular spasmolytics. 4. Effect of nitroprusside sodium, nitroglycerin, prenylamine and verapamil on the calcium uptake of microsomes of the smooth bascular muscles].

    PubMed

    Klinner, U; Ehlers, D; Fermum, R; Meisel, P

    1977-01-01

    Nitroprusside-sodium, nitroglycerol, and verapamil had no effect on the calcium uptake by microsomes from the carotid artery of cattle. Prenylamine reduced the passive binding and the active uptake and released already bound calcium. The basal Mg-dependendent ATPase and Ca-stimulatable Mg-ATPase were inhibited by prenylamine.

  18. Azepines and Piperidines with Dual Norepinephrine Dopamine Uptake Inhibition and Antidepressant Activity

    PubMed Central

    2012-01-01

    Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay. PMID:24900562

  19. Is non-insulin dependent glucose uptake a therapeutic alternative? Part 2: Do such mechanisms fulfil the required combination of power and tolerability?

    PubMed

    Wiernsperger, N F

    2005-12-01

    The worldwide burden of diabetes, the unavoidable worsening which is observed in long-term clinical trials despite treatment and the close link between glycaemia and microangiopathy appeal for much stronger treatment strategies. This, in turn, either requires polypharmacy (with new risks) or new, more powerful drugs to be invented. The first part of this review dealt with a thorough analysis of pros and cons for some selected pathways which could potentially increase glucose uptake without necessitating insulin. The choice of such targets for developing completely new drugs, however, requires a favourable background from existing tentatives with either drugs or cell biology approaches. Moreover, because vascular complications are what must ultimately be avoided when treating diabetic patients, we must be sure that increasing glucose uptake in a fashion which is no more controlled by normal physiology is compatible with the physiology of vascular cells (long-term tolerance). The aspect of drug side-effects must therefore be considered systematically. For reasons which are individually developed, it appears that each of the potential pathways analyzed either lacks sufficient power and/or is likely to induce side effects which are not acceptable for long-term application. The fact that GLUT-1 transporters are ubiquitously distributed even extends this cardinal question to the general principle of increasing glucose uptake. In conclusion a precise evaluation suggests that, although non-insulin dependent glucose uptake represents (3/4) of whole body glucose transport, it is difficult to consider such mechanisms able to generate a new treatment fulfilling the unavoidable request of combined efficacy and tolerability.

  20. Uptake of CO2, SO2, HNO3 and HCl on calcite (CaCO3) at 300 K: mechanism and the role of adsorbed water.

    PubMed

    Santschi, Ch; Rossi, M J

    2006-06-01

    All experimental observations of the uptake of the four title compounds on calcite are consistent with the presence of a reactive bifunctional surface intermediate Ca(OH)(HCO3) that has been proposed in the literature. The uptake of CO2 and SO2 occurs on specific adsorption sites of crystalline CaCO3(s) rather than by dissolution in adsorbed water, H2O(ads). SO2 primarily interacts with the bicarbonate moiety whereas CO2, HNO3 and HCl all react first with the hydroxyl group of the surface intermediate. Subsequently, the latter two react with the bicarbonate group to presumably form Ca(NO3)2 and CaCl2.2H2O. The effective equilibrium constant of the interaction of CO2 with calcite in the presence of H2O(ads) is kappa = deltaCO2/(H2O(ads)[CO2]) = 1.62 x 10(3) bar(-1), where CO2 is the quantity of CO2 adsorbed on CaCO3. The reaction mechanism involves a weakly bound precursor species that is reversibly adsorbed and undergoes rate-controlling concurrent reactions with both functionalities of the surface intermediate. The initial uptake coefficients gamma0 on calcite powder depend on the abundance of H2O(ads) under the present experimental conditions and are on the order of 10(-4) for CO2 and 0.1 for SO2, HNO3 and HCl, with gamma(ss) being significantly smaller than gamma0 for HNO3 and HCl, thus indicating partial saturation of the uptake. At 33% relative humidity and 300 K there are 3.5 layers of H2O adsorbed on calcite that reduce to a fraction of a monolayer of weakly and strongly bound water upon pumping and/or heating.

  1. Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel–cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines

    PubMed Central

    Ye, Jun; Xia, Xuejun; Dong, Wujun; Hao, Huazhen; Meng, Luhua; Yang, Yanfang; Wang, Renyun; Lyu, Yuanfeng; Liu, Yuling

    2016-01-01

    There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel–cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy. PMID:27601899

  2. Equilibrium uptake, sorption dynamics, process optimization, and column operations for the removal and recovery of malachite green from wastewater using activated carbon and activated slag

    SciTech Connect

    Gupta, V.K.; Srivastava, S.K.; Mohan, D.

    1997-06-01

    The waste slurry generated in fertilizer plants and slag (blast furnace waste) have been converted into low-cost adsorbents, activated carbon and activated slag, respectively, and these are utilized for the removal of malachite green (a basic dye) from wastewater. In the batch experiments, parameters studied include the effect of pH, sorbent dosage, adsorbate concentration, temperature, and contact time. Kinetic studies have been performed to have an idea of the mechanistic aspects and to obtain the thermodynamic parameters of the process. The uptake of the dye is greater on carbonaceous material than on activated slag. Sorption data have been correlated with both Langmuir and Freundlich adsorption models. The presence of anionic surfactants does not affect the uptake of dye significantly. The mass transfer kinetic approach has been applied for the determination of various parameters necessary for the designing of fixed-bed contactors. Chemical regeneration has been achieved with acetone in order to recover the loaded dye and restore the column to its original capacity without dismantling the same.

  3. Influence of exogenous urea on photosynthetic pigments, (14)CO 2 uptake, and urease activity in Elodea densa-environmental implications.

    PubMed

    Maleva, Maria; Borisova, Galina; Chukina, Nadezda; Nekrasova, Galina; Prasad, M N V

    2013-09-01

    This paper analyzes the effect of exogenous urea in increased concentration gradient (0, 100, 500 and 1,000 mg L(-1)) on photosynthetic pigments (measured spectrophotometrically), uptake of (14)CO2 (using radioisotope), and urease activity (by measuring ammonia with Nessler's reagent) in leaves of Elodea densa Planch. We have observed that low concentration of urea (100 mg L(-1)) stimulates the accumulation of photosynthetic pigments and intensifies photosynthesis in E. densa, whereas high concentration (1,000 mg L(-1)) suppresses these processes. Urease activity increased by approximately 2.7 and 8 fold when exogenous urea concentrations were 100 and 500 mg L(-1), respectively. However, exogenous urea in high concentration (1,000 mg L(-1)) decreased urease activity by 1.5 fold compared to the control. The necessity of mitigating urea and other nitrogen-containing compounds (NH3 from urea) in water bodies has been discussed with emphasis on the potential for phytoremediation of urea using common water weed viz. E. densa.

  4. Mechanism of ascaridole activation in Leishmania.

    PubMed

    Geroldinger, Gerald; Tonner, Matthias; Hettegger, Hubert; Bacher, Markus; Monzote, Lianet; Walter, Martin; Staniek, Katrin; Rosenau, Thomas; Gille, Lars

    2017-03-02

    values for Art. In a heme association assay Asc demonstrated a lower binding affinity to heme than Art. ICP-OES measurements revealed that in LtP the total iron concentrations were twice as high as values in J774 macrophages. Since low molecular iron was important in Asc activation we studied the influence of Asc on the labile iron pool (LIP) in LtP. Low temperature EPR experiments demonstrated that Asc shifts the redox balance of iron in the LIP to its oxidized state. These data demonstrate that univalent cleavage of Asc/Art in LtP is an essential part of their pharmacological mechanism. The structure of the EP determines whether activation by low molecular iron or heme is favored and the availability of these intracellular activators modulates their cytotoxicity. These findings may be helpful for synthesis of new Asc derivatives and understanding the action of EP in other cell types.

  5. Microwave irradiation of rats at 2. 45 GHz activates pinocytotic-like uptake of tracer by capillary endothelial cells of cerebral cortex

    SciTech Connect

    Neubauer, C.; Phelan, A.M.; Kues, H.; Lange, D.G. )

    1990-01-01

    Far-field exposures of male albino rats to 2.45-GHz microwaves (10-microseconds pulses, 100 pps) at a low average power density (10 mW/cm2; SAR approximately 2 W/kg) and short durations (30-120 min) resulted in increased uptakes of tracer through the blood-brain barrier (BBB). The uptake of systemically administered rhodamine-ferritin complex by capillary endothelial cells (CECs) of the cerebral cortex was dependent on power density and on duration of exposure. At 5 mW/cm2, for example, a 15-min exposure had no effect. Near-complete blockade of uptake resulted when rats were treated before exposure to microwaves with a single dose of colchicine, which inhibits microtubular function. A pinocytotic-like mechanism is presumed responsible for the microwave-induced increase in BBB permeability.

  6. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  7. Influence of mechanical activation of steel powder on its properties

    NASA Astrophysics Data System (ADS)

    Vaulina, O. Yu; Darenskaia, E. A.; Myachin, Y. V.; Vasilyeva, I. E.; Kulkov, S. N.

    2017-02-01

    It has been studied properties of stainless steel based powders after mechanical activation using planetary ball milling technique. It have been shown that after one minute mechanical activation porosity of sintered steel is less than 5%, which is less than the porosity of the sintered steel powder without mechanical activation. The sample without activation has austenite state, which changes after activation toaustenite and ferrite mixtures. X-ray analysis confirmed that the mechanical activation leads to a change in the phase state of the samples: the samples without activation of the FCC structure (γ-Fe), after activation - FCC (γ-Fe) and BCC (α-Fe). The hardness increases at increasing activation time from 800 MPa for the sample without mechanical activation to 1250 MPa for the sample with the activation time of 10 minutes.

  8. Effects of cerium oxide nanoparticles on soil enzymatic activities and wheat grass nutrients uptake

    NASA Astrophysics Data System (ADS)

    Li, Biting; Chen, Yirui; Bai, Lingyun; Jacobson, Astrid; Darnault, Christophe

    2015-04-01

    The US National Science Foundation estimated that the use of nanomaterials and nanotechnology would reach a global market value of 1 million this year. Concomitant with the wide applications of nanoparticles is an increasing risk of adverse effects to the environment and human health. As a common nanomaterial used as a fuel catalyst and polish material, cerium (IV) oxide nanoparticles (CeO2 NP) were tested for their potential impact on soil health and plant growth. Through exposure by air, water, and solid deposition, nanoparticles may accumulate in soils and impact agricultural systems. The objectives of this research were to determine whether CeO2 NPs affect the growth of wheat grass and selected soil enzyme activities chose as indicators of soil health. Wheat grass was grown in plant boxes containing CeO2 NPs mixed with agricultural soil at different concentrations. Two control groups were included: one consisting of soil with plants but no CeO2 NPs, and one containing only soil, i.e., no NP or wheat plants added. The plants were grown for 10 weeks and harvested every two weeks in a laboratory under sodium growth lights. At the end of the each growing period, two weeks, soils were assayed for phosphatase, β-glucosidase, and urease activities, and NPK values. Spectrophotometer analyses were used to assess enzyme activities, and NPK values were tested by Clemson Agricultural Center. Wheat yields were estimated by shoot and root lengths and weights.

  9. Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

  10. The high-affinity immunoglobulin E receptor (FcepsilonRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcepsilonRIbeta chain immunoreceptor tyrosine-based activation motif.

    PubMed

    Suzuki, Yoshihiro; Yoshimaru, Tetsuro; Inoue, Toshio; Nunomura, Satoshi; Ra, Chisei

    2008-04-01

    A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcepsilonRIbeta regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.

  11. Maximal oxygen uptake, ventilatory thresholds and mechanical power during cycling in tropical climate in Guadeloupean elite cyclists.

    PubMed

    Hue, O; Antoine-Jonville, S; Galy, O; Blonc, S

    2010-11-01

    The Tropical climate imposes a high level of physiological stress, which could modify the target heart rate in training load prescription, as the recommendations are often determined by maximal oxygen uptake testing in temperature-neutral laboratories. To test this hypothesis, 7 high-level cyclists performed two randomised maximal tests in neutral (19.2±0.9°C; 51.7±1.3% RH) and Tropical environment (25.8±1.1°C; 63.7±2.3% RH). Neither maximal oxygen uptake nor ventilatory threshold was influenced by the environmental conditions. However, ventilation (p<0.005) and the respiratory equivalent in O(2) (p<0.05) were significantly higher in the Tropical environment, whereas maximal power output and the time to attain maximal oxygen uptake were significantly lower (p<0.05 for both). Moreover, the ventilatory cost of cycling (expressed in LW(-1)) was significantly greater in the Tropical condition (0.40±0.03LW(-1) vs. 0.32±0.05LW(-1), in Tropical vs. Neutral; condition effect: p<0.005; condition × time: p<0.001). Rectal temperature was influenced by neither the environmental conditions nor exercise (36.7±0.1 and 37.0±0.1°C vs. 36.8±0.1 and 37.1±0.2°C, in Tropical vs. Neutral, before and after exercise) but was influenced by condition × time (p<0.05). The heart rate (HR) values usually used for training prescription were not significantly different (154±5bpm vs. 156±4bpm and 172±4bpm vs. 167±4bpm in Tropical vs. Neutral climate, for the first and second thresholds, respectively). We concluded that the usual parameters measured during maximal exercise to establish training programs are not impaired in moderate Tropical environment. Nevertheless, the thermal stress attested by the increased ventilatory cost of cycling could have prevented the cyclists from performing a true maximal test in Tropical conditions.

  12. Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

    PubMed Central

    Cunningham, Scott D.; Kapulnik, Yoram; Brewin, Nicholas J.; Phillips, Donald A.

    1985-01-01

    Six mutants of Rhizobium leguminosarum 3855 lacking uptake hydrogenase activity (Hup− phenotype) as a result of Tn5-mob mutagenesis of the hup-containing plasmid pRL6JI were tested for symbiotic performance on Pisum sativum L. and Vicia benghalensis L. Three pea cultivars and one vetch line, which induce four different levels of Hup activity in strain 3855, were grown to flowering under microbiologically controlled conditions in the absence of combined N. Direct Kjeldahl N measurements showed that in every case at least one Hup− mutant fixed as much N2 as the isogenic Hup+ strain. Measures of C2H2 reduction, H2 evolution, 3H2 incorporation, and plant dry weight were consistent with the interpretation that the oxidation of H2 produced by the nitrogenase enzyme complex was not necessarily associated with increased N2 fixation in these symbiotic associations. Tests with a smaller subset of the Hup− strains under four different root environments ranging from pH 5.0 to 8.2 likewise showed no significant advantage for the isogenic Hup+ strain. It was concluded that the improvements in symbiotic N2 fixation produced by pRL6JI are associated with some trait other than the Hup+ phenotype. PMID:16346912

  13. Prediction of oxygen uptake dynamics by machine learning analysis of wearable sensors during activities of daily living

    PubMed Central

    Beltrame, T.; Amelard, R.; Wong, A.; Hughson, R. L.

    2017-01-01

    Currently, oxygen uptake () is the most precise means of investigating aerobic fitness and level of physical activity; however, can only be directly measured in supervised conditions. With the advancement of new wearable sensor technologies and data processing approaches, it is possible to accurately infer work rate and predict during activities of daily living (ADL). The main objective of this study was to develop and verify the methods required to predict and investigate the dynamics during ADL. The variables derived from the wearable sensors were used to create a predictor based on a random forest method. The temporal dynamics were assessed by the mean normalized gain amplitude (MNG) obtained from frequency domain analysis. The MNG provides a means to assess aerobic fitness. The predicted during ADL was strongly correlated (r = 0.87, P < 0.001) with the measured and the prediction bias was 0.2 ml·min−1·kg−1. The MNG calculated based on predicted was strongly correlated (r = 0.71, P < 0.001) with MNG calculated based on measured data. This new technology provides an important advance in ambulatory and continuous assessment of aerobic fitness with potential for future applications such as the early detection of deterioration of physical health. PMID:28378815

  14. Prediction of oxygen uptake dynamics by machine learning analysis of wearable sensors during activities of daily living.

    PubMed

    Beltrame, T; Amelard, R; Wong, A; Hughson, R L

    2017-04-05

    Currently, oxygen uptake () is the most precise means of investigating aerobic fitness and level of physical activity; however, can only be directly measured in supervised conditions. With the advancement of new wearable sensor technologies and data processing approaches, it is possible to accurately infer work rate and predict during activities of daily living (ADL). The main objective of this study was to develop and verify the methods required to predict and investigate the dynamics during ADL. The variables derived from the wearable sensors were used to create a predictor based on a random forest method. The temporal dynamics were assessed by the mean normalized gain amplitude (MNG) obtained from frequency domain analysis. The MNG provides a means to assess aerobic fitness. The predicted during ADL was strongly correlated (r = 0.87, P < 0.001) with the measured and the prediction bias was 0.2 ml·min(-1)·kg(-1). The MNG calculated based on predicted was strongly correlated (r = 0.71, P < 0.001) with MNG calculated based on measured data. This new technology provides an important advance in ambulatory and continuous assessment of aerobic fitness with potential for future applications such as the early detection of deterioration of physical health.

  15. Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of nutrient uptake.

    PubMed

    Yip, Justine Y. H.; Vanlerberghe, Greg C.

    2001-07-01

    When wild type (wt) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) suspension cells were grown under phosphate (P) limitation, they contained large amounts of mitochondrial alternative oxidase (AOX). When these cells were resupplied with P, there was a large, immediate and sustained stimulation of respiration to support a period of rapid P uptake. Two lines of evidence suggest that the abundant level of AOX present in wt cells contributed to this stimulated rate of respiration. First, when P-limited transgenic antisense tobacco cells (AS8) lacking AOX were resupplied with P, the stimulation of respiration was much less dramatic even though these cells displayed similar rates of P uptake. Second, while the stimulated rate of respiration in AS8 cells was insensitive (as expected) to the AOX inhibitor n-propyl gallate (nPG), much of the stimulated rate of respiration in wt cells could be inhibited by nPG. Given the non-phosphorylating nature of AOX respiration, wt cells required higher rates of electron transport to O2 than AS8 cells to support similar rates of P uptake. The utilization of AOX by wt cells during P uptake was apparently not occurring because the cytochrome (Cyt) pathway alone could not fully support the rate of P uptake, as the respiration of cells lacking AOX (either untreated AS8 cells or wt cells treated with nPG) supported similar rates of P uptake as wt cells with abundant AOX. Rather, we provide in vivo evidence that the utilization of AOX during the period of high respiration supporting P uptake was to dampen the mitochondrial generation of active oxygen species (AOS).

  16. Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Gnanaguru, Gopalan; Choi, Ariel R.; Amarnani, Dhanesh; D'Amore, Patricia A.

    2016-01-01

    Purpose Accumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells. Methods Primary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring transepithelial resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays. Results Treatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in transepithelial resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity. Conclusions These data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression. PMID:27607416

  17. A Rice Mutant Defective in Si Uptake1

    PubMed Central

    Ma, Jian Feng; Tamai, Kazunori; Ichii, Masahiko; Wu, Guo Feng

    2002-01-01

    Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3 m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake. PMID:12481095

  18. COMBINED THEORETICAL AND EXPERIMENTAL INVESTIGATION OF MECHANISMS AND KINETICS OF VAPOR-PHASE MERCURY UPTAKE BY CARBONACOUES SURFACES

    SciTech Connect

    Radisav D. Vidic

    2002-05-01

    The first part of this study evaluated the application of a versatile optical technique to study the adsorption and desorption of model adsorbates representative of volatile polar (acetone) and non-polar (propane) organic compounds on a model carbonaceous surface under ultra high vacuum (UHV) conditions. The results showed the strong correlation between optical differential reflectance (ODR) and adsorbate coverage determined by temperature programmed desorption (TPD). ODR technique was proved to be a powerful tool to investigate surface adsorption and desorption from UHV to high pressure conditions. The effects of chemical functionality and surface morphology on the adsorption/desorption behavior of acetone, propane and mercury were investigated for two model carbonaceous surfaces, namely air-cleaved highly oriented pyrolytic graphite (HOPG) and plasma-oxidized HOPG. They can be removed by thermal treatment (> 500 K). The presence of these groups almost completely suppresses propane adsorption at 90K and removal of these groups leads to dramatic increase in adsorption capacity. The amount of acetone adsorbed is independent of surface heat treatment and depends only on total exposure. The effects of morphological heterogeneity is evident for plasma-oxidized HOPG as this substrate provides greater surface area, as well as higher energy binding sites. Mercury adsorption at 100 K on HOPG surfaces with and without chemical functionalities and topological heterogeneity created by plasma oxidation occurs through physisorption. The removal of chemical functionalities from HOPG surface enhances mercury physisorption. Plasma oxidation of HOPG provides additional surface area for mercury adsorption. Mercury adsorption by activated carbon at atmospheric pressure occurs through two distinct mechanisms, physisorption below 348 K and chemisorption above 348 K. No significant impact of oxygen functionalities was observed in the chemisorption region. The key findings of this study

  19. Activation of Ca2+ uptake and inhibition of reversal of the sarcoplasmic reticulum Ca2+ pump by aromatic compounds.

    PubMed

    Petretski, J H; Wolosker, H; de Meis, L

    1989-12-05

    The effects of aromatic compounds in sarcoplasmic reticulum Ca2+-ATPase were investigated. The solubility of the drugs in various organic solvents and water was measured. The ratio between the solubility in organic solvents and that in water (distribution coefficient) was used as an index of their hydrophobicity. The order found was triphenylphosphine greater than diphenylamine greater than 3-nitrophenol greater than 4-nitrophenol greater than 1,3-dihydroxybenzene. The effects observed on the Ca2+-ATPase were correlated with hydrophobicity of the drugs, activation and inhibition being obtained at a lower concentration the greater the distribution coefficient of the drug into organic solvent. In leaky vesicles, the effects of each compound on the ATPase activity varied depending on the Ca2+ concentration in the medium: it inhibited in the presence of 5 microM Ca2+ and activated when the Ca2+ concentration was raised to 2 mM. In intact vesicles, 3- and 4-nitrophenol, diphenylamine, and triphenylphosphine enhanced both the rate of ATP hydrolysis and the amount of Ca2+ accumulated by the vesicles. These four drugs inhibited Ca2+ uptake when ITP was used as substrate. 1,3-Dihydroxybenzene enhanced the amount of Ca2+ accumulated by the vesicles regardless of whether ATP or ITP was the substrate. All five compounds inhibited the phosphorylation of the enzyme by Pi, the efflux of Ca2+, and the synthesis of ATP measured during the reversal of the Ca2+ pump. The results indicate that the hydrophobic character of various organic compounds determines their access to sensitive domains of the membrane-bound calcium pump. Additional specific effects are then produced, depending on the structure of each compound.

  20. Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel.

    PubMed

    Pershadsingh, H A; Stubbs, E B; Noteboom, W D; Vorbeck, M L; Martin, A P

    1985-12-19

    The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.

  1. Drug-loading of poly(ethylene glycol methyl ether methacrylate) (PEGMEMA)-based micelles and mechanisms of uptake in colon carcinoma cells.

    PubMed

    Chang, Teddy; Gosain, Pallavi; Stenzel, Martina H; Lord, Megan S

    2016-08-01

    In this study polymeric micelles formed from poly(poly(ethylene glycol) methyl ether methacrylate)-block-poly(methyl methacrylate) (P(PEGMEMA75)-b-PMMA80) block copolymer of approximately 25nm in diameter were used to encapsulate the model drug, Nile Red, with a loading efficiency of 0.08wt% and a chemotherapeutic drug, doxorubicin (DOX), with an efficiency of 2.75wt%. The release of DOX from the micelles was sufficient to be cytotoxic to human colon carcinoma cells, WiDr, while Nile Red and the unloaded micelles were found not to be cytotoxic when exposed to the cells at polymer concentrations up to 200μg/mL. Nile Red loaded micelles were used to analyze uptake of the micelles into the cells which were rapidly internalized within minutes of exposure. The three major endocytotic pathways were involved in the internalization of micelles; however other passive mechanisms were also at play as the addition of inhibitors to all three pathways did not completely inhibit the uptake of these nanoparticles. These data demonstrate the potential of the P(PEGMEMA)75-b-PMMA80 block copolymer micelles to be rapidly internalized by carcinoma cells and deliver low doses of drugs intracellularly for controlled drug release.

  2. Effects of nitrogen fertilization on soil nutrient concentration and phosphatase activity and forage nutrient uptake from a grazed pasture system.

    PubMed

    Dillard, Sandra Leanne; Wood, Charles Wesley; Wood, Brenda Hall; Feng, Yucheng; Owsley, Walter Frank; Muntifering, Russell Brian

    2015-05-01

    Over a 3-year period, the effect of differing N-application regimes on soil extractable-P concentration, soil phosphatase activity, and forage P uptake in a P-enriched grazed-pasture system was investigated. In the fall of each year, six 0.28-ha plots were overseeded with triticale ( × Triticosecale rimpaui Wittm.) and crimson clover (Trifolium incarnatum) into a tall fescue (Lolium arundinacea)/bermudagrass (Cynodon dactylon) sod and assigned to 1 of 3 N-fertilizer treatments (n = 2): 100% of N recommendation in a split application (100N), 50% in a single application (50N), and 0% of N recommendation (0N) for triticale. Cattle commenced grazing the following spring and grazed until May. In the summer, plots were overseeded with cowpea (Vigna unguiculata), fertilized at the same rates by reference to N recommendations for bermudagrass, and grazed by cattle until September. There were no effects of N fertilization on soil phosphatase activity, electrical conductivity, or concentrations of water-soluble P. Concentrations of extractable P decreased in plots receiving 50N, but increasing N fertilization to 100N resulted in no further reduction in extractable P. Forage biomass, foliar P concentrations, and forage P mass were not affected by N fertilization rates at the plant-community level, but responses were observed within individual forage species. Results are interpreted to mean that N fertilization at 50% of the agronomic recommendation for the grass component can increase forage P mass of specific forages and decrease soil extractable P, thus providing opportunity for decreasing P losses from grazed pasture.

  3. Platelet Activation: The Mechanisms and Potential Biomarkers

    PubMed Central

    Yun, Seong-Hoon; Sim, Eun-Hye; Goh, Ri-Young; Park, Joo-In

    2016-01-01

    Beyond hemostasis and thrombosis, an increasing number of studies indicate that platelets play an integral role in intercellular communication, mediating inflammatory and immunomodulatory activities. Our knowledge about how platelets modulate inflammatory and immunity has greatly improved in recent years. In this review, we discuss recent advances in the pathways of platelet activation and potential application of platelet activation biomarkers to diagnosis and prediction of disease states. PMID:27403440

  4. Effects of H ₂SO₄ and O ₂ on Hg⁰ uptake capacity and reversibility of sulfur-impregnated activated carbon under dynamic conditions.

    PubMed

    Wei, Yuanyang; Yu, Danqing; Tong, Shitang; Jia, Charles Q

    2015-02-03

    Powder activated carbon (AC) injection is widely considered as the most viable technology for removing gaseous elemental mercury (Hg(0)) in flue gases of coal-fired power plants. However, sulfuric acid (H2SO4) can form on the external and internal surfaces of AC particles due to the presence of sulfur oxides, nitrogen oxides, oxygen, and moisture in flue gases. This work focuses on the effects of H2SO4 and O2 on the Hg(0) uptake capacity and reversibility of sulfur impregnated activated carbon (SIAC) under dynamic conditions. Experiments were conducted with 25 μg-Hg(0)/m(3) of nitrogen or air, using a semicontinuous flow fixed-bed reactor kept at 120 or 180 °C. H2SO4 had a profound hindering effect on Hg(0) uptake due to pore blockage. O2 significantly enhanced Hg(0) uptake and its reversibility, via the oxidation of Hg(0) which facilitated chemisorption and the subsequent physisorption onto chemically adsorbed Hg. Absorption of Hg in H2SO4 was unlikely a significant contributor, when Hg(0) concentrations were at levels of typical power plants (tens of ppb). The reversibility of and relative contributions of physisorption and chemisorption to Hg(0) uptake would change with Hg(0) concentrations in flue gases. These findings could be significant in developing a complete solution for Hg capture where the handling of spent sorbent materials and the possible secondary pollution need to be considered.

  5. A rapid, non-destructive methodology to monitor activity of sulfide-induced corrosion of concrete based on H2S uptake rate.

    PubMed

    Sun, Xiaoyan; Jiang, Guangming; Bond, Philip L; Wells, Tony; Keller, Jurg

    2014-08-01

    Many existing methods to monitor the corrosion of concrete in sewers are either very slow or destructive measurements. To overcome these limitations, a rapid, non-invasive methodology was developed to monitor the sulfide-induced corrosion process on concrete through the measurement of the H2S uptake rates of concrete at various corrosion stages. The H2S uptake rate for a concrete coupon was determined by measuring the gaseous H2S concentrations over time in a temperature- and humidity-controlled gas-tight reactor. The reliability of this method was evaluated by carrying out repeated tests on different concrete coupons previously exposed to 50 ppm of H2S, at 30 °C and 100% relative humidity for over 32 months. The H2S uptake measurements showed good reproducibility. It was also shown that a severely corroded coupon exhibited higher sulfide uptake rates than a less corroded coupon. This could be explained by the corrosion layer in the more corroded coupon having a higher biological sulfide oxidation activity than the less corroded coupon. Additionally, temperature changes had a stronger effect on the uptake rate of the heavily corroded coupon compared to the less corroded coupon. A corrosion rate of 8.9 ± 0.5 mm/year, estimated from the H2S uptake results, agreed well with the corrosion rate observed in real sewers under similar conditions. The method could be applied to investigate important factors affecting sulfide-induced concrete corrosion, particularly temperature, fluctuating gaseous H2S concentrations, oxygen concentrations, surface pH and relative humidity.

  6. Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β.

    PubMed

    Boye, Louise; Welsby, Iain; Lund, Lisbeth Drozd; Goriely, Stanislas; Frøkiaer, Hanne

    2016-11-01

    Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) -dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk-independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN-β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN-β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN-β and IL-12 production is inhibited by TLR stimulation from the plasma membrane.

  7. Mechanisms of cuticular uptake of xenobiotics into living plants: 1. Influence of xenobiotic dose on the uptake of three model compounds applied in the absence and presence of surfactants into Chenopodium album, Hedera helix and Stephanotis floribunda leaves.

    PubMed

    Forster, W Alison; Zabkiewicz, Jerzy A; Riederer, Markus

    2004-11-01

    This study determined the uptake of three model compounds, applied in the presence and absence of surfactants, into the leaves of three plant species (Chenopodium album L, Hedera helix L and Stephanotis floribunda Brongn). The results with 2-deoxy-D-glucose, 2,4-dichlorophenoxyacetic acid and epoxiconazole in the presence ofsurfactants (the polyethylene glycol monododecyl ethers C12EO3, C12EO6, C12EO10 and a trisiloxane ethoxylate with mean EO of 7.5, all used at one equimolar concentration and therefore different percentage concentrations) illustrate that the initial dose (nmol mm(-2)) of xenobiotic applied to plant foliage is a strong positive determinant of uptake. This held true for all the xenobiotics studied over a wide concentration range in the presence of these surfactants. Uptake on a unit area basis (nmol mm(-2)) could be related to the initial dose of xenobiotic applied per unit area (ID) by an equation of the form: Uptake = a [ID]b at time t = 24h. ID is given by the mass of xenobiotic applied, M divided by the droplet spread area, A. Total mass uptake is then calculated from an equation of the form: Total Uptake = a [ID]b x A.

  8. Improved in vitro anti-tumoral activity, intracellular uptake and apoptotic induction of gemcitabine-loaded pegylated unilamellar liposomes.

    PubMed

    Celia, Christian; Calvagno, Maria Grazia; Paolino, Donatella; Bulotta, Stefania; Ventura, Cinzia Anna; Russo, Diego; Fresta, Massimo

    2008-04-01

    Anaplastic thyroid carcinoma is one of the most aggressive and lethal solid carcinomas affecting humans. A major limit of the chemotherapeutic agents is represented by their low therapeutic index. In this work, we investigated the possibility of improving the anti-tumoral activity of gemcitabine by using pegylated unilamellar liposomes. Liposomes were made up of 1,2-dipalmitoyl-sn-glycero-3-phospocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio) and they were prepared with a pH gradient to improve the gemcitabine loading capacity. The anti-tumoral efficacy of the liposomal formulation was tested in vitro on human anaplastic thyroid carcinoma cells (ARO) in culture, comparing the effects with those of the free drug. Gemcitabine-loaded unilamellar liposomes had a mean size approximately 200 nm with a zeta potential approximately -2 mV. The liposomal carrier noticeably improved the anti-tumoral activity of gemcitabine against ARO cells in terms of both dose-dependent cytotoxic effect and of drug exposition effect. Namely, gemcitabine-loaded liposomes showed a cytotoxic effect (58.2% increase of cell mortality at 1 microM with respect to free drug) after 12 h incubation, while the free drug showed a significant activity only after 72 h incubation. Moreover, a significant effect on the cell mortality appeared at 0.1 microM and 100% mortality was detected at a concentration of 1 microM of gemcitabine-loaded liposomes, while the free drug elicited the same effect at a concentration of 100 microM. The improved anti-tumoral activity of gemcitabine determined by the liposomal carrier was due to a greater intracellular uptake. The intracellular gemcitabine levels as a function of time showed a sinusoidal profile with peaks after 2 h, 6 h and 11 h, related to the cellular cycle of ARO. PARP cleavage and DNA fragmentation analysis provided clear evidence of the apoptosis induction in

  9. Inositol uptake in rat aorta

    SciTech Connect

    Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl )

    1990-01-01

    {sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.

  10. Solid lipid nanoparticles (SLN) of Efavirenz as lymph targeting drug delivery system: Elucidation of mechanism of uptake using chylomicron flow blocking approach.

    PubMed

    Makwana, Vivek; Jain, Rashmi; Patel, Komal; Nivsarkar, Manish; Joshi, Amita

    2015-11-10

    The aim of the present work was to develop a lymph targeted SLN formulation of antiretroviral (ARV) drug and to have an understanding of its underlying mechanism of uptake by the lymphatics. The lymphatics are the inaccessible reservoirs of HIV in human body. Efavirenz (EFV) is a BCS class II, ARV drug that undergoes extensive first pass metabolism. The EFV SLN formulation was prepared using Gelucire 44/14, Compritol 888 ATO, Lipoid S 75 and Poloxamer 188 by hot homogenization technique followed by ultrasonication method, with mean particle size of 168 nm, polydispersity index (PDI) <0.220, and mean zeta potential of -35.55 mV. DSC and XRPD studies revealed change in crystallinity index of drug when incorporated into SLN. In vitro drug release was found to be prolonged and biphasic in PBS pH 6.8. There was no significant change in the mean particle size, PDI, zeta potential and entrapment efficiency of EFV SLN after storage at 30 ± 2°C/60 ± 5%RH for two months. The results from lymphatic transport and tissue distribution study indicate that a significant part of the EFV had by-passed portal system and was recovered in the lymph via chylomicron uptake mechanism. Reduction in the amount (44.70%) of the EFV reaching to liver indicates that major amount of EFV bypasses the liver and thereby, enhances the oral bioavailability of the EFV. A significant amount of EFV was found in spleen, a major lymphatic organ. EFV SLN seems to have potential to target the ARV to lymphatics for the better management of HIV.

  11. Recovery of rhythmic activity in a central pattern generator: analysis of the role of neuromodulator and activity-dependent mechanisms

    PubMed Central

    Zhang, Yili; Golowasch, Jorge

    2015-01-01

    The pyloric network of decapods crustaceans can undergo dramatic rhythmic activity changes. Under normal conditions the network generates low frequency rhythmic activity that depends obligatorily on the presence of neuromodulatory input from the central nervous system. When this input is removed (decentralization) the rhythmic activity ceases. In the continued absence of this input, periodic activity resumes after a few hours in the form of episodic bursting across the entire network that later turns into stable rhythmic activity that is nearly indistinguishable from control (recovery). It has been proposed that an activity-dependent modification of ionic conductance levels in the pyloric pacemaker neuron drives the process of recovery of activity. Previous modeling attempts have captured some aspects of the temporal changes observed experimentally, but key features could not be reproduced. Here we examined a model in which slow activity-dependent regulation of ionic conductances and slower neuromodulator-dependent regulation of intracellular Ca2+ concentration reproduce all the temporal features of this recovery. Key aspects of these two regulatory mechanisms are their independence and their different kinetics. We also examined the role of variability (noise) in the activity-dependent regulation pathway and observe that it can help to reduce unrealistic constraints that were otherwise required on the neuromodulator-dependent pathway. We conclude that small variations in intracellular Ca2+ concentration, a Ca2+ uptake regulation mechanism that is directly targeted by neuromodulator-activated signaling pathways, and variability in the Ca2+ concentration sensing signaling pathway can account for the observed changes in neuronal activity. Our conclusions are all amenable to experimental analysis. PMID:21573963

  12. Structural Studies of Bacterioferritin B from Pseudomonas aeruginosa Suggest a Gating Mechanism for Iron Uptake via the Ferroxidase Center

    SciTech Connect

    Weeratunga, Saroja K.; Lovell, Scott; Yao, Huili; Battaile, Kevin P.; Fischer, Christopher J.; Gee, Casey E.; Rivera, Mario

    2010-03-16

    the ferroxidase pore is the dominant entry route for the uptake of iron by Pa BfrB. These findings, which are clearly distinct from those made with Escherichia coli Bfr [Crow, A. C., Lawson, T. L., Lewin, A., Moore, G. R., and Le Brun, N. E. (2009) J. Am. Chem. Soc. 131, 6808-6813], indicate that not all bacterioferritins operate in the same manner.

  13. Potassium Uptake Modulates Staphylococcus aureus Metabolism

    PubMed Central

    Gries, Casey M.; Sadykov, Marat R.; Bulock, Logan L.; Chaudhari, Sujata S.; Thomas, Vinai C.; Bose, Jeffrey L.

    2016-01-01

    ABSTRACT As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. PMID:27340697

  14. DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium(II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity.

    PubMed

    Kalaivani, P; Prabhakaran, R; Dallemer, F; Poornima, P; Vaishnavi, E; Ramachandran, E; Padma, V Vijaya; Renganathan, R; Natarajan, K

    2012-01-01

    The coordination propensities of 4(N,N')-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones (H(2)L(1-4)) were investigated by reacting with an equimolar amount of [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various spectroscopic techniques. The structure determination of the complexes [Pd(DeaSal-tsc)(PPh(3))] (1), [Pd(DeaSal-mtsc)(PPh(3))] (2) and [Pd(DeaSal-etsc)(PPh(3))] (3) by X-ray crystallography showed that ligands are coordinated in a dibasic tridentate ONS donor fashion forming stable five and six membered chelate rings. The binding ability of complexes (1-4) to calf-thymus DNA (CT DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed. The protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using lysozyme as a model protein. As determined by MTT assays, complex 3 exhibited a higher cytotoxic effect towards human lung cancer cell line (A549) and liver cancer cells (HepG2). LDH, NO assay and cellular uptake of the complexes have been studied. Further, antibacterial activity studies of the complexes have been screened against the pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, MIC50 values of the complexes showed that the complexes exhibited significant activity against the pathogens and among the complexes, 3 exhibited higher activity.

  15. The intestinal uptake of particles and the implications for drug and antigen delivery.

    PubMed Central

    O'Hagan, D T

    1996-01-01

    A number of researchers from different scientific disciplines have independently described the uptake of a variety of particulates across the gastrointestinal tract in animal models. The reports of particle uptake are briefly reviewed and the alternative mechanisms and proposed sites of uptake are discussed. Following these observations, some researchers have exploited the phenomenon of particulate uptake by using microparticles and nanoparticles as oral delivery systems for active agents, such as drugs and vaccines. The potential use of particulate carrier systems as drug and vaccine delivery systems is also briefly discussed. PMID:8982819

  16. APPLICATION OF MECHANICAL ACTIVATION TO PRODUCTION OF PYROCHLORE CERAMIC CONTAINING SIMULATED RARE-EARTH ACTINIDE FRACTION OF HLW

    SciTech Connect

    Stefanovsky, S.V.; Kirjanova, O.I.; Chizhevskaya, S.V.; Yudintsev, S.V.; Nikonov, B.S.

    2003-02-27

    Samples of zirconate pyrochlore ceramic (REE)2(Zr,U)2O7 (REE = La-Gd) containing simulated REE-An fraction of HLW were synthesized by two routes: (1) conventional cold compaction of oxide mixtures in pellets under pressure of 200 MPa and sintering of the pellets at 1550 C for 24 hours; and (2) using preliminary mechanical activation of oxide powders in a linear inductive rotator (LIV-0.5E) and a planetary mill - activator with hydrostatic yokes (AGO-2U) for 5 or 10 min. All the samples sintered at 1550 C were monolithic and dense with high mechanical integrity. As follows from X-ray diffraction (XRD) data, the ceramic sample produced without mechanical activation is composed of pyrochlore as major phase but contains also minor unreacted oxides. The samples prepared from pre-activated mixtures are composed of the pyrochlore structure phase only. Scanning electron microscopy (SEM) data also show higher structural and compositional homogeneity of the samples prepared from mechanically activated batches. The samples produced from oxide mixtures mechanically activated in the LIV for 10 min were slightly contaminated with iron resulting in formation of minor perovskite structure phase not detected by XRD but seen on SEM-images of the samples. Comparison of the samples prepared from non-activated and activated batches showed higher density, lower open porosity, water uptake, and elemental leaching for the samples fabricated from mechanically activated oxide mixtures.

  17. Physiologic mechanism of the ultrasonically activated scalpel.

    PubMed

    McCarus, S D

    1996-08-01

    An ultrasonically activated scalpel was developed and used clinically to provide hemostatic cutting in laparoscopic surgery. Results of experimental work with the ultrasonic scalpel blades were compared with those of electrosurgery and lasers. Some features that distinguish this energy form may confer specific advantages in various surgical procedures.

  18. The novel mechanism of lenalidomide activity.

    PubMed

    Fink, Emma C; Ebert, Benjamin L

    2015-11-19

    Lenalidomide acts by a novel drug mechanism-modulation of the substrate specificity of the CRL4(CRBN) E3 ubiquitin ligase. In multiple myeloma, lenalidomide induces the ubiquitination of IKZF1 and IKZF3 by CRL4(CRBN). Subsequent proteasomal degradation of these transcription factors kills multiple myeloma cells. In del(5q) myelodysplastic syndrome, lenalidomide induces the degradation of CK1α, which preferentially affects del(5q) cells because they express this gene at haploinsufficient levels. In the future, modulation of ubiquitin ligase function may enable us to target previously "undruggable" proteins.

  19. Optimal Control of Active Recoil Mechanisms

    DTIC Science & Technology

    1977-02-01

    forces from 25 to 2.5% for lower zones and cavitation was avoided for zone 8. Tachometer feedback was shown to be effective for low zones. The...concept of feedback control system coupled with optimization procedure to design recoil mechanisms was demonstrated to be an efficient and very effective ...122o •nl260 .01300 .01340 .01380 • ouzo #01460 •01500 •01540 •01580 •0162" .0166 i 309o,6 504P.6 9964.5 10075,9 39121.5 75397.3

  20. Atorvastatin prevents cell damage via modulation of oxidative stress, glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation.

    PubMed

    Vandresen-Filho, Samuel; Martins, Wagner C; Bertoldo, Daniela B; Mancini, Gianni; Herculano, Bruno A; de Bem, Andreza F; Tasca, Carla I

    2013-06-01

    Oxygen-glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.

  1. AMPK activators: mechanisms of action and physiological activities

    PubMed Central

    Kim, Joungmok; Yang, Goowon; Kim, Yeji; Kim, Jin; Ha, Joohun

    2016-01-01

    AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, which coordinates metabolic pathways and thus balances nutrient supply with energy demand. Because of the favorable physiological outcomes of AMPK activation on metabolism, AMPK has been considered to be an important therapeutic target for controlling human diseases including metabolic syndrome and cancer. Thus, activators of AMPK may have potential as novel therapeutics for these diseases. In this review, we provide a comprehensive summary of both indirect and direct AMPK activators and their modes of action in relation to the structure of AMPK. We discuss the functional differences among isoform-specific AMPK complexes and their significance regarding the development of novel AMPK activators and the potential for combining different AMPK activators in the treatment of human disease. PMID:27034026

  2. Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium.

    PubMed

    Phillips, D A; Kapulnik, Y; Bedmar, E J; Joseph, C M

    1990-04-01

    Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P activity and should

  3. Effects and Mechanisms of Mechanical Activation on Hydrogen Sorption/ Desorption of Nanoscale Lithium Nitrides

    SciTech Connect

    Shaw, Leon, L.; Yang, Gary, Z.; Crosby, Kyle; Wwan, Xufei. Zhong, Yang; Markmaitree, Tippawan; Osborn, William; Hu, Jianzhi; Kwak, Ja Hun

    2012-04-26

    The objective of this project is to investigate and develop novel, mechanically activated, nanoscale Li3N-based and LiBH4-based materials that are able to store and release {approx}10 wt% hydrogen at temperatures near 100 C with a plateau hydrogen pressure of less than 10 bar. Four (4) material systems have been investigated in the course of this project in order to achieve the project objective. These 4 systems are (i) LiNH2+LiH, (ii) LiNH2+MgH2, (iii) LiBH4, and (iv) LiBH4+MgH2. The key findings we have obtained from these 4 systems are summarized below. *The thermodynamic driving forces for LiNH2+LiH and LiBH4 systems are not adequate to enable H2 release at temperatures < 100 C. *Hydrogen release in the solid state for all of the four systems is controlled by diffusion, and thus is a slow process. *LiNH2+MgH2 and LiBH4+MgH2 systems, although possessing proper thermodynamic driving forces to allow for H2 release at temperatures < 100 C, have sluggish reaction kinetics because of their diffusion-controlled rate-limiting steps. *Reducing particles to the nanometer length scale (< 50 nm) can improve the thermodynamic driving force to enable H2 release at near ambient temperature, while simultaneously enhancing the reaction kinetics as well as changing the diffusion-controlled rate-limiting step to gas desorption-controlled rate-limiting step. This phenomenon has been demonstrated with LiBH4 and offers the hope that further work along this direction will make one of the material systems, i.e., LiBH4, LiBH4+MgH2 and LiNH2+MgH2, possess the desired thermodynamic properties and rapid H2 uptake/release kinetics for on-board applications. Many of the findings and knowledge gained from this project have been published in archival refereed journal articles [1-15] and are accessible by general public. Thus, to avoid a bulky final report, the key findings and knowledge gained from this project will be succinctly summarized, particularly for those findings and knowledge

  4. Lipid Dependent Mechanisms of Protein Pump Activity

    DTIC Science & Technology

    1989-05-23

    properties which result form the colligative interactions of many lipid molecules. Important materials properties include . . . i I I II II I i I 1 the...d identify by olock number) *This project is aime at investigating if a lipid elastic property , known as the spontaneous radius of curvature Ro’, is...a regulated membrane property and if its value modulates membrane protein activity. Specific aims reported on here include: 1) Correlation of ion pump

  5. Uptake of 2,4-Dichlorophenoxyacetic Acid by Pseudomonas fluorescens

    USGS Publications Warehouse

    Wedemeyer, Gary

    1966-01-01

    Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of “non-metabolic” uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by pH, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at pH values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carrier-mediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranyl ion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.

  6. Uptake of 2, 4-Dichlorophenoxyacetic acid by Pseudomonas fluorescens

    USGS Publications Warehouse

    Wedemeyer, G.A.

    1966-01-01

    Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of “nonmetabolic” uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by pH, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at pH values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carriermediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranylion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.

  7. High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

    PubMed

    Ray, Balmiki; Bailey, Jason A; Simon, Jay R; Lahiri, Debomoy K

    2012-07-01

    Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

  8. Mechanisms of Cl(-) uptake in rainbow trout: cloning and expression of slc26a6, a prospective Cl(-)/HCO3(-) exchanger.

    PubMed

    Boyle, David; Clifford, Alexander M; Orr, Elizabeth; Chamot, Danuta; Goss, Greg G

    2015-02-01

    In fresh waters, fishes continuously acquire ions to offset diffusive losses to a more dilute ambient environment and to maintain acid-base status. The objectives of the present study were to clone slc26a6, a prospective Cl(-)/HCO3(-) exchanger from rainbow trout, investigate its expression patterns in various tissues, at different developmental stages and after differential salinity exposure, and probe the mechanisms of Cl(-) uptake in rainbow trout embryos during development using a pharmacological inhibitor approach combined with (36)Cl(-) unidirectional fluxes. Results showed that the cloned gene encoded a 783 amino acid protein with conserved domains characteristic of the SLC26a family of anion exchange proteins. Phylogenetic analysis of this sequence against all subfamilies of the SLC26a family demonstrated that this translated protein shared a common ancestor with other actinopterygii and mammalian SLC26a6 isoforms and thus confirmed the identity of the cloned gene. Expression of slc26a6 was detected in all tissues and developmental stages assayed but was highest in the gill of juvenile trout. In trout embryos, Cl(-) uptake increased significantly post-hatch and was demonstrated to be mediated via an anion exchanger specific (DIDS sensitive) pathway that was also sensitive to hypercapnia. This parallels well with the predicted function of slc26a6, and the detection of the transcript in embryos and tissues of trout. In conclusion, this study is the first report of slc26a6 in rainbow trout and functional and expression analyses indicate its likely involvement in Cl(-)/HCO3(-) exchange in two life stages of rainbow trout.

  9. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.

  10. The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

    PubMed

    Flaherty, Briana R; Wang, Yuxiao; Trope, Edward C; Ho, Tienhuei G; Muralidharan, Vasant; Kennedy, Eileen J; Peterson, David S

    2015-01-01

    Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

  11. Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

    PubMed Central

    Carlsen, Eric D.; Hay, Christie; Henard, Calvin A.; Popov, Vsevolod; Garg, Nisha Jain

    2013-01-01

    Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis. PMID:23918780

  12. Mechanism for Clastogenic Activity of Naphthalene

    SciTech Connect

    Buchholz, Bruce A.

    2015-09-29

    Naphthalene incubations form DNA adducts in vitro in a dose dependent manner in both mouse and rat tissues. Rodent tissue incubations with naphthalene indicate that naphthalene forms as many DNA adducts as Benzo(a)pyrene, a known DNA binding carcinogen. The mouse airway has the greatest number of DNA adducts, corresponding to the higher metabolic activation of naphthalene in this location. Both rat tissues, the rat olfactory (tumor target) and the airways (non-tumor target), have similar levels of NA-DNA adducts, indicating that short term measures of initial adduct formation do not directly correlate with sites of tumor formation in the NTP bioassays.

  13. Mechanism for Clastogenic Activity of Naphthalene

    SciTech Connect

    Buchholz, Bruce A.

    2016-06-24

    Naphthalene incubations form DNA adducts in vitro in a dose dependent manner in both mouse and rat tissues. Rodent tissue incubations with naphthalene indicate that naphthalene forms as many DNA adducts as Benzo(a)pyrene, a known DNA binding carcinogen. The mouse airway has the greatest number of DNA adducts, corresponding to the higher metabolic activation of naphthalene in this location. Both rat tissues, the rat olfactory (tumor target) and the airways (non-tumor target), have similar levels of NA-DNA adducts, indicating that short term measures of initial adduct formation do not directly correlate with sites of tumor formation in the NTP bioassays.

  14. Hydrogen peroxide-dependent uptake of iodine by marine Flavobacteriaceae bacterium strain C-21.

    PubMed

    Amachi, Seigo; Kimura, Koh; Muramatsu, Yasuyuki; Shinoyama, Hirofumi; Fujii, Takaaki

    2007-12-01

    The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I-). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, 125I-. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.

  15. Mechanisms of tissue uptake and retention of paclitaxel-coated balloons: impact on neointimal proliferation and healing

    PubMed Central

    Granada, Juan F; Stenoien, Mark; Buszman, Piotr P; Tellez, Armando; Langanki, Dan; Kaluza, Greg L; Leon, Martin B; Gray, William; Jaff, Michael R; Schwartz, Robert S

    2014-01-01

    Background The efficacy of paclitaxel-coated balloons (PCB) for restenosis prevention has been demonstrated in humans. However, the mechanism of action for sustained drug retention and biological efficacy following single-time drug delivery is still unknown. Methods and results The pharmacokinetic profile and differences in drug concentration (vessel surface vs arterial wall) of two different paclitaxel coating formulations (3 µg/mm2) displaying opposite solubility characteristics (CC=crystalline vs AC=amorphous) were tested in vivo and compared with paclitaxel-eluting stents (PES). Also, the biological effect of both PCB formulations on vascular healing was tested in the porcine coronary injury model. One hour following balloon inflation, both formulations achieved similar arterial paclitaxel levels (CC=310 vs AC=245 ng/mg; p=NS). At 24 h, the CC maintained similar tissue concentrations, whereas the AC tissue levels declined by 99% (p<0.01). At this time point, arterial levels were 20-fold (CC) and 5-fold (AC) times higher compared to the PES group (p<0.05). At 28 days, arterial levels retained were 9.2% (CC) and 0.04% (AC, p<0.01) of the baseline levels. Paclitaxel concentration on the vessel surface was higher in the CC at 1 (CC=36.7% vs AC=13.1%, p<0.05) and 7 days (CC=38.4% vs AC=11%, p<0.05). In addition, the CC induced higher levels of neointimal inhibition, fibrin deposition and delayed healing compared with the AC group. Conclusions The presence of paclitaxel deposits on the vessel surface driving diffusion into the arterial tissue in a time-dependent fashion supports the mechanism of action of PCB. This specific pharmacokinetic behaviour influences the patterns of neointimal formation and healing. PMID:25332821

  16. Mechanism of antibacterial activity of copper nanoparticles

    NASA Astrophysics Data System (ADS)

    Chatterjee, Arijit Kumar; Chakraborty, Ruchira; Basu, Tarakdas

    2014-04-01

    In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu2+ ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously.

  17. Mechanism of antibacterial activity of copper nanoparticles.

    PubMed

    Chatterjee, Arijit Kumar; Chakraborty, Ruchira; Basu, Tarakdas

    2014-04-04

    In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu(2+) ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously.

  18. Mechanism of photodynamic activity of pheophorbides.

    PubMed

    Tanielian, C; Kobayashi, M; Wolff, C

    2001-04-01

    Plasmid DNA is efficiently photocleaved by sodium pheophorbides (Na-Phdes) a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na-Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na-Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures and involves two types of reactions: (i) direct electron transfer between DNA bases (especially guanine) and pheophorbide singlet excited state, and (ii) indirect reactions mediated by reactive oxygen species, including singlet oxygen whose production from molecular oxygen is sensitized by the Na-Phdes triplet state. A preliminary report has appeared in "Photodynamic Therapy of Cancer II," Proc. SPIE 2325, 416-424 (1994).

  19. Cannabinoids prevent the amyloid β-induced activation of astroglial hemichannels: A neuroprotective mechanism.

    PubMed

    Gajardo-Gómez, Rosario; Labra, Valeria C; Maturana, Carola J; Shoji, Kenji F; Santibañez, Cristian A; Sáez, Juan C; Giaume, Christian; Orellana, Juan A

    2017-01-01

    The mechanisms involved in Alzheimer's disease are not completely understood and how astrocytes and their gliotransmission contribute to this neurodegenerative disease remains to be fully elucidated. Previous studies have shown that amyloid-β peptide (Aβ) induces neuronal death by a mechanism that involves the excitotoxic release of ATP and glutamate associated to astroglial hemichannel opening. We have demonstrated that synthetic and endogenous cannabinoids (CBs) reduce the opening of astrocyte Cx43 hemichannels evoked by activated microglia or inflammatory mediators. Nevertheless, whether CBs could prevent the astroglial hemichannel-dependent death of neurons evoked by Aβ is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aβ alone or in combination with the following CBs: WIN, 2-AG, or methanandamide (Meth). Hemichannel activity was monitored by single channel recordings and by time-lapse ethidium uptake while neuronal death was assessed by Fluoro-Jade C staining. We report that CBs fully prevented the hemichannel activity and inflammatory profile evoked by Aβ in astrocytes. Moreover, CBs fully abolished the Aβ-induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aβ. Consequently, this work opens novel avenues for alternative treatments that target astrocytes to maintain neuronal function and survival during AD. GLIA 2016 GLIA 2017;65:122-137.

  20. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation.

    PubMed

    Hawley, Simon A; Ross, Fiona A; Chevtzoff, Cyrille; Green, Kevin A; Evans, Ashleigh; Fogarty, Sarah; Towler, Mhairi C; Brown, Laura J; Ogunbayo, Oluseye A; Evans, A Mark; Hardie, D Grahame

    2010-06-09

    A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.

  1. The interactive effects of exercise and feeding on oxygen uptake, activity levels, and gastric processing in the graceful crab Cancer gracilis.

    PubMed

    McGaw, Iain J

    2007-01-01

    Exercise and digestive processes are known to elevate the metabolic rate of organisms independently. In this study, the effects of simultaneous exercise and digestion were examined in the graceful crab Cancer gracilis. This species exhibited resting oxygen uptake levels between 29 and 42 mg O(2) kg(-1) h(-1). In postprandial crabs, oxygen uptake was approximately double that of unfed crabs. During exercise, oxygen uptake increased three- to fourfold, reaching maximal levels of more than 130 mg O(2) kg(-1 ) h(-1). However, there was no difference in oxygen uptake during activity between unfed and postprandial animals. There was also no difference in exercise endurance levels between unfed and postprandial animals; both sets of animals were unable to right themselves after being turned on their backs, reaching exhaustion after 13-15 attempts. To determine whether increased activity affected gastric processes, the passage of a meal through the digestive system was followed using a fluoroscope. Passage of digesta through the gut system was slower in active animals than in resting crabs. Resting crabs cleared the foregut after approximately 18 h, which was significantly faster than the 34.5 h for constantly active animals. Likewise, the midgut region of resting animals was cleared at a faster rate than that of active animals. Because of residual amounts of digesta remaining in the hindgut, no difference in clearance rates of this section of the gut was evident. The slower clearance times of the foregut were due to a significantly slower rate of mastication of food, as evidenced by a lower cardiac stomach contraction rate. Contraction of the pyloric region of the foregut functions to move the digesta along the midgut, and there was a direct correlation between slower contraction rates of this region and the increased time of passage for digesta through the midgut of active animals. Because increased activity levels affected gastric processing, the crabs exhibited a

  2. Genetic characterization, nickel tolerance, biosorption, kinetics, and uptake mechanism of a bacterium isolated from electroplating industrial effluent.

    PubMed

    Nagarajan, N; Gunasekaran, P; Rajendran, P

    2015-04-01

    Electroplating industries in Madurai city produce approximately 49,000 L of wastewater and 1200 L of sludge every day revealing 687-5569 ppm of nickel (Ni) with other contaminants. Seventeen Ni-tolerant bacterial strains were isolated from nutrient-enriched effluents. Among them one hyper Ni accumulating strain was scored and identified as Bacillus cereus VP17 on the basis of morphology, biochemical tests, 16S rDNA gene sequencing, and phylogenetic analysis. Equilibrium data of Ni(II) ions using the bacterium as sorbent at isothermal conditions (37 °C) and pH 6 were best adjusted by Langmuir (R(2) = 0.6268) and Freundlich models (R(2) = 0.9505). Experimental validation reveals Ni sorption takes place on a heterogeneous surface of the biosorbent, and predicted metal sorption capacity is 434 ppm. The pseudo-second-order kinetic model fitted the biosorption kinetic data better than the pseudo-first-order kinetic model (R(2) = 0.9963 and 0.3625). Scanning electron microscopy, energy dispersive X-ray, and Fourier transform infrared spectroscopy studies of the bacterial strain with and without Ni(II) ion reveals the biosorption mechanism. The results conclude possibilities of using B. cereus VP17 for Ni bioremediation.

  3. On the mechanisms that limit oxygen uptake during exercise in acute and chronic hypoxia: role of muscle mass

    PubMed Central

    Calbet, José A L; Rådegran, Göran; Boushel, Robert; Saltin, Bengt

    2009-01-01

    Peak aerobic power in humans () is markedly affected by inspired O2 tension (). The question to be answered in this study is what factor plays a major role in the limitation of muscle peak in hypoxia: arterial O2 partial pressure () or O2 content ()? Thus, cardiac output (dye dilution with Cardio-green), leg blood flow (thermodilution), intra-arterial blood pressure and femoral arterial-to-venous differences in blood gases were determined in nine lowlanders studied during incremental exercise using a large (two-legged cycle ergometer exercise: Bike) and a small (one-legged knee extension exercise: Knee) muscle mass in normoxia, acute hypoxia (AH) () and after 9 weeks of residence at 5260 m (CH). Reducing the size of the active muscle mass blunted by 62% the effect of hypoxia on in AH and abolished completely the effect of hypoxia on after altitude acclimatization. Acclimatization improved Bike peak exercise from 34 ± 1 in AH to 45 ± 1 mmHg in CH (P < 0.05) and Knee from 38 ± 1 to 55 ± 2 mmHg (P < 0.05). Peak cardiac output and leg blood flow were reduced in hypoxia only during Bike. Acute hypoxia resulted in reduction of systemic O2 delivery (46 and 21%) and leg O2 delivery (47 and 26%) during Bike and Knee, respectively, almost matching the corresponding reduction in . Altitude acclimatization restored fully peak systemic and leg O2 delivery in CH (2.69 ± 0.27 and 1.28 ± 0.11 l min−1, respectively) to sea level values (2.65 ± 0.15 and 1.16 ± 0.11 l min−1, respectively) during Knee, but not during Bike. During Knee in CH, leg oxygen delivery was similar to normoxia and, therefore, also in spite of a of 55 mmHg. Reducing the size of the active muscle mass improves pulmonary gas exchange during hypoxic exercise, attenuates the Bohr effect on oxygen uploading at the lungs and preserves sea level convective O2 transport to the active muscles. Thus, the altitude-acclimatized human has potentially a similar exercising capacity as at sea level when the

  4. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  5. Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO2 Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica1

    PubMed Central

    Nobel, Park S.; Hartsock, Terry L.

    1983-01-01

    The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H+ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C3 photosynthesis. The total nocturnal H+ accumulation and CO2 uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H+ accumulation and 4 moles per square meter per day for CO2 uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H+ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period. A minimum ratio of H+ accumulated to CO2 taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H+-to-CO2 ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H+ accumulation and CO2 uptake decreased, but the H+-to-CO2 ratio increased. A ratio of two H+ per CO2 is consistent with the H+ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO2 uptake is strongly favored over respiratory activity. PMID:16662802

  6. Relationship of changing delta 4-steroid 5 alpha-reductase activity to (125I)iododeoxyuridine uptake during regeneration of involuted rat prostates

    SciTech Connect

    Kitahara, S.; Higashi, Y.; Takeuchi, S.; Oshima, H. )

    1989-04-01

    To elucidate the phenotypic expression of proliferating prostatic cells, rats were castrated, and the regenerating process of involuted ventral prostates during testosterone propionate (TP) administration was investigated by examining morphology, (5-{sup 125}I)iododeoxyuridine ({sup 125}I-UdR) uptake, DNA content, weight, acid phosphatase, and delta 4-steroid 5 alpha-reductase (5 alpha-reductase) activities. Morphologically, TP treatment initially increased the number of epithelial cells lining glandular lobules and subsequently restored the shape of epithelial cells. {sup 125}I-UdR uptake peaked on Day 3 of TP treatment and stayed at higher levels than for uncastrated controls until Day 14 of treatment. Prostatic weight, protein content, acid phosphatase, and DNA content returned to uncastrated control levels by Day 14 of TP treatment. TP administration markedly stimulated prostatic 5 alpha-reductase activity, which peaked on the Day 5 of treatment and decreased to uncastrated control levels by Day 14 of treatment. It is concluded that TP administration to castrated rats initially induced active mitotic division of the remaining stem cells, followed by formation of differentiated functional epithelial cells. Prostatic 5 alpha-reductase was highly active at the initial phase of active mitotic cell division. The major portion of the increased enzyme activity can be regarded as a phenotypic expression of stem or transient cells of prostatic epithelium.

  7. Effect of coumarin and xanthotoxin on mitochondrial structure, oxygen uptake, and succinate dehydrogenase activity in onion root cells.

    PubMed

    Kupidlowska, E; Dobrzynska, K; Parys, E; Zobel, A M

    1994-10-01

    At concentrations in which they occur on the plant surface and retard mitosis, coumarin and xanthotoxin lowered uptake of oxygen (by 60 and 30%, respectively) by meristematic cells ofAllium cepa root tips. They caused changes in the structure of the mitochondrial matrix to become dense, and protrusions of mitochondrial membranes were visible parallelling their hypertrophy, indicating alteration in the structure and physiology of these organelles. Coumarin and, to a lesser extent, xanthotoxin increased succinate dehydrogenase production in mitochondria and also in the cytoplasm, indicating changes in membrane permeability. Changes in oxygen uptake and mitochondrial structure, in addition to the retardation of mitosis, may be the reason these compounds act as allelochemicals after they have been removed from the plant surface and reach the root meristem.

  8. NOTE: The specific uptake size index for quantifying radiopharmaceutical uptake

    NASA Astrophysics Data System (ADS)

    Fleming, John S.; Bolt, Livia; Stratford, Jennifer S.; Kemp, Paul M.

    2004-07-01

    Quantitative indices of radionuclide uptake in an object of interest provide a useful adjunct to qualitative interpretation in the diagnostic application of radionuclide imaging. This note describes a new measure of total uptake of an organ, the specific uptake size index (SUSI). It can either be related in absolute terms to the total activity injected or to the specific activity in a reference region. As it depends on the total activity in the object, the value obtained will not depend on the resolution of the imaging process, as is the case with some other similar quantitative indices. This has been demonstrated in an experiment using simulated images. The application of the index to quantification of dopamine receptor SPECT imaging and parathyroid thyroid subtraction planar scintigraphy is described. The index is considered to be of potential value in reducing variation in quantitative assessment of uptake in objects with applications in all areas of radionuclide imaging.

  9. Sensory active piperine analogues from Macropiper excelsum and their effects on intestinal nutrient uptake in Caco-2 cells.

    PubMed

    Obst, Katja; Lieder, Barbara; Reichelt, Katharina V; Backes, Michael; Paetz, Susanne; Geißler, Katrin; Krammer, Gerhard; Somoza, Veronika; Ley, Jakob P; Engel, Karl-Heinz

    2017-03-01

    The phytochemical profile of Macropiper excelsum (G.Forst.) Miq. subsp. excelsum (Piperaceae), a shrub which is widespread in New Zealand, was investigated by LC-MS-guided isolation and characterization via HR-ESI-TOF-MS and NMR spectroscopy. The isolated compounds were sensorily evaluated to identify their contribution to the overall taste of the crude extract with sweet, bitter, herbal and trigeminal impressions. Besides the known non-volatile Macropiper compounds, the lignans (+)-diayangambin and (+)-excelsin, four further excelsin isomers, (+)-diasesartemin, (+)-sesartemin, (+)-episesartemin A and B were newly characterized. Moreover, piperine and a number of piperine analogues as well as trans-pellitorine and two homologues, kalecide and (2E,4E)-tetradecadienoic acid N-isobutyl amide were identified in M. excelsum, some of them for the first time. Methyl(2E,4E)-7-(1,3-benzodioxol-5-yl)hepta-2,4-dienoate was identified and characterized for the first time in nature. Sensory analysis of the pure amides indicated that they contributed to the known chemesthetic effects of Macropiper leaves and fruits. Since the pungent piperine has been shown to affect glucose and fatty acid metabolism in vivo in previous studies, piperine itself and four of the isolated compounds, piperdardine, chingchengenamide A, dihydropiperlonguminine, and methyl(2E,4E)-7-(1,3-benzodioxol-5-yl)hepta-2,4-dienoate, were investigated regarding their effects on glucose and fatty acid uptake by enterocyte-like Caco-2 cells, in concentrations ranging from 0.1 to 100 μM. Piperdardine showed the most pronounced effect, with glucose uptake increased by 83 ± 18% at 100 μM compared to non-treated control cells. An amide group seems to be advantageous for glucose uptake stimulation, but not necessarily for fatty acid uptake-stimulating effects of piperine-related compounds.

  10. Influence of dehydroepiandrosterone on G-6-PD activity and /sup 3/H-thymidine uptake of human lymphocytes in vitro

    SciTech Connect

    Ennas, M.G.; Laconi, S.; Dessi, S.; Milia, G.; Murru, M.R.; Manconi, P.E.

    1987-01-01

    Dehydroepiandrosterone (DHEA) was found to inhibit experimental cancer development in mouse and rat lung, colon and mammary gland. Since DHEA is a potent inhibitor of mammalian G-6-PD, the hypothesis that the compound could inhibit cell proliferation through an inhibition of the pentose phosphate pathway has been formulated. We studied the effects of DHEA on the proliferation in vitro of human lymphocytes induced by several mitogens (PHA, ConA and PWM), measuring /sup 3/H-thymidine uptake. DHEA inhibited /sup 3/H-thymidine uptake of mitogen-stimulated cells from both G-6-PD+ and G-6-PD- (mediterranean type deficiency) individuals in a dose-dependent and reversible fashion. The inhibitory effect was found even if DHEA was added to cells in the last hours of culture, simultaneously with the addition of /sup 3/H-thymidine. These data suggest that the inhibition of thymidine uptake induced by DHEA on human lymphocytes probably does not depend on the inhibition of G-6-PD.

  11. Mechanical Activation of Construction Binder Materials by Various Mills

    NASA Astrophysics Data System (ADS)

    Fediuk, R. S.

    2016-04-01

    The paper deals with the mechanical grinding down to the nano powder of construction materials. During mechanical activation a composite binder active molecules cement minerals occur in the destruction of the molecular defects in the areas of packaging and breaking metastable phase decompensation intermolecular forces. The process is accompanied by a change in the kinetics of hardening of portland cement. Mechanical processes during grinding mineral materials cause, along with the increase in their surface energy, increase the Gibbs energy of powders and, respectively, their chemical activity, which also contributes to the high adhesion strength when contacting them with binders. Thus, the set of measures for mechanical activation makes better use of the weight of components filled with cement systems and adjust their properties. At relatively low cost is possible to provide a spectacular and, importantly, easily repeatable results in a production environment.

  12. Investigation of oxidation process of mechanically activated ultrafine iron powders

    NASA Astrophysics Data System (ADS)

    Lysenko, E. N.; Nikolaev, E. V.; Vlasov, V. A.; Zhuravkov, S. P.

    2016-02-01

    The oxidation of mechanically activated ultrafine iron powders was studied using X- ray powder diffraction and thermogravimetric analyzes. The powders with average particles size of 100 nm were made by the electric explosion of wire, and were subjected to mechanical activation in planetary ball mill for 15 and 40 minutes. It was shown that a certain amount of FeO phase is formed during mechanical activation of ultrafine iron powders. According to thermogravimetric analysis, the oxidation process of non-milled ultrafine iron powders is a complex process and occurs in three stages. The preliminary mechanical activation of powders considerably changes the nature of the iron powders oxidation, leads to increasing in the temperature of oxidation onset and shifts the reaction to higher temperatures. For the milled powders, the oxidation is more simple process and occurs in a single step.

  13. Processes of nickel and cobalt uptake by a manganese oxide forming sediment in Pinal Creek, Globe mining district, Arizona

    USGS Publications Warehouse

    Kay, J.T.; Conklin, M.H.; Fuller, C.C.; O'Day, P. A.

    2001-01-01

    A series of column experiments was conducted using manganese oxide coated sediments collected from the hyporheic zone in Pinal Creek (AZ), a metal-contaminated stream, to study the uptake and retention of Mn, Ni, and Co. Experimental variables included the absence (abiotic) and presence (biotic) of active Mn-oxidizing bacteria, the absence and presence of dissolved Mn, and sediment manganese oxide content. Uptake of Mn under biotic conditions was between 8 and 39% higher than under abiotic conditions. Continuous uptake of Mn due to biotic oxidation was evident from extraction of column sediments. Manganese uptake is hypothesized to initially occur as adsorption, which led to subsequent surface and/or microbial oxidation. Complete breakthrough of Ni within 100 pore volumes indicated no process of continuous uptake and was modeled as an equilibrium adsorption process. Nickel uptake in the presence of dissolved Mn was 67-100% reversible. Sediment extractions suggest that Ni uptake occurred through weak and strong adsorption. Continuous uptake of cobalt increased with sediment manganese oxide content, and Co uptake was up to 75% greater under biotic than abiotic conditions. Cobalt uptake was controlled by both existing and newly formed manganese oxides. Only a small amount of Co uptake was reversible (10-25%). XANES spectral analysis indicated that most Co(II) was oxidized to Co(III) and probably incorporated structurally into manganese oxides. Although manganese oxides were the primary phase controlling uptake and retention of Mn, Ni, and Co, the mechanisms varied among the metals.

  14. Bavachin from Psoralea corylifolia Improves Insulin-Dependent Glucose Uptake through Insulin Signaling and AMPK Activation in 3T3-L1 Adipocytes.

    PubMed

    Lee, Hyejin; Li, Hua; Noh, Minsoo; Ryu, Jae-Ha

    2016-04-08

    The fruit of Psoralea corylifolia L. (Fabaceae) (PC), known as "Bo-Gol-Zhee" in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO) cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4) translocation by activating the Akt and 5'AMP-activated protein kinase (AMPK) pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways.

  15. Bavachin from Psoralea corylifolia Improves Insulin-Dependent Glucose Uptake through Insulin Signaling and AMPK Activation in 3T3-L1 Adipocytes

    PubMed Central

    Lee, Hyejin; Li, Hua; Noh, Minsoo; Ryu, Jae-Ha

    2016-01-01

    The fruit of Psoralea corylifolia L. (Fabaceae) (PC), known as “Bo-Gol-Zhee” in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO) cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα). Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4) translocation by activating the Akt and 5′AMP-activated protein kinase (AMPK) pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways. PMID:27070585

  16. Antibacterial activity of phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual mechanisms.

    PubMed

    Ning, Yawei; Yan, Aihong; Yang, Kun; Wang, Zhixin; Li, Xingfeng; Jia, Yingmin

    2017-08-01

    Phenyllactic acid (PLA), a phenolic acid phytochemical, is considered to be a promising candidate for use as a chemical preservative due to its broad antimicrobial activity. The antibacterial target of PLA has rarely been reported, thus investigations were performed to elucidate the antibacterial mechanism of PLA against Listeria monocytogenes and Escherichia coli. Flow cytometry analysis stained with propidium iodide (PI) demonstrated that PLA could damage the membrane integrity of L. monocytogenes, while it could not disrupt that of E. coli. The uptake of 1-N-phenylnaphthylamine (NPN) indicated that PLA interrupted the outer membrane permeability of E. coli. Scanning electron microscopy (SEM) observation visualized the damage caused by PLA as morphological changes in L. monocytogenes and E. coli. Fluorescence assays demonstrated that PLA could interact with bacterial genomic DNA in the manner of intercalation. This finding suggested dual antibacterial targets of PLA, namely membrane and genomic DNA.

  17. Lipophilic 2'-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery.

    PubMed

    Biscans, Annabelle; Bertrand, Jean-Rémi; Dubois, Josephine; Rüger, Jacqueline; Vasseur, Jean-Jacques; Sczakiel, Georg; Dupouy, Christelle; Debart, Françoise

    2016-11-03

    The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi-based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2'-O-phenylisobutyryloxymethyl (PiBuOM)-modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM-modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)-modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM-modified siRNA and of substantial target suppression in human cells in serum-containing medium.

  18. Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity.

    PubMed

    Stoffel, Sabine A; Rodenko, Boris; Schweingruber, Anne-Marie; Mäser, Pascal; de Koning, Harry P; Schweingruber, M Ernst

    2006-02-01

    Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.

  19. Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration.

    PubMed

    Akita, Hidetaka; Nakatani, Taichi; Kuroki, Kimiko; Maenaka, Katsumi; Tange, Kota; Nakai, Yuta; Harashima, Hideyoshi

    2015-07-25

    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold.

  20. Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death.

    PubMed

    Soler, David C; Ohtola, Jennifer; Sugiyama, Hideaki; Rodriguez, Myriam E; Han, Ling; Oleinick, Nancy L; Lam, Minh; Baron, Elma D; Cooper, Kevin D; McCormick, Thomas S

    2016-06-08

    Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin