Home About us Contact
|
Home About us Contact | ||
|
|
||
Uptake Experiments (uptake + experiment)
Selected AbstractsUptake Mechanism of Oppositely Charged Fluorescent Nanoparticles in HeLa CellsMACROMOLECULAR BIOSCIENCE, Issue 12 2008Julia Dausend Abstract The endocytotic mechanisms involved in the uptake of charged polystyrene nanoparticles into HeLa cells were investigated. Uptake experiments were done in the presence or absence of drugs known to inhibit various factors in endocytosis. Independent of the particle charge, endocytosis is highly dependent on dynamin, F-actin, and tyrosine-specific protein kinases, which suggests a dynamin-dependent and lipid raft-dependent mechanism. However, cholesterol depletion did not hinder particle uptake. Regarding positively charged particles, macropinocytosis, the microtubule network, and cyclooxygenases are also involved. The clathrin-dependent pathway plays a minor role. [source] Transcellular transport of genistein, a soybean-derived isoflavone, across human colon carcinoma cell line (Caco-2)BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2001Masataka Oitate Abstract Genistein, a soybean-derived isoflavone, is thought to have an anticarcinogenic action, but little is known about the cellular mechanisms of its intestinal absorption. This study was designed to investigate the absorption mechanisms of genistein using human colon carcinoma cell line, Caco-2 cells. The apical-to-basolateral transcellular transport of genistein across a Caco-2 cell monolayer was significantly greater than that in the opposite direction. An uptake experiment revealed that cellular uptake of genistein by Caco-2 cells was concentrative. The transcellular transport of genistein was saturable and temperature-dependent, and was inhibited by other flavonoids such as rutin, quercetin, (+)-catechin and (,)-epicatechin. These results suggest that genistein is transported across Caco-2 cells by a carrier-mediated system, located on the apical membrane. Copyright © 2001 John Wiley & Sons, Ltd. [source] Accumulation of 137Cs by larvae of the midge Chironomus riparius from sediment: Effect of potassiumENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2003Lieven Bervoets Abstract We studied the effect of potassium on the uptake of radiocesium from sediment by larvae of the midge Chironomus riparius. Sediment ingestion rate was determined for one week. After 24 h the gut content remained constant, indicating that equilibrium was reached between sediment ingestion and sediment elimination. These data were used to account for radiocesium present in the gut in subsequent uptake experiments. Reference sediment was equilibrated with solutions containing different concentrations of potassium: 1, 10, 100, and 1,000 ,M. Adsorption of 137Cs to the sediment was investigated. Three different radiocesium levels (0.3, 0.6, and 1.2 KBq/ml) were applied at the four different potassium levels. In all cases more than 94% of all radiocesium was adsorbed to the sediment within 48 h. The sediment, equilibrated with the four different potassium levels, was spiked with a constant amount of 296 Bq/ml 137Cs. Accumulation by midge larvae was followed for one week, and subsequently elimination was followed for another week. No significant differences in radiocesium levels in midge larvae among the treatments were found after one week of exposure. However, using a one-compartment accumulation model, a small but significant effect of potassium in water and sediment on the uptake and elimination rate constants (ka and ke) was found. These results indicate that although differences were rather small, radiocesium accumulation decreased with increasing potassium level in the sediment. [source] Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitiveEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Antonia Marín-Burgin Abstract The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at ,,43 °C and ,,53 °C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1,10 ,m) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1,10 ,m) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 ,m) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site. [source] Length-Dependent Uptake of DNA-Wrapped Single-Walled Carbon Nanotubes,ADVANCED MATERIALS, Issue 7 2007L. Becker A length threshold for cell uptake of DNA-wrapped single-walled carbon nanotubes (SWNTs) by human lung fibroblasts (IMR90) is identified. Competitive uptake experiments with well-defined and characterized length fractions show that SWNTs above the length threshold are excluded from the cell, whereas SWNTs labeled with Cy3-derivatized DNA below the threshold are able to access the cell interior, as shown in the fluorescence image and on the cover. [source] High glucose inhibits fructose uptake in renal proximal tubule cells: Involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Su Hyung Park The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l -glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14,22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs. © 2004 Wiley-Liss, Inc. [source] Protein adsorption kinetics in charged agarose gels: Effect of agarose content and modelingAICHE JOURNAL, Issue 2 2009Emily B. Schirmer Abstract The adsorption kinetics of myoglobin in charged gels of varying agarose content have been measured macroscopically, through batch uptake experiments, and microscopically, using light microscopy with gels supported in microfluidics chips. The apparent effective pore diffusivities, determined by fitting either set of rate data to the shrinking core model, were greater than the free solution diffusivity and concentration-dependent. Moreover, the microscopically derived concentration profiles were qualitatively different from the predicted ones. Therefore, a new model taking into account an assumed favorable partitioning of the protein in the pore liquid is proposed to describe the adsorption kinetics. The new model yields effective pore diffusivities that are in approximate agreement with the values determined chromatographically under nonbinding conditions and with hindered diffusion theory. In addition, it predicts concentration profiles in the gel that are consistent with those observed microscopically. The overall increase in mass transfer is attributed to the favorable partitioning of the protein in the pores at low ionic strength, which results in a greater diffusional driving force. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source] Identification of a GM1/Sodium,Calcium exchanger complex in the nuclear envelope of non-neuronal cellsJOURNAL OF NEUROCHEMISTRY, Issue 2002X. Xie Our previous studies identified a Na,Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108-15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non-neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti-NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non-nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti-NCX antibody and the precipitated proteins separated on SDS,PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS,PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1. Acknowledgements:, supported by NIH grant NS 33912. [source] Transungual iontophoretic transport of polar neutral and positively charged model permeants: Effects of electrophoresis and electroosmosisJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2008Jinsong Hao Abstract Transungual iontophoretic transport of model neutral permeants mannitol (MA), urea (UR), and positively charged permeant tetraethylammonium ion (TEA) across fully hydrated human nail plates at pH 7.4 were investigated in vitro. Four protocols were involved in the transport experiments with each protocol divided into stages including passive and iontophoresis transport of 0.1 and 0.3 mA. Water and permeant uptake experiments of nail clippings were also conducted to characterize the hydration and binding effects of the permeants to the nails. Iontophoresis enhanced the transport of MA and UR from anode to cathode, but this effect (electroosmosis) was marginal. The transport of TEA was significantly enhanced by anodal iontophoresis and the experimental enhancement factors were consistent with the Nernst,Planck theory predictions. Hindered transport was also observed and believed to be critical in transungual delivery. The barrier of the nail plates was stable over the time course of the study, and no significant electric field-induced alteration of the barrier was observed. The present results with hydrated nail plates are consistent with electrophoresis-dominant (the direct field effect) transungual iontophoretic transport of small ionic permeants with small contribution from electroosmosis. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:893,905, 2008 [source] Changes in zinc uptake in response to ascorbic acid and folic acid in rat liver slices under normal and oxidative stress conditionsBIOFACTORS, Issue 1 2007R.S. Tupe Abstract Zinc plays a dual role, as an integral part of metabolic machinery and in defense against reactive oxygen species. Hepatocytes are important sites for zinc metabolism for synthesis of zinc metalloproteins and maintaining its homeostasis. However, the factors influencing post absorptive zinc metabolism under normal and oxidative stress (OS) conditions are not well understood. Using rat liver slices, we conducted a series of four in vitro zinc uptake experiments to study influence of ascorbic acid and folic acid in normal and oxidative stress conditions with Zn concentrations representing deficient to excess states (7.7,30.7 millimole/L). Zinc uptakes under OS at these four zinc levels were lower than the normal conditions. Folic acid showed significant inhibitory effect on zinc uptake under both normal and OS conditions in a dose response manner. Nevertheless, dose response of ascorbic acid at four zinc levels indicated its marked enhancing effect under OS condition. Differences in zinc uptake trend lines between the normal and OS conditions for interaction of both the vitamins narrowed down as the zinc levels increased. Our results suggest that folic acid causes inhibitory effect, while ascorbic acid may be protective in OS with reference to zinc uptake. [source] Differential inhibitory effects of drugs acting at the noradrenaline and 5-hydroxytryptamine transporters in rat and human neocortical synaptosomes,BRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2009M Mantovani Background and purpose:, Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. Experimental approach:, Synaptosomes prepared from fresh human and rat neocortical tissues were used for [3H]-5-HT and [3H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). Key results:, In saturation experiments on synaptosomal [3H]-5-HT and [3H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram , duloxetine = fluvoxamine , fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. Conclusions and implications:, This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile. [source] Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: The possible contribution of 86Rb+ efflux and 45Ca2+ uptakeCELL BIOCHEMISTRY AND FUNCTION, Issue 8 2008M. Töpfer Abstract Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+ - and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1 -receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5,- N -ethylcarboxyamidoadenosine) at high concentration (10,µM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10,nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6 -cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50,µM) can be increased by 10,µM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1 -receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues. Copyright © 2008 John Wiley & Sons, Ltd. [source] High Throughput Screening for the Design and Optimization of Chromatographic Processes: Assessment of Model Parameter Determination from High Throughput Compatible DataCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 12 2008A. Susanto Abstract Chromatographic processes can be optimized in various ways, and the two most prominent approaches are based either on statistical data analysis or on experimentally validated simulation models. Both strategies rely heavily on experimental data, the generation of which usually imposes a significant bottleneck on rational process design. The latter approach is followed in this work, and the utilizability of high throughput compatible experiments for the determination of model parameters which are required for in silico process optimization, is assessed. The unknown parameter values are estimated from batch uptake experiments on a robotic platform and from dynamic breakthrough experiments with miniaturized chromatographic columns. The identified model is then validated with respect to process optimization by comparison of model predictions with experimental data from a preparative scale column. In this study, a strong cation exchanger Toyopearl SP-650M and lysozyme solved in phosphate buffer (pH 7), is used as the test system. The utilization of data from miniaturized and high throughput compatible experiments is shown to yield sufficiently accurate results, and minimizes efforts and costs for both parameter estimation and model validation. [source] | |||||||||||||||||||