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Uptake Studies (uptake + studies)
Selected AbstractsNucleoside transporter expression and function in cultured mouse astrocytesGLIA, Issue 1 2005Liang Peng Abstract Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755,760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1,4°C to study diffusional uptake and 1,60-min uptakes at 37°C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na+ -independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na+ -dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine. © 2005 Wiley-Liss, Inc. [source] Localization and functional characterization of the human NKCC2 isoformsACTA PHYSIOLOGICA, Issue 3 2010I. Carota Abstract Aim:, Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na+/K+/2Cl, cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. Methods:, RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. Results:, All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl, affinity as determined by 86Rb+ uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. Conclusion:, The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function. [source] Protein Kinase C Regulation of Rat Jejunal Transport Systems: Mechanisms Involved in Lactate MovementEXPERIMENTAL PHYSIOLOGY, Issue 6 2002Marisa Tosco We examined whether protein kinase C (PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+ -lactate symport, as well as on apical Na+ -glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4-,-PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+,K+ -ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis. [source] Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27FEBS JOURNAL, Issue 18 2006Cornelia Schwarzenlander Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 µg DNA·(mg protein),1·min,1, demonstrating an extremely efficient binding and uptake rate of 40 kb·s,1·cell,1. Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments. [source] A study in the uranyl ions uptake on acrylic acid and acrylamide copolymeric hydrogelsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2008Ghanshyam S. Chauhan Abstract A series of copolymeric hydrogels comprising of acrylic acid and acrylamide and crosslinked with trimethylolpropane triacrylate (TMPTA) were prepared using ammonium persulfate (APS) as initiator. The hydrogels were functionalized further by partial hydrolysis and were characterized by SEM, FTIR, nitrogen analysis, and also by water uptake studies as a function of time, temperature, pH, NaCl, and concentration of sodium dodecyl sulfate (SDS). These hydrogels were used as sorbents for the uranyl ions uptake in the presence of 5% NaCl, which was studied as function of time, temperature, pH, and ion strength. The uranyl uptake was found to be affected both by the structural aspects of the hydrogels as well as by the external environmental factors. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Pectin and acrylamide based hydrogels for environment management technologies: Synthesis, characterization, and metal ions sorptionJOURNAL OF APPLIED POLYMER SCIENCE, Issue 4 2007Ghanshyam S. Chauhan Abstract The present article describes various aspects of preparation and characterization of hydrogels based on pectin and three different amide monomers viz: acrylamide (AAm), N -isopropyl acrylamide (N -i-PAAm), and 2-acrylamido-2-methyl-1-propane sulfonic acid (AAmPSA). Hydrogels have been prepared by graft copolymerization as well as in the presence of crosslinker N,N -Methylene bisacrylamide ((N,N -MBAAm) and initiated by redox system comprising of ammonium peroxysulphate,ferrous ammonium sulfate (APS: FAS) at two temperatures. Hydrogels thus synthesized have been characterized by SEM, FTIR, and water uptake studies. The later has been carried as a function of time, temperature, pH, crosslinker concentration, and temperature at which the hydrogels were prepared. Candidate hydrogels have been used for the sorption of some common metal ions pollutants found in soil, industrial, and mining water bodies. Biodegradability studies have been carried by soil burial method to investigate the effect of chemical modification on biodegradability of pectin and to understand the possibility of eco-friendly nature and to explore the scope of reusability of the hydrogels and waste minimization. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007 [source] Phosphate and calcium are required for TGF,-mediated stimulation of ANK expression and function during chondrogenesisJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Paulina Oca The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGF,. The purpose of this study was to determine whether TGF, stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGF, increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na+/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGF, required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGF, on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGF,. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGF,-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (,1C) inhibited the TGF, stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGF, stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation. J. Cell. Physiol. 224: 540,548, 2010. © 2010 Wiley-Liss, Inc. [source] Cholangiocyte bile salt transporters in cholesterol gallstone,susceptible and resistant inbred mouse strainsJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 10 2008Julia J Liu Abstract Background and Aim:, We investigated the dietary and gender influences on the expression and functionality of cholangiocyte bile salt transporters and development of biliary hyperplasia in cholesterol gallstone-susceptible C57L/J and resistant AKR/J mice. Methods:, C57L and AKR mice were fed chow, a lithogenic diet, or a cholic acid-containing diet for 14 days. Expression of cholangiocyte bile salt transporter proteins ASBT (SLC10A2), ILBP (FABP6), and MRP3 (ABCC3) were studied by Western blot analysis. Taurocholate uptake studies were performed using microperfusion of isolated bile duct units. The pre- and post-perfusion taurocholate concentrations were analyzed by high performance liquid chromatography. Biliary proliferation in liver sections was scored. Results:, The lithogenic diet induced ductular proliferation in C57L mice. On chow, SLC10A2 and ABCC3 were overexpressed in male and female C57L compared to AKR mice. A lithogenic diet reduced the expressions of FABP6 in both male and female C57L mice, SLC10A2 in female C57L mice, and ABCC3 in male C57L mice. These alterations in transporter expressions were not associated with changes in taurocholate uptake. The lithogenic diet induced biliary hyperplasia and reduced bile salt transporter expressions in C57L mice. Conclusions:, Although bile salt uptake was not increased in the bile duct unit, we speculate that the biliary hyperplasia on the lithogenic diet may lead to an increase in intrahepatic bile salt recycling during cholesterol cholelithogenesis. [source] Comparison of monofunctional and multifunctional monomers in phosphate binding molecularly imprinted polymersJOURNAL OF MOLECULAR RECOGNITION, Issue 6 2008Xiangyang Wu Abstract In this study, molecularly imprinted polymers (MIPs) prepared using a multifunctional and a monofunctional monomer were compared with respect to their affinities, selectivities, and imprinting efficiencies for organophosphates. This is of interest because multifunctional monomers have higher affinities than traditional monofunctional monomers for their target analytes and thus should yield MIPs with higher affinities and selectivities. However, polymers containing multifunctional monomer may also have a higher number of unselective, non-templated binding sites. This increase in background binding sites could lead to a decrease in the magnitude of the imprinting effect and in the selectivity of the MIP. Therefore, phosphate selective imprinted and non-imprinted polymers (NIPs) were prepared using a novel multifunctional triurea monomer. The binding properties of these polymers were compared with polymers prepared using a monofunctional monourea monomer. The binding affinities and selectivities of the monomers, imprinted polymers, and NIPs were characterized by NMR titration, binding uptake studies, and binding isotherms. MIPs prepared with the triurea monomer showed higher binding affinity and selectivity for the diphenylphosphate anion in organic solvents than the MIPs prepared with the monofunctional monomer. Surprisingly, the binding properties of the NIPs revealed that the polymers prepared using the multifunctional and monofunctional monomers were very similar in affinity and selectivity. Thus, the multifunctional monomers increase not only the affinity of the MIP but also enhance the imprinting effect. Copyright © 2008 John Wiley & Sons, Ltd. [source] Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides,JOURNAL OF MOLECULAR RECOGNITION, Issue 1 2005Johannes Oehlke Abstract In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure,activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg3,Orn7 -SP and its D -amino acid analog, all- D -Arg3,Orn7 -SP, as well as by electron microscopic autoradiography using 3H-labelled SP and 125I-labelled all- D -SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature. Copyright © 2004 John Wiley & Sons, Ltd. [source] Comparison of the effects of chemical permeation enhancers on the lipoidal pathways of human epidermal membrane and hairless mouse skin and the mechanism of enhancer actionJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2007Doungdaw Chantasart Abstract Previously, the effects of chemical permeation enhancers upon the permeability of the lipoidal pathway of hairless mouse skin (HMS) were investigated and a quantitative structure enhancement relationship was established. The present study was to study the effects of these enhancers on human epidermal membrane (HEM) using the same experimental method employed in the previous HMS studies. The effects of enhancers on the permeability coefficients of the lipoidal pathways of HEM and HMS for corticosterone were found to be essentially the same. In the equilibrium uptake studies of the enhancers and ,-estradiol, it was found that the amounts of enhancers taken up and the partitioning of ,-estradiol into the HEM stratum corneum (SC) intercellular lipid under the E,=,10 conditions were different from those of HMS. Despite these differences, the HEM data show a correlation between the intercellular lipid/PBS partition coefficients of the enhancers and the enhancer n-octanol/PBS partition coefficients. This correlation is consistent with the observed chemical microenvironment of the site of enhancer action in the HMS SC in previous studies. Therefore, provided with proper experimental protocols, HMS can be a reliable model for the evaluation of the effects of skin permeation enhancers on the lipoidal pathway of HEM. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2310,2326, 2007 [source] Uptake and apoplastic retention of EDTA- and phytosiderophore-chelated chromium(III) in maizeJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 6 2007Bülent E. Erenoglu Abstract Increasing the mobilization and root uptake of chromium (Cr) by synthetic and plant-borne chelators might be relevant for the design of phytoremediation strategies on Cr-contaminated sites. Short-term uptake studies in maize roots supplied with 51CrCl3 or 51Cr(III)-EDTA led to higher apoplastic Cr contents in plant roots supplied with 51CrCl3 and in Fe-sufficient plants relative to Fe-deficient plants, indicating that Fe stimulated co-precipitation of Cr. Concentration-dependent retention of Cr in a methanol:chloroform-treated cell-wall fraction was still saturable and in agreement with the predicted tendency of Cr(III) to precipitate as Cr(OH)3. To investigate a possible stimulation of Cr(III) uptake by phytosiderophores, Fe-deficient maize roots were exposed for 6 d to Cr(III)-EDTA or Cr(III)-DMA (2'-deoxymugineic acid). Relative to plants without Cr supply, the supply of both chelated Cr species in a subtoxic concentration of 1 µM resulted in alleviation of Fe deficiency,induced chlorosis and higher Cr accumulation. Long-term Cr accumulation from Cr(III)-DMA was similar to that from Cr(III)-EDTA, and Cr uptake from both chelates was not altered in the maize mutant ys1, which is defective in metal-phytosiderophore uptake. We therefore conclude that phytosiderophores increase Cr solubility similar to synthetic chelators like EDTA, but do not additionally contribute to Cr(III) uptake from Cr-contaminated sites. [source] Evaluation of the Effect of Ethanol's Toxic Metabolite Acetaldehyde on the Gastrointestinal Oligopeptide Transporter, PEPT1: In Vitro and in Vivo StudiesALCOHOLISM, Issue 1 2008Scott J. Fisher Background:, The effects of alcohol consumption and its subsequent metabolism on drug transport, absorption and pharmacokinetics are poorly understood. This study examines the effects of the ethanol metabolite, acetaldehyde, on the clinically relevant drug transporter, PEPT1. The metabolism of ethanol and the following acetaldehyde formation is thought to modulate the uptake capacity of PEPT1 within the gastrointestinal tract for a variety of clinically important peptidomimetic drug compounds. Methods:, Glycylsarcosine ([3H]-GlySar), a nonhydrolysable PEPT1 specific substrate was used in our studies. In vitro uptake studies were performed in the Caco-2 and Chinese hamster ovary (CHO)-hPEPT1 cell models, measuring cellular uptake of labeled compound against increasing levels of unlabeled compound in the presence of acetaldehyde. In vivo absorption of [3H]-GlySar was measured in male Sprague,Dawley rats that were treated with oral dose of ethanol/disulfiram (5 g/kg / 100 mg/kg) for 6 days. These results were compared to control rats treated with saline, ethanol alone or disulfiram alone. Results:, In vitro uptake of [3H]-GlySar in CHO-hPEPT1 cells treated with 1 mM acetaldehyde was significantly decreased (p < 0.05) as compared to untreated controls. The uptake of [3H]-GlySar in Caco-2 cell monolayers treated with 1 mM acetaldehyde was also significantly decreased as compared to the untreated control cells. In vivo absorption of [3H]-GlySar in ethanol treated rats, as measured by AUC0,12 hours were decreased by approximately 50% versus the control rat group. Conclusion:, The effects of acetaldehyde due to consumption of ethanol on the uptake and bioavailability of therapeutic drug compounds transported by the PEPT1 oligopeptide transporter have not been documented. In the present studies, we demonstrate that acetaldehyde significantly modulates PEPT1 function and, thereby, affects drug bioavailability. To our best knowledge, this is the first report on the effects of an ethanol metabolite on substrate absorption in the gastrointestinal tract, rather than interactions in the liver, which is an under-represented area of research in alcohol pathophysiology. [source] | ||||||||||||||||