scholarly journals Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide

1996 ◽  
Vol 319 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Krikor TOROSSIAN ◽  
Marie AUDETTE ◽  
Richard POULIN
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Polyamine Transport ◽  
Substrate Protection

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11±4 and 96±16 µM after 30 min at 22 °C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and Km for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 °C respectively. Spermine (10 µM) almost fully protected against spermidine-transport inactivation by EDC at 22 °C, and decreased the rate of inactivation at 37 °C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 °C with EC50 values estimated at 10, 1 and less than 1 µM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89±3 compared with 44±5%), whereas the converse was true for 100 µM DCC (81±3 compared with 92±5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na+-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to cross-link a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

scholarly journals Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Abstract Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

scholarly journals Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 206-213 ◽  
Author(s):  
P Mayer ◽  
C Lam ◽  
H Obenaus ◽  
E Liehl ◽  
J Besemer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
White Blood Cells ◽  
Chinese Hamster ◽  
Coli Strain ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Gm Csf ◽  
Cell Counts

The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.

Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

2007 ◽  
Vol 292 (5) ◽  
pp. F1583-F1591 ◽  
Author(s):  
Ryan M. Pelis ◽  
Yodying Dangprapai ◽  
Theresa M. Wunz ◽  
Stephen H. Wright
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Inorganic Mercury ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Tubular Secretion ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Transport Activity ◽  
Concentration Dependent Manner ◽  
Quadruple Mutant

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO2-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg2+(as HgCl2) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [3H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl2, with an IC50of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl2caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg2+, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl2than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl2effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg2+with these residues causes reduced TEA transport activity.

Stereoselective Interaction of Ketamine with Recombinant [micro sign], [small kappa, Greek], and [small delta, Greek] Opioid Receptors Expressed in Chinese Hamster Ovary Cells 

1999 ◽  
Vol 90 (1) ◽  
pp. 174-182 ◽  
Author(s):  
Kazuyoshi Hirota ◽  
Hirobumi Okawa ◽  
Balraj L. Appadu ◽  
David K. Grandy ◽  
Lakshmi A. Devi ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Room Temperature ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Racemic Ketamine ◽  
Dose Dependent

Background The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). Methods CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. Results Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. Conclusions Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.

scholarly journals A Real-Time, Fluorescence-Based Assay for Measuring µ-Opioid Receptor Modulation of Adenylyl Cyclase Activity in Chinese Hamster Ovary Cells

2013 ◽  
Vol 19 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Alisa Knapman ◽  
Fe Abogadie ◽  
Peter McIntrye ◽  
Mark Connor
Keyword(s):  
Adenylyl Cyclase ◽  
Real Time ◽  
Opioid Receptor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Simultaneous Application ◽  
Ovary Cells ◽  
Receptor Modulation

Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential–sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK ( pEC50, 7.3) or calcitonin ( pEC50, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO ( pEC50, 7.4; Emax, 56%), morphine ( pEC50, 7.0; Emax, 61%); and buprenorphine ( pEC50, 8.6; Emax, 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.

Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation

2004 ◽  
Vol 287 (3) ◽  
pp. C737-C745 ◽  
Author(s):  
Horia Vais ◽  
Rugang Zhang ◽  
William W. Reenstra
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Channel Activity ◽  
Ovary Cells ◽  
Channel Opening ◽  
Excised Patches ◽  
A Site ◽  
R Domain

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities ( Po), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased Po, whereas Ser-to-Ala mutations at 737 and 768 increased Po. In general, differences in Po were due to differences in mean closed time. For selected constructs with either high or low values of Po, channel activity was measured in excised patches. With 1 mM ATP, Po was similar to that observed in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated Po values. ATP-dependent increases in Po were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.

Anionic amino acid uptake by microvillous membrane vesicles from human placenta

1989 ◽  
Vol 257 (5) ◽  
pp. C1005-C1011 ◽  
Author(s):  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamate Uptake ◽  
Membrane Vesicles ◽  
Amino Acid Uptake ◽  
Maternal Blood ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Concentration Dependent Manner

The transport mechanisms for anionic amino acids in trophoblast microvillous (maternal facing) membrane were investigated by characterization of L-[3H]aspartate and L-[3H]glutamate uptake in membrane vesicles. Uptake of the anionic amino acids was by a single high-affinity Na+-dependent K+-stimulated cotransporter that is pH sensitive and electrogenic. A second Na+-dependent transporter could not be discriminated, and there was no observable Na+-independent uptake. An outwardly directed K+ gradient (100 mM KCl inside) resulted in a 5- to 10-fold stimulation in glutamate uptake in the presence of Na+. Intravesicular KCl had no effect on transporter affinity but increased transporter velocity in a concentration-dependent manner. Inhibition of Na+-K+-dependent uptake of L-aspartate and L-glutamate (20 mM, 30 s) by 2 mM unlabeled amino acids demonstrated stereoselectivity for L-glutamate but not for L-aspartate. The neutral amino acids (L-alanine, L-threonine, L-serine, L-cysteine, L-phenylalanine) were not effective inhibitors. These data are consistent with an anionic amino acid transporter in the microvillous membrane of the trophoblast, which has characteristics qualitatively similar to the X-AG system found in other epithelia. This system may mediate the concentrative placental uptake of anionic amino acids from maternal blood in utero.

Sialyltransferase specificity in selectin ligand formation

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3618-3625 ◽  
Author(s):  
Lesley G. Ellies ◽  
Markus Sperandio ◽  
Gregory H. Underhill ◽  
James Yousif ◽  
Michael Smith ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Neuraminidase Treatment ◽  
Ovary Cells ◽  
Rolling Velocity ◽  
Selectin Ligands ◽  
Selectin Ligand

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor α (TNF-α)–induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin–dependent manner was decreased while E-selectin–dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.

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