scholarly journals Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method

1984 ◽  
Vol 224 (3) ◽  
pp. 1001-1008 ◽  
Author(s):  
A R P Paterson ◽  
E R Harley ◽  
C E Cass
Keyword(s):  
Cellular Uptake ◽  
Cultured Cells ◽  
Flow System ◽  
Kinetic Constants ◽  
Cell Water ◽  
Short Intervals ◽  
Adenosine Uptake ◽  
Uptake Rates ◽  
Time Courses ◽  
Initial Uptake

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.

scholarly journals Cellular Uptake and Intracellular Levels of the Bcl-2 Antisense G3139 in Cultured Cells and Treated Patients with Acute Myeloid Leukemia

2005 ◽  
Vol 11 (8) ◽  
pp. 2998-3008 ◽  
Author(s):  
Guowei Dai ◽  
Kenneth K. Chan ◽  
Shujun Liu ◽  
Dale Hoyt ◽  
Susan Whitman ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cellular Uptake ◽  
Myeloid Leukemia ◽  
Cultured Cells ◽  
Acute Myeloid

scholarly journals Bovine endometrium-derived cultured cells are suitable for lipofection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mai Shiokawa ◽  
Ryotaro Miura ◽  
Aki Okubo ◽  
Yujiro Hagita ◽  
Itaru Yoshimura ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Transfection Efficiency ◽  
Cell Populations ◽  
Kidney Cells ◽  
Reporter Assay ◽  
Vimentin Expression ◽  
Bovine Kidney ◽  
Short Intervals ◽  
Veterinary Research

AbstractBovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.

scholarly journals Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent

2000 ◽  
Vol 352 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Tadao HASEGAWA ◽  
Hengyi XIAO ◽  
Fumiyasu HAMAJIMA ◽  
Ken-ichi ISOBE
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Cultured Cells ◽  
Similar Region ◽  
C Terminus ◽  
Mouse Tissues ◽  
Time Courses ◽  
Shock Proteins ◽  
Two Hybrid

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ134.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the γ134.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase

1983 ◽  
Vol 3 (1) ◽  
pp. 82-90
Author(s):  
M B Puziss ◽  
R M Wohlhueter ◽  
P G Plagemann
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
High Capacity ◽  
Rate Equations ◽  
Cell Water ◽  
Adenine Phosphoribosyltransferase ◽  
Novikoff Hepatoma ◽  
L929 Cells ◽  
Equilibrium Exchange ◽  
Time Courses

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).

scholarly journals A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake in Streptococcus mutans

2006 ◽  
Vol 188 (23) ◽  
pp. 8005-8012 ◽  
Author(s):  
Alexander J. Webb ◽  
Arthur H. F. Hosie
Keyword(s):  
Streptococcus Mutans ◽  
Abc Transporter ◽  
Atp Binding ◽  
Diverse Range ◽  
Adenosine Uptake ◽  
Uptake Rates ◽  
Genes Encoding ◽  
Uptake Transporters ◽  
Atp Binding Cassette

ABSTRACT Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2-14C]cytidine or [2-14C]uridine and had significantly reduced [8-14C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity (Km = 1 to 2 μM) transporter of cytidine, uridine, and adenosine. The inhibition of [14C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily.

Ethanol metabolism and inhibition of nucleoside uptake lead to increased extracellular adenosine in hepatocytes

1992 ◽  
Vol 262 (5) ◽  
pp. C1175-C1180 ◽  
Author(s):  
L. E. Nagy
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Ethanol Metabolism ◽  
Nucleoside Transporter ◽  
Extracellular Adenosine ◽  
Mediated Effects ◽  
Adenosine Uptake ◽  
Nucleoside Uptake ◽  
Acute And Chronic Effects

Recent evidence suggests that adenosine mediates many of the acute and chronic effects of ethanol in both cultured cells and whole animals. These adenosine-mediated effects of ethanol result from ethanol-induced increases in extracellular adenosine. Acute exposure of primary cultures of rat hepatocytes to 12.5-200 mM ethanol increased extracellular adenosine concentrations by 20-35%. Pretreatment of hepatocytes with 100 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, completely blocked ethanol-induced increases in extracellular adenosine at 12.5 and 25 mM ethanol. However, even in the presence of 4-methylpyrazole, ethanol at concentrations greater than 50 mM still increased extracellular adenosine concentrations. This increase appears to be due to ethanol inhibition of adenosine uptake via the nucleoside transporter (50% inhibitory concentration, 28 mM). After chronic treatment with 100 mM ethanol for 48 h, acute challenge with ethanol no longer inhibited adenosine uptake, i.e., the nucleoside transporter had become tolerant to ethanol. Moreover, in these chronically treated cells, ethanol-induced increases in extracellular adenosine were completely blocked by treatment with 4-methylpyrazole at all concentrations of ethanol. Taken together, these results suggest that increased extracellular adenosine in hepatocytes is dependent on both ethanol oxidation and inhibition of adenosine uptake via the nucleoside transporter.

scholarly journals Adenosine deaminase of cultured brain cells

1975 ◽  
Vol 152 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Eberhard G. Trams ◽  
Carl J. Lauter
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Adenosine Deaminase ◽  
Cultured Cells ◽  
Soluble Form ◽  
Monolayer Cultures ◽  
Adenosine Uptake ◽  
Atp Pool ◽  
Human Astrocytoma ◽  
Nervous System Function

Two types of adenosine deaminase (EC 3.5.4.4) were found in cultured cells of central-nervous-system origin. The predominant and more active enzyme was obtained in soluble form from the cytosol of mouse neuroblastoma (N-18), neonatal hamster astrocytes (NN), human oligodendroglioma (HOL) and human astrocytoma (Cox Clone). Particulate adenosine deaminase was probably associated with the plasma membrane. When radioactive adenosine was added to superfusates of monolayer cultures it was rapidly converted into inosine and hypoxanthine. The metabolic conversion required adenosine uptake by the cells, a probable transition through the intracellular ATP pool(s) and a rapid excretion into the superfusate of the catabolic products. We discuss the evidence that points to adenosine and its derivatives as neurohumoral modulators of central-nervous-system function.

scholarly journals Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase.

1983 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
M B Puziss ◽  
R M Wohlhueter ◽  
P G Plagemann
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
High Capacity ◽  
Rate Equations ◽  
Cell Water ◽  
Adenine Phosphoribosyltransferase ◽  
Novikoff Hepatoma ◽  
L929 Cells ◽  
Equilibrium Exchange ◽  
Time Courses

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).

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