Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: Impact of 1 mM AICAR on folate influx and net uptake in HeLa cells. Transport of 0.5 μM [3H]MTX (left), [3H]5-formyl-THF (center), and [3H]5-methyl-THF (right) in the presence and absence of 1 mM AICAR. AICAR was added 10 min before addition of folates and was present during the transport measurements. Top, initial rates of folate uptake (influx). Bottom, the time course of the net uptake of folates. The horizontal interrupted line in bottom left represents MTX tightly bound to dihydrofolate reductase. The different between bound and total intracellular MTX is the component free within the intracellular water (see Materials and Methods). The data are from an experiment representative of three independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques:

Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: Roles of RFC and PCFT in AICAR potentiation of net folate transport. Top, net uptake of 0.5 μM [3H]MTX (left) and [3H]5-formyl-THF (right) in the presence and absence of 1 mM AICAR in cells that express only RFC (HeLa-RFC6). Bottom, net uptake of 0.5 μM [3H]MTX (left) and [3H]5-formyl-THF (right) in the presence and absence of 1 mM AICAR in cells that express only PCFT (HeLa-PCFT). AICAR was added 10 min before addition of folates and was present during the transport measurements. The data are representative of three independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques:

Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: Impact of AICAR on RFC influx kinetics. HeLa cells were incubated in the presence or absence of 1 mM AICAR for 10 min after which influx of [3H]MTX was assessed in the presence of this agent. The line is best fit to the Michaelis-Menten equation (V = Vmax[S]/(Kt+[S]). Kt is in micromoles per liter and represents the extracellular concentration (MTXe) at which MTX influx is half of maximum; Vmax is in picomoles per milligram of protein per minute. Results are the mean ± S.E.M. from three independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques: Incubation, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: The impact of 5-iodotubercidin and the reversibility of AICAR stimulation of RFC-mediated transport. A, cells were preincubated for 10 min with 1 mM AICAR and then were incubated for 30 min in AICAR-free buffer after which uptake of 0.5 μM [3H]5-formyl-THF was assessed. Results are the mean ± S.E.M. from three independent experiments. The inset amplifies the initial uptake over 5 min. B, cells were preincubated for 10 min with 1 mM AICAR and then were exposed to AICAR-free buffer at time 0, and uptake of [3H]5-formyl-THF was assessed. The inset amplifies uptake over the first 15 min. Results are the mean ± S.E.M. from three independent experiments. C, time course of uptake of 0.5 μM [3H]5-formyl-THF in HeLa cells preincubated for 10 min with 1 mM AICAR in the presence and absence (control) of 1 μM 5-iodotubercidin. Data are the mean ± S.E.M. from three independent experiments. D, initial rate of 1 mM [3H]AICAR uptake in HeLa cells as a function of 5-iodotubercidin concentration. Data are the mean ± S.E.M. from three independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques: Incubation, Control, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: Impact of AICAR on trans-stimulation mediated by 5-formyl-THF. HeLa cells were loaded for 20 min with 1 mM AICAR, 100 μM 5-formyl-THF, or both. The cells were then washed in 0°C HBS after which influx of 0.5 μM [3H]5-methyl-THF was assessed. Data are the mean ± S.E.M. from four independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques:

Journal: Molecular Pharmacology

Article Title: Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

doi: 10.1124/mol.112.078642

Figure Lengend Snippet: Inhibitory effect of ZMP on [3H]MTX influx and efflux. Top panel, influx of 0.5 μM [3H]MTX was assessed in HeLa cells in the absence or presence of nonlabeled ZMP at the indicated concentrations. Bottom panel, impact of AICAR on [3H]MTX efflux. HeLa cells were incubated for 30 min with 0.5 μM [3H]MTX in the presence or absence of 1 mM AICAR after which 100 μM 5-formyl-THF was added to block [3H]MTX influx, and the decline of intracellular [3H]MTX was monitored. Data are the mean ± S.E.M. from six independent experiments.

Article Snippet: Tritiated (6 S )5-formyltetrahydrofolate (5-formyl-THF), (6 S )5-methyltetrahydrofolate (5-methyl-THF), MTX, and AICAR were obtained from Moravek Biochemicals (Brea, CA) with purity established and monitored by high-performance liquid chromatography (HPLC) as described previously ( Zhao et al., 2000 ).

Techniques: Incubation, Blocking Assay

Journal: Developmental Cell

Article Title: A GBF1-Dependent Mechanism for Environmentally Responsive Regulation of ER-Golgi Transport

doi: 10.1016/j.devcel.2019.04.006

Figure Lengend Snippet:

Article Snippet: Aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR) (Enzo #BML-E1330-0050) in HGM was added to cells for 24 hours in the concentration stated.

Techniques: Recombinant, Luciferase, Software