Figure 1.
Figure 1. ADA is rapidly induced by hypoxia. (A) Microarray analysis of individual nucleoside/nucleotide deaminases in response to hypoxia (ADA, CDA, AMPD2, DCTD, and AMPD1). Confluent HMEC-1s were exposed to normoxia (pO2 147 mm Hg, 18 hours) or hypoxia (24-hour exposure to pO2 20 mm Hg) and the relative expression of individual deaminases was quantified from total RNA by microarray analysis. *Increased fluorescence, P < .01. #Decreased fluorescence, P < .01. Note the dramatic induction of ADA with hypoxia (47.5-fold). (B) Confluent HMEC-1 or HUVEC monolayers were exposed to normoxia (pO2 147 mm Hg, 18 hours) or hypoxia (pO2 20 mm Hg) as indicated. Total RNA was isolated, and ADA mRNA levels were determined by real-time RT-PCR. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold increase over normoxia ± SD at each indicated time. Results are derived from 3 experiments in each condition (*P < .01). (C) Human saphenous veins were obtained from patients undergoing aorto-coronary bypass surgery and exposed ex vivo to ambient normoxia (pO2 147 mm Hg, 24 hours) or hypoxia (pO2 20 mm Hg for 2, 8, or 24 hours). After total RNA isolation, real-time PCR was performed to investigate ADA inducibility by hypoxia. Data were calculated relative to an internal control (β-actin) and are expressed as fold increase over normoxia ± SD at each indicated time. Results are derived from 3 experiments in each condition (*P < .01). (D) Increase in total ADA protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia (0-72 hours), washed, and lysed. Lysates were resolved by SDS-PAGE, and resultant Western blots were probed with a polyclonal goat antibody directed against human ADA. A representative experiment of 3 is shown. (E) Increase in surface ADA protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia (0-48 hours), monolayers were washed, surface proteins were biotinylated, and cells were lysed. ADA was immunoprecipitated with a polyclonal goat antibody directed against human ADA. Immunoprecipitates were resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. A representative experiment of 3 is shown.

ADA is rapidly induced by hypoxia. (A) Microarray analysis of individual nucleoside/nucleotide deaminases in response to hypoxia (ADA, CDA, AMPD2, DCTD, and AMPD1). Confluent HMEC-1s were exposed to normoxia (pO2 147 mm Hg, 18 hours) or hypoxia (24-hour exposure to pO2 20 mm Hg) and the relative expression of individual deaminases was quantified from total RNA by microarray analysis. *Increased fluorescence, P < .01. #Decreased fluorescence, P < .01. Note the dramatic induction of ADA with hypoxia (47.5-fold). (B) Confluent HMEC-1 or HUVEC monolayers were exposed to normoxia (pO2 147 mm Hg, 18 hours) or hypoxia (pO2 20 mm Hg) as indicated. Total RNA was isolated, and ADA mRNA levels were determined by real-time RT-PCR. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold increase over normoxia ± SD at each indicated time. Results are derived from 3 experiments in each condition (*P < .01). (C) Human saphenous veins were obtained from patients undergoing aorto-coronary bypass surgery and exposed ex vivo to ambient normoxia (pO2 147 mm Hg, 24 hours) or hypoxia (pO2 20 mm Hg for 2, 8, or 24 hours). After total RNA isolation, real-time PCR was performed to investigate ADA inducibility by hypoxia. Data were calculated relative to an internal control (β-actin) and are expressed as fold increase over normoxia ± SD at each indicated time. Results are derived from 3 experiments in each condition (*P < .01). (D) Increase in total ADA protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia (0-72 hours), washed, and lysed. Lysates were resolved by SDS-PAGE, and resultant Western blots were probed with a polyclonal goat antibody directed against human ADA. A representative experiment of 3 is shown. (E) Increase in surface ADA protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia (0-48 hours), monolayers were washed, surface proteins were biotinylated, and cells were lysed. ADA was immunoprecipitated with a polyclonal goat antibody directed against human ADA. Immunoprecipitates were resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. A representative experiment of 3 is shown.

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