Hybridomas were selected by addition of hypoxanthine (Acros Organics, Cat #1220100) and
azaserine (Sigma, Cat #A4142) at a final concentration of 200 [micro]M and 11.5 [micro]M respectively in RPMI 1640 (Sigma, Cat #R6504) containing 10% FBS, 23.8 mM sodium bicarbonate (Fisher Scientific, Cat #BP328), 7.5 mM HEPES (Amresco, Cat #0485), 170 [micro]M Penicillin G (Tokyo Chemical Industry, Cat #P1770), 137 [micro]M Streptomycin (Sigma, Cat #S9137), 50 [micro]M [beta]-mercaptoethanol, and 1mM sodium pyruvate (ThermoFisher, Cat #11360070).
Udayasankar et al., "Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS406 and
azaserine result in reduced islet amyloid formation in vitro," The American Journal of Physiology: Cell Physiology, vol.
For instance,
azaserine can directly inhibit N[H.sub.4.sup.+] uptake by inhibiting glutamate synthase in Symbiodinium (Rahav et al., 1989), and herbicides like DCMU (3-(3,4-dichlorophenyl)-l,1-dimethylurea) can inhibit photosynthetic electron transport (Calvayrac et al., 1979), or cessation of N[H.sub.4.sup.+] uptake can be induced in darkness (Muscatine and D'Elia, 1978); under such conditions symbiotic corals release N[H.sub.4.sup.+] (Rahav et al., 1989).
The cells were finally suspended in culture medium containing hypoxanthine and
azaserine and dispensed into 10 48-well plates.
Certain synthetic retinoids, administered in the diet for 1 year at a dose of 0.5-2 mmol/kg, have chemopreventive potential, reducing the progression of pancreatic carcinomas induced in rats by five weekly injections of
azaserine (Longnecker et al.
The next day, 1 mL of medium was replaced by 1 mL of selecting medium (RPMI-1640 20%FCS containing 5.8 [micro]mol/L
azaserine and 0.1 mmol/L hypoxanthine).
At present, GS but not GOGAT has been detected in zooxathellae (Summons and Osmond, 1981; Wilkerson and Muscatine, 1984), although azaserine, a potent inhibitor of GOGAT, prevents ammonium uptake in intact corals (Rahav et al., 1989), and the presence of GS/GOGAT activity is supported by the data of Summons et al.
Methionine sulphoximine (MSX), an inhibitor of GS (Ranzio et al., 1969; Meister, 1974), and azaserine (AZS), an inhibitor of 2-oxoglutarate amido transferase (GO-GAT) (Wallsgrove et al., 1977; Elrifi and Turpin, 1986), were purchased from the Sigma Chemical Company.
Azaserine is an inhibitor of the rate-limiting enzyme, GFAT.