Ornithine decarboxylase assays

Ornithine Decarboxylase Assays. The double-chamber assay system of Moskal and Basu (59) was used to measure enzyme activity in the form of L C] carbon dioxIHe evolution. The assay conditions of O Brien and Diamond (60) were used and consisted of the following components (in micromoles, unless otherwise stated) in a total volume of 100 jul sodium phosphate buffer, pH 7.2, 5.0 EDTA, 1.0 dithiothreitol, 5.0 pyridoxal-5 -monophosphate, 0.2 L-ornithine (specific activity 0.5 x 10° cpm/-jumole), 0.1 and protein, 0.1-0.5 mg. Incubations were carried out at 37°C for 60 min, and the reactions were terminated by the addition of 200 jul of 2M sodium citrate followed by a post-incubation period of 3 hours at 37°C to insure maximal release of radiolabeled carbon dioxide. 

Epstein-Barr virus early antigen induction. Methanol extract of the dried leaf, in cell culture at a concentration of 1 pg/mL, was inactive. The assay was designed for tumor-promoting activity . Two diastere-oisomers of 2,7,1 l-cembratriene-4,6-diol (a- and 3-CBT) from the neutral fractions of cigarette smoke condensate, in Raji cells, produced potent inhibitory effects on the induction of Epstein-Barr virus (EBV)-EA by 12-0-tetradecanoylphorbol-13-acetate (TPA). The doses of a- and P-CBT required for 50% inhibition of EBV-EA induction by TPA were 7.7 and 6.7 mg/mL, respectively. Application of a- and P-CBT to mouse skin before treatment with TPA, inhibited TPA-induced ornithine decarboxylase activity in a dose-dependent manner. Application of 16.5 pM/mouse of a- and p-CBT resulted... 

RLV/Fischer rat assay without the addition of an exogenous metabolic activation system. In a single study, mouse JB6 epidermal cells were transformed by di(2-ethyl-hexyl) phthalate without activation and in one of two studies a weak response was reported in the CSHIOT A cell transformation assay with di(2-ethylhexyl) phthalate in either the absence or presence of exogenous metabolic activation. BALB/c-3T3 cells were not transformed by di(2-ethylhexyl) phthalate with or without metabolic activation. Di(2-ethylhexyl) phthalate inhibited gap-junctional intercellular communication in Chinese hamster V79 cells in six of seven studies, but not in one study of liver cells of cynomolgus monkeys in vivo. Di(2-ethylhexyl) phthalate treatment of Syrian hamster embryo cells in a two-stage exposure with 12-O-tetradecanoylphorbol 13-acetate resulted in superinduction of ornithine decarboxylase, an early event in morphological transformation no effect was seen after a one-stage treatment with di(2-ethylhexyl) phthalate alone. 

Fig. 7. Changes in the contents of specific mRNAs and nuclear receptors in mouse kidney after exposure to testosterone. Female NCS mice were given a single intraperitoneal dose of 10 mg of testosterone at time zero. Thereafter, nuclear androgen receptors were measured, The specific mRNAs of three proteins were also assayed, namely for ornithine decarboxylase (- -), KAP (-0-) and /3-glucuronidase (- -). Redrawn from Catterall et al. [26],...

The reaction mixture contained in a final volume of 1 mL 20 mAf sodium phosphate buffer (pH 5.0), 2.5 mAf dithiothreitol, 0.1 mAf pyridoxal 5 -phosphate, 0.4 /xCi L-[2,3-3H]ornithine, 100 mAf ornithine hydrochloride, and 0.3 mL of either E. coli ornithine decarboxylase or tissue homogenate. The E. coli enzyme was assayed at pH 5.0 and 37°C the pH values of reaction mixtures were adjusted to pH 7.3 before assaying homogenates of mammalian tissues. The reaction was stopped by injection onto the column. 

The high concentration of OCT is in hepatic tissue relative to other tissues this leads to this enzyme being regarded as a liver-specific enzyme. OCT is localized in the periportal mitochondria and is the second of the five enzymes in the urea cycle. The enzyme measurement should not be confused with ornithine decarboxylase (ODC), which is of interest in studies of polyamine metabolism. The use of the method has been limited by some of technical requirements for the older assays, but newer enzymatic methods may increase its use (Peltenburg et al. 1991 Isobe, Matsuzawa, and Nagamura 1993 Ishikawa et al. 2002). In a number of rat studies, ALT was found to be more predictive of hepatotoxicity than OCT (Bondy et al. 2000). 

In an effort to determine the cancer chemopreventive activity of tea compounds, we conducted several in vitro mechanistic assays and in vitro cell-transformation assays to screen for efficacy of these compounds. The mechanistic assays measure (a) inhibition of DNA adduct formation, (b) inhibition of free-radical formation, and (c) enhancement of glutathione (GSH), glutathione-S-transferase (GST), ornithine decarboxylase (ODC), and NAD(P)H quinone reductase (QR) activity. No extracts were positive in all six assays, but several were positive in four to five assays. 

The assay described for amino acid decarboxylase can be used to quantitate the substrates and products associated with the decarboxylation of arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine. 

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