Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

Preclinical studies suggest ropinirole (a D2/D3 dopamine agonist) may be neuroprotective in Parkinson's disease (PD), and a pilot clinical study using (18)F-dopa positron emission tomography (PET) suggested a slower loss of striatal dopamine storage with ropinirole compared with levodopa. This prospective, 2-year, randomized, double-blind, multinational study compared the rates of loss of dopamine-terminal function in de novo patients with clinical and (18)F-dopa PET evidence of early PD, randomized 1 to 1 to receive either ropinirole or levodopa. The primary outcome measure was reduction in putamen (18)F-dopa uptake (Ki) between baseline and 2-year PET. Of 186, 162 randomized patients were eligible for analysis. A blinded, central, region-of-interest analysis showed a significantly lower reduction (p = 0.022) in putamen Ki over 2 years with ropinirole (-13.4%; n = 68) compared with levodopa (-20.3%; n = 59; 95% confidence interval [CI], 0.65-13.06). Statistical parametric mapping localized lesser reductions in (18)F-dopa uptake in the putamen and substantia nigra with ropinirole. The greatest Ki decrease in each group was in the putamen (ropinirole, -14.1%; levodopa, -22.9%; 95% CI, 4.24-13.3), but the decrease was significantly lower with ropinirole compared with levodopa (p < 0.001). Ropinirole is associated with slower progression of PD than levodopa as assessed by (18)F-dopa PET.

No abstract

Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables, such as broccoli, that has been identified as a potent inducer of glutathione S-transferase activities in laboratory animals. The present studies were carried out to elucidate the metabolic fate of SFN in the rat. Particular emphasis was placed on glutathione (GSH)-dependent pathways because conjugation with GSH is a major route by which many isothiocyanates are eliminated in mammals. Male Sprague-Dawley rats were administered a single dose of SFN (50 mg kg-1 ip), and bile and urine were collected over ascorbic acid. Analysis of biological fluids was carried out by ionspray LC-MS/MS using the neutral loss (129 Da) and precursor ion (m/z 164) scan modes to detect GSH and N-acetylcysteine (NAC) conjugates, respectively. In bile, five thiol conjugates (designated M1-M5) were detected. Metabolites M2 and M4 were identified as the GSH conjugates of SFN and erucin (ERN, the sulfide analog of SFN), respectively, by comparing their LC-MS/MS properties with those of standards obtained by synthesis. M1 was characterized as the GSH conjugate of a desaturated metabolite of SFN (tentatively assigned the structure of delta 1-SFN), suggesting that the parent compound also undergoes oxidative metabolism. Metabolites M3 and M5 were identified as the NAC conjugates of SFN and ERN, respectively, and together with the NAC conjugate of delta 1-SFN, these species also were detected in urine. Quantitative determination of the former two mercapturates in urine indicated that approximately 60% and approximately 12% of a single dose of SFN is eliminated in 24 h as the NAC conjugates of SFN and ERN, respectively. The corresponding figures in rats dosed with ERN were approximately 67% and approximately 29%. When the GSH conjugate of SFN was incubated with phosphate buffer (pH 7.4, 37 degrees C), < 1% of the conjugate dissociated to liberate free SFN. On the other hand, the conjugate underwent a facile thiol exchange reaction (> 70% conversion) when incubated in the presence of excess cysteine, thereby acting as an effective carbamoylating agent. It is concluded that SFN undergoes metabolism by S-oxide reduction and dehydrogenation and that GSH conjugation is the major pathway by which the parent compound and its phase I metabolites are eliminated in the rat.

Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.