Response of Holstein and Brown Swiss cows fed alfalfa hay-based diets to supplemental methionine at two stages of lactation.

In this study, we evaluated the effects of dietary supplementation at two stages of lactation with various levels of Mepron85 (M85) and M85 plus DL-methionine (DL-Met) on milk production and composition of Holstein and Brown Swiss cows fed an alfalfa-hay and corn grain-based diet. In experiment 1, control diets were formulated to supplement, in early lactation [days in milk (DIM) = 73.2], concentrations of metabolizable methionine at 104% of the estimated requirements based on the Cornell Net Carbohydrate and Protein System. Treatment groups were fed the control diet plus 10, 20, or 30 g/d of M85 at 116, 128, or 139% of the estimated requirements for metabolizable methionine. The supplementation with 10 g/d in Brown Swiss and 30 g/d of M85 in Holstein cows increased milk yields and fat percentage, but had no effects on protein percentage. These data suggested that the estimated postruminal supply of metabolizable methionine in the control ration was limiting for maximum milk fat synthesis. Conversely, in experiment 2, the cosupplementation with M85 (15 g/d) plus DL-Met (15 g/d) to cows in midlactation (DIM = 140.5) did not influence fat percentage, but increased protein yield and percentage (+0.1%) in both Holstein and Brown Swiss, and lactose percentage (+0.18%) in Holstein cows. The supplementation with 15 g/d of M85 reduced milk and protein yields, whereas 15 g/d of DL-Met reduced protein percentage in four of the five experimental weeks for Holstein cows. We conclude that supplementation with M85, alone or in combination with DL-Met, may be used to influence milk composition, but these effects are influenced by dosage and type of supplemental methionine, breed, and stage of lactation.

Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells.

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G2.

The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a] pyrene (B[ a]P), and its metabolite, BPDE (7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1 levels and cell cycle kinetics were determined in MCF-7 breast cancer cells. Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5 microM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 microM), the loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this checkpoint was transient since cells resumed to G2/M after 12 hours with reduced levels of BRCA-1. In these cells, levels of p53 were increased, whereas the cellular content of p21 remained unaltered. In contrast, the co-treatment with the AhR antagonist, alpha-naphthoflavone (ANF), abrogated the deleterious effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53 and disruption of cell cycle profile. These findings suggest that the AhR mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA levels. The negative effects of BPDE on BRCA-1 expression were not transient since removal of BPDE did not allow complete reversal of the repression. These cumulative data suggest that the B[a]P metabolite, BPDE, may play a key role in disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial cells.