Characterization of the enzymatic and nonenzymatic reaction of 13-oxooctadecadienoic acid with glutathione

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.     

Failure of femoral head fixation: a cadaveric analysis of lag screw cut-out with the gamma locking nail and AO dynamic hip screw

The most commonly reported failure mode of sliding hip screws in published literature is cut-out of the lag screw. This study investigates the resistance to failure of the femoral head, with lag screws used in two types of sliding hip screws, the gamma locking nail (Howmedica) and the dynamic hip screw (DHS) (Synthes). The investigation consisted of biomechanical tests under static loading conditions on 12 pairs of cadaveric femoral heads, to establish the failure loads due to screw cut-out for the two implant lag screws. The gamma nail appeared to reduce the tendency to cut-out in the osteoporotic bone (soft) associated with elderly patients in whom these devices are commonly used (p < 0.05). In high density bone (hard) the gamma lag screw also appeared to be stronger, because the DHS showed a tendency to bend. The larger diameter of the gamma nail lag screw resists bending and appears to reduce the risk of cut-out compared with the DHS.     

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.     

Dietary variety via sweetening and voluntary feed intake of lactating dairy cows

The effects of choice of diets on feed intake were studied using 12 lactating Holstein cows. A switchback design was used that had three periods and two sequential blocks. Diets were 1) a control total mixed ration (TMR), which consisted of alfalfa haylage, corn silage, and a concentrate mixture based on ground corn and soybean meal (25:25:50 on a dry matter basis) and 2) a sweetened TMR in which a brown sugar food product constituted 1.5% of the dietary dry matter. Treatments consisted of the control TMR fed on both sides of divided feed bunks, the sweetened TMR fed on both sides of divided feed bunks, or both TMR fed on alternating (daily) sides of divided feed bunks in tie stalls. Periods were 2 wk in duration, and cows averaged 67 and 53 d of lactation at the start of blocks 1 and 2, respectively. The dry matter intake, body weight, milk yield, and percentages of milk fat, protein, and solids not fat were similar when either TMR was fed alone. A choice of control TMR or sweetened TMR did not affect any of these variables. The dry matter intake, body weight, milk yield, and milk protein percentage were affected by block; however, these effects were probably caused by differences between the blocks in environment and stage of lactation. The results of this experiment might have been affected by the composition of the control TMR, its similarity to the sweetened TMR, availability of both diets simultaneously when a choice was offered, and use of a TMR instead of separate feeds or simpler mixtures.     

图形测试:多工位模拟和混合信号器件并行测试效率的关键

1介绍多工位测试是大多数模拟和混合信号器件生产厂家大批量测试的基石.随着并行测试工位数的提高,模拟和混合信号器件测试系统的设计人员需要     

Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.