Purification and characterization of the hepatic microsomal monooxygenase system from the coastal marine fish Stenotomus chrysops

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution August 1983Three cytochrome P-450 forms were highly purified (8-12 nmol/mg) from the hepatic microsomes of the untreated coastal marine fish Stenotomus chrysops (scup) by detergent solubilization and column chromatography. Scup cytochromes P-450A, P-450B and P-450E (labeled in order of elution from the first ion exchange column) had distinct spectroscopic properties, substrate profiles and minimum molecular weights on SDS-polyacrylamide gels (52.7, 45.9 and 54.3 K, respectively). The three purified cytochrome P-450 isozymes yielded different peptide maps when digested with a-chymotrypsin or S. aureus V8 protease. An additional hemoprotein fraction called cytochrome P-450 fraction D was also resolved and partially purified. This cytochrome P-450 preparation was characterized by a red shift in the CO-ligated, reduced difference spectrum with a chromophore near 451 nm. The scup NADPH-cytochrome P-450 reductase was purified to electrophoretic homogeneity (Mr = 82.6 K), had a specific activity of 45-60 U/mg with cytochrome c and contained both FAD and FMN. Scup cytochrome P-450E (Mr = 54.3 K) is the major aryl hydrocarbon hydroxylating form in untreated hepatic microsomes as judged by both its abundance (30-50% of the total resolved cytochromes P-450) and catalytic activity with benzo[a]pyrene (turnover number = 0.9 nmol product/nmol P-450/min). Further, reconstituted cytochrome P-450E was inhibited 70-80% by 100 uM 7, 8-benzoflavone in catalytic assays, similar to the 80-90% inhibition of benzo[a]pyrene hydroxylase in microsomal incubations. Analysis of benzo[a]pyrene products derived from reconstitutions of purified cytochrome P-450E revealed that greater than 50% of the oxidation occurred at benzo-ring positions, like the product profile observed in incubations with microsomes. The molecular weight of the purified cytochrome P-450E is identical to the major microsomal hemoprotein induced by 3-methylcholanthrene pretreatment, suggesting cytochrome P-450E is the major aromatic hydrocarbon-inducible cytochrome P-450 form in scup. Rabbit antisera raised against purified scup cytochrome P-450E reacts in Ouchterlony diffusion analysis with cytochrome P-450E antigenic determinants in microsomes but not purified cytochrome P-450A. Further, the antisera cross-reacts with an apparent 3-methylcholanthrene-inducible cytochrome P-450 isozyme purified from trout liver (TLM-4a; Williams and Buhler, Compo Biochem. Physiol. 7SC: 25-32, 1983), giving a pattern of fusion without visible-spurring in Ouchterlony analysis. These observations imply common antigenic determinants for the apparent major 3-methylchoianthrene-inducible cytochrome P-450 forms in trout and scup. Monooxygenase reconstitution experiments indicated that purified scup cytochrome P-450A actively hydroxylated testosterone at the 6ß position (turnover number = 0.8 nmol/min/nmol cytochrome P-450). Reconstituted cytochrome P-450B oxidized testosterone at two different sites tentatively identified as the 2-a and l5-a positions (total turnover number = 0.07 min-1). Cytochrome P-450 fraction D produced several metabolites upon reconstitution (sum turnover number = 0.2 min-1) including two chromatographically similar to 16a- and 16ß-hydroxytestosterone. Reconstituted cytochrome P-450E was a poor catalyst of testosterone hydroxylase but the only detectable product was 6 ß-hydroxytestosterone (turnover number = 0.04 min-1). However, besides benzo[a]pyrene, reconstituted cytochrome P-450E was active with 7-ethoxycoumarin, acetanilide and 7,8-benzoflavone as substrates. Addition of purified scup cytochrome b5 to monooxygenase reconstitutions had a stimulatory effect on some catalytic rates. The magnitude of the cytochrome b5 stimulation depended on the P-450 isozyme and the substrate used in the reconstitution; cytochrome P-450A was generally influenced by the presence of cytochrome b5. This rate stimulation was greater than the effect obtained with purified rabbit liver cytochrome b5. In an extreme example, cytochrome P-450E was completely inactive in reconstitutions of 7-ethoxyresorufin O-deethylase (an activity associated in microsomes with aromatic hydrocarbon induction) in the presence or absence of rabbit cytochrome b5 but the addition of scup cytochrome b5 to the reconstitution led to an observed O-deethylation rate of 7.0 min-1. It is uncertain whether these cytochrome b5 effects are exhibited in microsomes or in vivo but the stimulation in reconstitutions appears to be important in the evaluation of catalytic activity with purified isozymes.Financial support for my investigations was gleaned in part from a NSF Pre-doctoral Fellowship, the WHOI Education Office, WHOI Coastal Research Center grant 67.08, NSF grant OCE 80-18569 (J. S.) and NIH grant GM 21643 (C. W.)

Anomalous pressure dependence of the atomic displacements in the relaxor ferroelectric PbMg1/3_{1/3}Ta2/3_{2/3}O3_3

The crystal structure of the PbMg$_{1/3}$Ta$_{2/3}$O$_3$ (PMT) relaxor ferroelectric was studied under hydrostatic pressure up to $\sim 7$ GPa by means of powder neutron diffraction. We find a drastic pressure-induced decrease of the lead displacement from the inversion centre which correlates with an increase by $\sim$ 50 % of the anisotropy of the oxygen temperature factor. The vibrations of the Mg/Ta are, in contrast, rather pressure insensitive. We attribute these changes being responsible for the previously reported pressure-induced suppression of the anomalous dielectric permittivity and diffuse scattering in relaxor ferroelectrics

Intrauterine repair of gastroschisis in fetal rabbits

Objective: Infants with gastroschisis (GS) still face severe morbidity. Prenatal closure may prevent gastrointestinal organ damage, but intrauterine GS repair (GSR) has not been established yet. Methods: In New Zealand White rabbits we developed and compared GS versus GSR: creation of GS was achieved by hysterotomy, right-sided laparotomy of the fetus and pressure on the abdominal wall to provoke evisceration. GSR was accomplished by careful reposition of eviscerated organs and a running suture of the fetal abdominal wall. For study purposes, 18 animals were divided equally into 3 groups: GS, GS with GSR after 2 h, and unmanipulated controls (C). Vitality was assessed by echocardiography. After 5 h all animals were sacrificed. Results: GSR inflicted no increased mortality, because all fetuses survived GS or GS with GSR. All fetuses with GS demonstrated significant evisceration of abdominal organs. In contrast, the abdominal wall of the fetuses from GSR was intact. Conclusion:The present animal model demonstrated the technical feasibility and success of an intrauterine repair of GS for the first time. However, further long-term studies (leaving GS and GSR in utero for several days) will be necessary to compare survival rates and intestinal injury, motility or absorption. The clinical application of GSR in utero remains a vision so far. Copyright (C) 2003 S. Karger AG, Basel

Reliable quantification of 1,2-dihydroxynaphthalene in urine using a conjugated reference compound for calibration.

After environmental and occupational exposure to naphthalene, 1,2-dihydroxynaphthalene (1,2-DHN) was shown to be one major metabolite in human naphthalene metabolism. However, the instability of free 1,2-DHN complicates the reliable determination of this promising biomarker in urine. To solve this stability problem, glucuronide conjugates of 1,2-DHN and the corresponding isotopically labelled D-6-1,2-dihydroxynaphthalene (D-6-1,2-DHN) were synthesised and applied as reference material and internal standard in a gas chromatographic-tandem mass spectrometric (GC-MS/MS) method. The determination of 1- and 2-naphthol (1-MHN, 2-MHN) was included in the procedure to enable a comprehensive assessment of naphthalene metabolism and exposure. The results of the validation showed a high reliability and sensitivity of the method. The detection limits range from 0.05 to 0.16 mu g/L. Precision and repeatability were determined to range from 1.4 to 6.6% for all parameters. The simultaneous determination of 1- and 2-MHN as additional parameters besides 1,2-DHN enables the application of the method for further metabolism and kinetic studies on naphthalene. The use of glucuronide-derivative reference substances and the application of structurally matched isotopic-labelled internal standards for each substance guarantee a reliable quantification of the main naphthalene metabolites 1,2-DHN and 1- and 2-MHN

Strong electrically tunable exciton g-factors in an individual quantum dots due to hole orbital angular momentum quenching

Strong electrically tunable exciton g-factors are observed in individual (Ga)InAs self-assembled quantum dots and the microscopic origin of the effect is explained. Realistic eight band k.p simulations quantitatively account for our observations, simultaneously reproducing the exciton transition energy, DC Stark shift, diamagnetic shift and g-factor tunability for model dots with the measured size and a comparatively low In-composition of x(In)~35% near the dot apex. We show that the observed g-factor tunability is dominated by the hole, the electron contributing only weakly. The electric field induced perturbation of the hole wavefunction is shown to impact upon the g-factor via orbital angular momentum quenching, the change of the In:Ga composition inside the envelope function playing only a minor role. Our results provide design rules for growing self-assembled quantum dots for electrical spin manipulation via electrical g-factor modulation

Search for neutrinos from transient sources with the ANTARES telescope and optical follow-up observations

The ANTARES telescope has the opportunity to detect transient neutrino sources, such as gamma-ray bursts, core-collapse supernovae, flares of active nuclei... To enhance the sensitivity to these sources, we have developed a new detection method based on the optical follow-up of "golden" neutrino events such as neutrino doublets coincident in time and space or single neutrinos of very high energy. The ANTARES Collaboration has therefore implemented a very fast on-line reconstruction with a good angular resolution. These characteristics allow to trigger an optical telescope network; since February 2009. ANTARES is sending alert trigger one or two times per month to the two 25 cm robotic telescope of TAROT. This follow-up of such special events would not only give access to the nature of the sources but also improves the sensitivity for transient neutrino sources.Comment: 3 pages, 3 figures, Proceedings of the 31st ICRC, Lodz, Polan, July 200

Asymmetric optical nuclear spin pumping in a single uncharged quantum dot

A highly asymmetric dynamic nuclear spin pumping is observed in a single self assembled InGaAs quantum dot subject to resonant optical pumping of the neutral exciton transition leading to a large maximum polarization of 54%. This dynamic nuclear polarization is found to be much stronger following pumping of the higher energy Zeeman state. Time-resolved measurements allow us to directly monitor the buildup of the nuclear spin polarization in real time and to quantitatively study the dynamics of the process. A strong dependence of the observed dynamic nuclear polarization on the applied magnetic field is found, with resonances in the pumping efficiency being observed for particular magnetic fields. We develop a model that fully accounts for the observed behaviour, where the pumping of the nuclear spin system is due to hyperfine-mediated spin flip transitions between the states of the neutral exciton manifold.Comment: published version; 4+ pages, 3 figures (eps