2,121 research outputs found
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 PbMgTaO
The crystal structure of the PbMgTaO (PMT) relaxor
ferroelectric was studied under hydrostatic pressure up to 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 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
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