Pascale Debey

Use of Percoll density gradient centrifugation for preparing isolated rat hepatocytes having long-term viability

Abstract A method for preparing suspensions of hepatocytes with long-term viability is described. Its originality lies in the use, after conventional preparation, of a Percoll density gradient centrifugation which allows a complete and rapid separation of cell debris and released proteases from the intact hepatocytes. A series of functional reactions characterizing drug metabolism ( P -450 level, ethoxycoumarin hydroxylation, methylumbelliferone conjugation) and membrane transport and integrity (α-aminoisobutyric acid transport and its stimulation by both insulin and glucagon) were conducted, in parallel to the trypan blue exclusion test, to evaluate the metabolic activity of both conventional and Percoll preparations. This investigation clearly demonstrates that the use of Percoll density gradient centrifugation significantly increases (from 5–6 to 30–35 h) the half-life of freshly isolated hepatocyte suspensions.

Dynamics of chromatin changes in live one-cell mouse embryos: A continuous follow-up by fluorescence microscopy

Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.

Highly purified microsomal P-450: the oxyferro intermediate stabilized at low temperature.

Abstract The oxyferro intermediate of highly purified microsomal P-450 from rabbit liver was formed and stabilized at −30°C in a mixture of aqueous buffer and glycerol ( 1 1 ). Absolute and difference (Fe2·+O2-Fe3+) spectra of this intermediate appear to be very similar to those obtained under either steady state kinetics or stopped flow conditions on the same cytochrome as well as on bacterial P-450cam. (Absolute and difference spectra present maxima at 420 and 557–558 nm and a broad maximum at 442 nm respectively). As temperature increases the oxyferro intermediate autoxidizes and ferric cytochrome P-450 is restored. This reaction appears to follow biphasic first order kinetics. The rate constant of both phases decreases with temperature and increases with protons concentrations.

Presence of Permanently Activated Signal Transducers and Activators of Transcription in Nuclear Interchromatin Granules of Unstimulated Mouse Oocytes and Preimplantation Embryos

Abstract We previously described that mouse oocytes and preimplantation embryos express the two subunits of interferon-gamma receptor. We now report that, despite the presence of STAT1 (signal transducer and activator of transcription 1) at both the mRNA and protein levels, interferon γ (IFNγ) as well as IFNα are unable to trigger massive nuclear translocation of STAT1 in these cells, even at high cytokine concentrations. Conversely, nuclear accumulation of STAT1 was readily observed in murine L929 somatic cells under the same conditions. However, in the absence of any stimulation, both tyrosine (Y701p) and serine (S727p) phosphorylated forms of STAT1 were already detected in the nuclei of oocytes and early embryos. Phosphorylated STAT1 appeared concentrated in large nuclear dots, which were identified by indirect immunofluorescence and electron microscopy as clusters of interchromatin granules (IGCs or speckles). A similar distribution was also observed for the serine (S727p) phosphorylated form of STAT3 as well as for tyrosine (Y689p) phosphorylated STAT2. Western blot analysis confirmed that STAT factors present in mouse oocytes are predominantly phosphorylated. In parallel, we showed that the transcription of two IFNγ-target genes, namely interferon regulatory factor-1 (IRF-1) and suppressor of cytokine signaling-1 (SOCS-1) is indeed increased in two-cell embryos in response to IFNγ. Altogether, our results suggest that, despite the lack of massive nuclear accumulation of STAT1 in response to exogenous IFNs and the permanent presence of phosphorylated STATs in the nucleus, JAK/ STAT pathways are functional during early development.

Low temperature column chromatography: Application to microsomal hydroxylating system

Abstract A chromatography device working at subzero temperature is described. Some results concerning resolution of microsomal hydroxylating system are given, after solubilisation and chromatography at −20°C.

In vitro DNA fluorescence after in vitro fertilization (IVF) failure.

PurposeA pilot study was performed to test the diagnostic value of in vitro DNA fluorescence in oocytes that failed to fertilize after IVF. Ten patients with a cleavage rate less than 20% after IVF were included.ResultsUncleaved oocytes were observed by fluorescence microscopy after incubation with the DNA fluorescent dye Hoechst 33342. Four main causes which may have contributed to the low cleavage rate were found: (1) sperm incapacity to penetrate the oocyte despite the absence of the usual criteria for male infertility, (2) oocyte immaturity, (3) delayed fertilization, and (4) oocyte abnormalities revealed by aberrations in the morphology of the female chromatin.ConclusionsThe possibility of a rapid and detailed analysis of the maturational status of unfertilized oocytes, the morphology of the female chromatin, the presence and quantity of spermatozoa tightly bound to the zona pellucida, and sperm penetration into the oocyte without subsequent pronucleus formation, using DNA fluorescence, allows us to clarify further the cause of fertilization failure and to orient infertility treatment toward the male, the female, or both partners.

Sub-zero temperature studies of microsomal cytochrome P-450: interaction of Fe2+ with oxygen.

Part of the mystery underlying the cycle of redox states undergone by the microsomal cytochrome P-450 during drug hydroxylation was solved by the earlier description by Estabrook et al. [l] of a new spectral intermediate appearing during the steadystate drug oxidation metabolism and attributed to the oxy-ferrous cytochrome P-450. This compound has a maximum at 440 nm in a difference spectrum and was obtained in the presence of hexobarbital as hydroxylisable substrate. Later on, an increasing interest was oriented towards the ability of cytochrome P-450 to bind and use organic hydroperoxides as combined source of electron and oxygen, either in the microsomes or in a reconstituted system [2-41. The problem of the interaction of oxygen with reduced cytochrome P-450 was reactivated by the recent work of Guengerich et al. [S] describing the binding kinetics of O2 to highly purified preparation of reduced cytochrome and the obtention of at least two distinguishable compounds. The present work describes two spectrally detectable complexes, very similar to thoses of Guengerich et al. and obtained by the addition of O2 to completely reduced intact microsomes at sub-zero temperatures. The advantages of such an experimental procedure is to allow a slowing down of reaction rates and the ‘uncoupling’ of certain processes from the preceding or following ones within a multi-reactions system. This procedure was already applied to study in the microsomes some reactions involving the cytochrome P-450, and previously described [6-81.

Subzero temperature studies of microsomol cytochrome P-450: O-Dealkylation of 7-ethoxycoumarin coupled to single turnover

We reported with intact microsomes the formation and partial stabilization of a ferrous-oxy intermediate of cytochrome P-450 in fluid mixed solvents at subzero temperature [ 11. The data compare well with those from stopped flow measurements on purified cytochrome above O’C [2] and steady state recordings on intact microsomes [3]. The subzero temperature experiments offered two advantages, particularly with intact microsomes: (1) Uncoupling of the oxygen reaction from the other steps of the cycle; (2) Therefore single turnover of enzyme since the first electron is blocked at 2 -20°C [4,5]. The decomposition of this ferrous-oxy compound was also shown to be accompanied by the discharge of possible ‘leaks’ of oxidizing species, indirectly detected by the oxidative luminescence of luminol [S]. Here we use the same temperature programme to show that enzymatic U-dealkylation of the substrate 7ethoxycoumarin occurs during the single redox cycle of cytochrome P-450. This substrate was mainly chosen for the easy fluorometric detection of its product 7-hydroxycoumarin (umbelliferone, 7-OH coumarin) [6].

Low temperature studies of microsomal cytochrome P450. Release of oxidizing species

It is known that the functionning of the multienzyme hydroxylating system of rat liver microsomes produces oxidizing species [l-3] identified as 02 and HzOz [3-51, but it remains unknown whether such species are normally or incidentally released by the NADPH cytochrome Ph50 reductase [6] , or by cytochrome Pd5,, , or both. The demonstration that such a release of oxidizing species can occur from cytochrome PgsO would give decisive evidence of its oxygenated intermediate, as has been postulated by Eastabrook [7] . The present paper shows that, after enzymic reduction of cytochrome PgsO at room temperature, and subsequent cooling (which inhibits the enzymatic electron transport activity of the system), addition of oxygen in the presence of luminol determines an emission of luminescence due to the thermal decomposition of an oxygenated intermediate of cytochrome P 450.

Cytochrome P-450 oxygen intermediates and reactivity at subzero temperatures

Abstract The oxy-ferro compound is the last stable intermediate of the cytochrome P-450 reaction cycle leading to substrate hydroxylation. It has been stabilized at subzero temperatures for two systems, the bacterial camphor hydroxylase and the microsomal drug hydroxylase. Their stability, spectra, decomposition and reactivity are compared.