Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119.

Using UV-Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV-Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo-ferric complex of CYP119, undergoes a 'peroxide shunt' pathway during gradual annealing at 170-195 K and returns to the low-spin ferric form.

Crystallization and preliminary X-ray diffraction analysis of a cytochrome P450 (CYP119) from Sulfolobus solfataricus.

CYP119 is a cytochrome P450 with a molecular weight of 43 kDa which has been isolated from the thermophilic archaeon Sulfolobus solfataricus. This enzyme is extremely stable to high temperature and high pressure. The first crystallization and preliminary crystallographic study of CYP119 is reported here. Crystals of CYP119 were obtained by the sitting-drop vapour-diffusion method using a precipitant solution containing 20%(w/v) PEG 4000 and 0.2 M sodium thiocyanate at pH 6.4. Using synchrotron radiation, the CYP119 crystal diffracted to 1.84 A resolution. It belongs to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 86.17 (0.07), c = 221.11 (0.04) A, in which the numbers in parentheses describe the standard deviations. Assuming two molecules of the CYP119 per asymmetric unit, the calculated molar volume (V(m)) is 2.38 A(3) Da(-1). Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem irons. The complete crystallographically defined structure is currently in progress using MIR (multiple isomorphous replacement) and MAD (multiwavelength anomalous diffraction) techniques.

Characterization of a cytochrome P450 from the acidothermophilic archaea Sulfolobus solfataricus.

We report the cloning, expression, purification, and molecular characterization of a cytochrome P450 (CYP119) from the thermophilic archaea Sulfolobus solfataricus. This protein displays an absorption spectra in the reduced, oxidized, and carbonyl adduct analogous to those of other P450 enzymes. We demonstrate that P450 (CYP119) exhibits remarkable thermo- and pressure stability, with a melting temperature 40 degrees higher than that of the extensively studied cytochrome P450cam (CYP101) and an optical spectra completely resistant to the formation of the inactive P420 by hydrostatic pressure up to 2 kbar. CO flash photolysis experiments, as well as construction of a CYP119 homology model, suggest an open active site with greater solvent access than P450 (CYP101) and similar to that of P450 (CYP102). This communication represents the first molecular characterization of an extremophilic cytochrome P450.

A group therapy format for the simultaneous treatment of marital and sexual dysfunctions: a case illustration.

This report describes a brief, 15-session couples group therapy format developed by a university-affiliated human sexuality clinic for the simultaneous treatment of marital and sexual dysfunctions. The major marital and sexual themes addressed in this group treatment design, an overview and description of the structure of the cognitive-behavioral approach, and a case illustration are presented.

Tachistoscopic and questionnaire methods for the measurement of psychological defences.

Only few attempts have been made to compare different methods aiming at quantifying defensive strategies. In this study the tachistoscopic Defence Mechanism Test (Kragh, 1960b and 1985) is compared to paper-and-pencil tests of defence. There were virtually no correlations between the DMT and the other measures of defence. This may indicate that the concept of defence is a highly complex one. It is suggested that the Defence Mechanism Test may be measuring primary defence while defence questionnaires may be assessing more secondary forms of defence. The two questionnaires used for assessing defence correlated significantly, while a scale for social desirability showed no correlation with DMT or the two defence questionnaires.

The transmission of bluetongue virus by embryo transfer in sheep.

The susceptibility of sheep to intrauterine infection with bluetongue virus (BTV) was established by introducing 10(4) plaque-forming units of BTV type 10 into the uterine lumen of two seronegative ewes in a simulated embryo transfer operation. Both ewes became viraemic and underwent seroconversion to BTV. Embryos recovered from seronegative superovulated donor ewes were incubated in vitro for 8 h with BTV type 10. After incubation the embryos were thoroughly rinsed by repeated transfer to sterile culture medium, and 12 of these embryos were then transferred to the uterus or oviduct of seronegative, synchronized recipients. Viraemia and seroconversion were detected in nine recipient ewes. Embryos recovered from eight viraemic ewes were transferred to 15 seronegative, synchronized recipients. Viraemia and seroconversion were detected in 2 of the recipients, both of which also became pregnant. A lamb born to a ewe becoming infected at the time of embryo transfer was clinically normal, and no evidence of BTV infection was obtained at postmortem examination of the lamb after slaughter.

Interferon production in lymphocyte cultures after rubella infection in humans.

When stimulated with rubella virus irradiated with ultraviolet light, cultures of lymphocytes from individuals immune to rubella responded with production of 12-14 times more interferon than did cultures of lymphocytes from individuals susceptible to rubella. This property may be retained throughout life, since it was seen in individuals who had had rubella in their recent and remote pasts. Peak levels of interferon were detected six days after stimulation of lymphocytes with rubella virus antigen. In contrast, phytohemagglutinin induced production of interferon equally well in cultures of lymphocytes from persons immune and persons susceptible to rubella, with levels peaking three days after stimulation. Since there was a positive correlation among titers of hemagglutination-inhibiting antibody to rubella virus, incorporation of [14C]thymidine, and production of interferon after stimulation of lymphocyte cultures by rubella virus, interferon synthesis may be considered to be another measurable parameter of cell-mediated immunity in rubella.