Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure.

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

Remifentanil with morphine transitional analgesia shortens neurological recovery compared to fentanyl for supratentorial craniotomy.

PURPOSE: To compare the recovery profiles, efficacy and safety of remifentanil and morphine for transitional analgesia with fentanyl in patients undergoing elective craniotomy for supratentorial mass lesions. METHODS: Ninety-one patients were enrolled in this prospective, randomized, multicentre study. Anesthesia was induced with thiopental and remifentanil (1.0 micro g x kg(-1) bolus and a 1 micro g x kg(-1) x min(-1) infusion) or fentanyl (1 micro g x kg(-1) bolus and a 1.0 micro g x kg(-1) x min(-1) infusion). The opioid infusion continued until the level of anesthesia was deemed appropriate for intubation. Anesthesia was maintained with N(2)O/O(2), isoflurane 0.5 MAC and remifentanil 0.2 micro g x kg(-1) x min(-1) or fentanyl 0.04 micro g x kg(-1) x min(-1). At bone flap replacement, either morphine 0.08 mg x kg(-1) (remifentanil group) or saline (fentanyl group) was given. RESULTS: Systolic blood pressure was greater in those receiving fentanyl during induction (145.6 +/-17.5 mmHg vs 128.8 +/-18.3 mmHg; P = 0.006) and intubation (126.9 +/-17.1 vs 110.9 +/-16.5 mmHg; P < 0.001). Median time to tracheal extubation was similar but less variable in the remifentanil group (remifentanil = 8 min: range = 2-44 min; fentanyl = 8 min: range = 1-732 min). The fentanyl patients required a longer time to achieve the first normal neurological score (fentanyl = 38.0 min; remifentanil = 26.0 min; P = 0.035). Both the anesthesiologists and the recovery room nurses rated remifentanil better with respect to level of consciousness. Analgesics were required earlier in patients receiving remifentanil; median time 0.5 vs 1.08 hr, P < 0.001. CONCLUSIONS: Remifentanil is a suitable alternative to fentanyl in supratentorial craniotomy. Time to preoperative neurological recovery is faster and morphine provides some transitional analgesia without compromising the quality of recovery.

Two polymorphs of lysozyme nitrate: temperature dependence of their solubility.

Two crystallographic forms of lysozyme nitrate are known, namely monoclinic and triclinic. Having previously determined the temperature dependence of the solubility of the monoclinic form (0.2 M NaNO (3) solutions at pH = 4.5) [Legrand et al. (2001). J. Crystal Growth 232, 244-249], we focus here on the solubility of the triclinic form. The temperature dependence of the solubility of this crystallographic form has been measured with a static light device developed in our laboratory. This device allows to observe of the dissolution of one phase and/or the occurrence of a new one by varying the temperature with a sweep rate as low as 0.6 degree/hour. The new solubility data are complemented with crystallographic data of the triclinic form for the sake of completeness. The faces of a triclinic crystal are indexed. The crystallisation enthalpy of the triclinic form is deduced from these new results. These new solubility data allow us now to discuss (1). the published protocols used to obtain the monoclinic and triclinic forms of lysozyme nitrate and (2). the phase transformation.

Tomato (Lycopersicon esculentum) pectin methylesterase and polygalacturonase behaviors regarding heat- and pressure-induced inactivation.

The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degrees C to 105 degrees C, and from 0.1 to 800 MPa, respectively. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the P/T plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.

Crystallization within agarose gel in microgravity improves the quality of thaumatin crystals.

To prevent crystals from moving in orbit and sedimenting upon their return to earth, the model protein thaumatin was crystallized in agarose gel in the Advanced Protein Crystallization Facility during the eight-day Space Shuttle mission STS-95 (November 1998). The quality of tetragonal crystals grown in microgravity was compared with that of controls prepared in parallel in the laboratory. On the basis of their diffraction properties, microgravity crystals were more ordered than crystals grown in gel on earth (the latter being, on average, better than reference crystals obtained in solution on earth). It is concluded that protein crystallization within a gel in microgravity may yield crystals of superior quality by combining the advantages of both environments. A possible explanation for the positive effect of microgravity on protein crystallization in gels involving the better quality of the nucleus is discussed.

Study of nucleation-related phenomena in lysozyme solutions. Application to gel growth.

Two populations of aggregates are generally indentified in supersaturated solutions of biological macromolecules: small aggregates of a size which is less than 5 nm and large aggregates, the largest of which are at least one order of magnitude bigger. In order to understand the role played by the microporous network of a gel in the growth and behaviour of these different species in the prenucleation period, an in situ observation of nucleation has been carried out using either free solutions or solutions trapped in agarose gels. In a previous study, free solutions were investigated by small-angle neutron scattering (SANS) to identify the small aggregates. Optical observations, made under the same conditions, revealed the formation of an amorphous precipitate which disappeared at the end of the experiment. The sedimentation of this phase, which occurs in free solution but never occurs in gelled solution, depletes the solution bulk and this could explain why the nucleation density is higher in agarose gel than in free solution. The case of silica gel, the behaviour of which is completely different with respect to nucleation, will be discussed.

A gel-mediated feeding technique for protein crystal growth from hanging drops.

A procedure which allows an investigator to supply a crystal with fresh mother material without inducing significant growth defects is described. This technique requires that the crystal is grown in a gelled hanging or sitting drop. An example concerning a model macromolecule, hen egg-white lysozyme, is given. Extension of this procedure to other macromolecules is discussed.

Systemic Candida infection in pediatric BM autotransplantation: clinical signs, outcome and prognosis.

Of the 393 children who underwent BM autotransplantation in the pediatric oncology unit of the Institut Gustave Roussy between February 1979 and September 1991, 14 (3.56%) developed disseminated Candida infection within 3 months. This incidence was far lower than in other published series. Eleven of the 14 patients recovered from the infection, giving a far higher survival rate (78%) than among adult BM transplant recipients (usually < 30%). All 14 patients had four or more risk factors and persistent BM aplasia (median, 25 days); six had Candida tropicalis infection. Four cases of deep visceral involvement were documented, two of which were lethal. Clinical signs were relatively uniform and included secondary high-grade fever (> 40 degrees C) for 8 days; half the patients developed cardiovascular impairment, respiratory distress, neurological disturbances (altered consciousness or delirium) and severe diarrhoea, within as little as 10 days after transplantation. Blood cultures rapidly became positive after the onset of clinical signs and this permitted prompt treatment with a combination of amphotericin B and 5-fluorocytosine; in addition, central catheters were removed. Blood cultures became sterile within 4 days of treatment in 10 of the 14 cases. The generally favourable outcome in this series could be related to the young age of the patients, the absence of GVHD, the absence of total body irradiation in the conditioning regimen, and early antifungal treatment.

[Acute disseminated multinodular non-recurrent adiponecrosis. Cutaneous manifestations of recent toxoplasmosis]. / Adiponécrose multinodulaire disséminée aiguë non récidivante. Manifestation cutanée d'une toxoplasmose récente.

Acute disseminated multinodular non suppurative panniculitis in a 3 year-old boy is reported. The diagnosis was histologically confirmed. Detection of high levels of specific serum IgM favored a diagnosis of acute toxoplasmosis. The relationship between panniculitis and acute toxoplasmosis is discussed.

[Rupture of the left subclavian artery associated with fracture of the 1st rib]. / Rupture de l'artère sous-clavière gauche associée à une fracture de la première côte.

Treatment in a case of partial rupture of the subclavicular artery, provoking subacute ischemia of the upper limb and associated with a fracture of the first rib, was by sternoclavicular disinsertion and resection-suture. The importance of systematic investigation of possible subclavicular lesions in patients with fractured first ribs is emphasized, both during the initial and follow-up examinations. The presence of a fracture of this type has little influence on the classical surgical attitude adapted, apart from the possible need for resection of the first rib.