Biological activities and structural properties of the atypical bacteriocins mesenterocin 52b and leucocin b-ta33a.

The antibacterial spectra and modes of action of synthetic peptides corresponding to mesenterocin 52B and leucocin B-TA33a greatly differ despite their high sequence homology. Circular dichroism experiments establish the capacity of each of these two peptides to partly fold into an amphiphilic helix that might be crucial for their adsorption at lipophilic-hydrophilic interfaces.

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution.

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.

Crystallization and preliminary X-ray diffraction studies of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus.

The homotetrameric holo-D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus has been crystallized in the presence of NADP+ using the hanging-drop vapour-diffusion method. Crystals grew from a solution containing 2-methyl-2,4-pentanediol and magnesium acetate. A native data set has been collected to 2.1 A using synchrotron radiation and cryocooling. Diffraction data have been processed in the orthorhombic system (space group P21212) with unit-cell dimensions a = 136.7, b = 153.3, c = 74.9 A and one tetramer per asymmetric unit.

Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer.

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridinium si face requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out on Escherichia coli GAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant, Biochimie 72, 545-554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung, Protein Pept. Lett. 1, 19-24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors the syn orientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH from E. coli (E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg, J. Mol. Biol. 257, 814-838, 1996). The Kd constants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between the syn and anti forms occurs in the [Thr313-Gly317] double mutant with a preference for the anti conformer. Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with an anti orientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.

Conformational studies of a synthetic peptide from the putative lipid-binding domain of bovine milk component PP3.

In bovine milk, a glycosylated phosphoprotein, component PP3, is known for its remarkable emulsifying properties and its capability to inhibit lipolytic activities. The determination of its primary structure is not sufficient to explain these properties. Secondary structure predictions of component PP3 and of its homologous proteins were achieved using a combination of multiple predictive methods. Based on this study, the f 119-135 region of component PP3 was proposed to be likely to adopt an amphipathic helical conformation, which is a lipid-binding motif. The conformation of the synthetic peptide corresponding to the C-terminal f 119-135 part of bovine component PP3 was analyzed by circular dichroism experiments using various media. The circular dichroism data indicated that the peptide was able to form an amphipathic alpha-helix structure in trifluoroethanol as well as in the presence of sodium dodecyl sulfate or acidic and neutral lipids, but not in water. Moreover, the conformation of this peptide is solvent dependent because it was found to adopt a beta-sheet structure for low concentrations of sodium dodecyl sulfate or a low molar ratio of acidic lipid to peptide. Tensiometric measurements showed that the amphipathic C-terminal region of component PP3 is highly tensioactive and, thus, must be responsible for the particular behavior of the protein in emulsions.

A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+.

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.

Phosphorus-31 nuclear magnetic resonance studies on coenzyme binding and specificity in glyceraldehyde-3-phosphate dehydrogenase.

Binding of NAD(P)+ to wild type and a series of mutants of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by 31P NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP+ 2'-phosphate pKa deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pKas ranging from 7.7 for the L187A-P188S mutant to < 5.7 for the D32G-L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP+ is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pKa of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pKa, it is suggested that the 2'-phosphate ionization state of bound NADP+ in chloroplastic GAPDH is dianionic. The NADP+ dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D32G-L187A-P188S, are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or conformational change upon binding.

Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]-enkephalin in aqueous solution as revealed by the combined use of 17O-NMR and Fourier-transform infrared spectroscopy.

Solvent-induced and temperature-induced 17O chemical shifts of [17O-Gly2, Leu5]-enkephalin and [17O-Gly3, Leu5]-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu5]-enkephalin and [1-13C-Gly2, Leu5]-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 17O-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.

17O NMR and FT-IR study of the ionization state of peptides in aprotic solvents. Application to Leu-enkephalin.

The ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions.

Structure of tBuCO-Val psi [NH-CO]NHtBu.

N-Isobutylidenedipivalamide, C14H28N2O2, Mr = 256.39, orthorhombic, P2(1)2(1)2(1), a = 16.656 (2), b = 9.751 (2), c = 10.583 (2) A, V = 1718.8 A3, Z = 4, D chi = 0.99 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 4.56 cm-1, mu Rmax much less than 1, F(000) = 568, T = 293 K, R = 0.081 for 887 observed reflections. The geometrical parameters of this retro-peptide molecule are quite similar to the standard values for peptides. Conformational angles are phi = -101 (1), phi' = 99 (1) degrees.

Structure of MeCO-Gly psi [NH-CO]NHMe.

N,N'-Methylenediacetamide, C5H10N2O2, Mr = 130.15, orthorhombic, Pna2(1), a = 17.218 (1), b = 4.489 (1), c = 18.124 (1) A, V = 1400.8 A3, Z = 8, D chi = 1.23 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu Rmax much less than 1, mu = 7.19 cm-1, F(000) = 560, T = 293 K, R = 0.044 for 1318 observed reflections. This retro-peptide molecule assumes two nearly identical conformational states (A phi = 93.2, phi' = 77.0; B phi = 90.6, phi' = 79.6 degrees) with planar trans amide functions. The bond lengths and bond angles are very close to the standard dimensions of the peptide group.

Structure of MeCO-psi[NH-CO]Val-NHMe.

N,N'-Dimethylisopropylmalonamide, C8H16N2O2, Mr = 172.23, orthorhombic, Pbcm, a = 4.859 (1), b = 13.523 (2), c = 15,469 (2) A, V = 1016.4 A3, Z = 4, Dx = 1.12 g cm-3, lambda(Cu K alpha) = 1.5418 A, microRmax much less than 1, mu = 5.88 cm-1, F(000) = 376, T = 293 K, R = 0.060 for 665 observed reflections. Dimensions of this retropeptide molecule are quite similar to the standard values for peptides. The C alpha and C beta atoms are in a mirror (z = 1/4), so conformational angles are psi' = -psi = -110.4 degrees (2).

Immediate and mid-term results of percutaneous mitral commissurotomy.

The results of percutaneous mitral commissurotomy (PMC) were assessed in a series of 600 patients (pts) with mitral stenosis. Their mean age was 43 +/- 15 years (13-86). One hundred and eight had had a previous surgical commissurotomy; 464 were in NYHA class III or IV; atrial fibrillation was present in 188. One hundred and fifty-nine had valvular calcification and angiography disclosed a mild regurgitation (MR) (1/4) in 255. Technical failure occurred in 19 pts. In the remainder, PMC improved valve function: valve area (VA) increased from 1.1 +/- 0.3 cm2 to 2.2 +/- 0.5 cm2 (P less than 0.0001) as assessed by haemodynamics, and from 1 +/- 0.2 to 2 +/- 0.4 cm2 (P less than 0.0001) as assessed by two-dimensional echocardiography. Complications were as follows: death (0.5%), haemopericardium (0.8%), severe MR (3.8%), embolism (3.3%), atrial shunt (14%). Secondary surgery for complications following PMC was necessary in 4.8% of cases. There were poor results (VA less than 1.5 cm2 and/or MR greater than 2/4) in 13%; their predictors being valve anatomy (P less than 0.001), initial valve area (P less than 0.01) and previous surgical commissurotomy (P less than 0.05). Among the 437 pts resident in France, 98% were followed-up 15 +/- 11 months after PMC (range 1-48). After 42 months, the actuarial rates of survival, freedom from need for reoperation and good functional results were respectively: 87 +/- 6%, 81 +/- 3% and 72 +/- 6%.(ABSTRACT TRUNCATED AT 250 WORDS)

[Björk-Shiley-Monostrut prosthesis of the aortic valve. Mid-term development]. / Prothèse de Björk-Shiley-Monostrut en position aortique. Evolution à moyen terme.

From July 84 to June 88, 100 patients underwent an isolated aortic valve replacement by a Monostrut-Björk-Shiley prosthesis. Fifty-one had calcified aortic stenosis, 24% aortic insufficiency, 25% mixed aortic lesions. Pre-operatively, their mean age was 57 years, 68% were male, 46% were in NYHA class II or IV, 43% had angina, all were in sinus rhythm. Operative mortality was 4%. All the survivors were followed-up for a mean period of 22 months (6 to 58) with a cumulative follow-up of 183 patients-years. All patients were placed on a long-term regimen of anticoagulant therapy. Two late deaths occurred (1 myocardial infarction, 1 cerebral hemorrhage). The 4 years survival rate was 94%. Postoperative functional results were excellent. Nearly all patients were asymptomatic. Concerning valve related complications: the 4 years actuarial rate of patients free from thromboembolism, hemorrhage, valve thrombosis, periprosthetic leakage and endocarditis were respectively 97%, 97%, 100%, 100% and 100%. No patient were reoperated on. Valvular function was evaluated by mean transprothetic gradient on echo-doppler (61 cases) and by the calculation of the valvular area with transseptal catheterization (21 cases). For small sizes prosthesis (annulus diameter of 19 or 21 mm), medium size prosthesis (23 or 25 mm), large size prosthesis (27 to 29 mm), mean transprothetic gradient were respectively 16, 10 and 6.9 mmHg and valvular aortic area were respectively 1.5, 1.9 and 2.4 cm2. Mid term results of the Monostrut-Björk-Shiley prosthesis on aortic position are good with a low rate of valve related complications and good hemodynamic results, even with the small valve size.

[Eisenmenger syndrome and pregnancy. Apropos of a case]. / Syndrome d'Eisenmenger et grossesse. A propos d'un cas.

A case of Eisenmenger syndrome associated with an ongoing pregnancy and maternal death one month after cesarean section is reported. The review of the world literature shows the very high maternal risk of this condition, which should be prevented by tubal sterilization or therapeutic abortion in the very early pregnancy.

[Echography in surgical mitral insufficiency. Pathologic diagnosis and provision of the surgical procedure]. / L'échographie des insuffisances mitrales chirurgicales. Diagnostic lésionnel et prévision du type de chirurgie.

The aim of this study was to determine the reliability of preoperative transthoracic and transesophageal echocardiography compared with the surgical findings in pure or dominant severe mitral regurgitation with respect to: the evaluation of the lesions, mechanism and etiology; the provision of the type of surgery (valve replacement or reconstruction); One hundred and fifty patients were divided into two groups: Group I (N = 120) in which preoperative assessment included transthoracic echo-Doppler coupled with color Doppler in the last 32 patients; Group II (N = 30) operated recently who underwent both transesophageal and transthoracic echo-Doppler examination. In Group I, the sensitivity of transthoracic echo in the evaluation of the etiological was 86% overall [100% in rheumatic valve disease (N = 28), 86% in degenerative or dystrophic valves (N = 72), 44% in endocarditis (N = 9), 87% in ischaemic dysfunction (N = 8)]. The echo evaluation of the mechanism of the regurgitation was also reliable with the exception of ruptured chordae in which direct visualisation of the rupture was only possible in 19 of the 64 cases (30%). The type of surgery predicted by echo was practiced in 87% of cases.

[Value of transesophageal echocardiography. From a preliminary experience of 532 cases]. / Intérêt de l'échographie cardiaque par voie transoesophagienne. A partir d'une expérience préliminaire de 532 cas.

Transesophageal echocardiography (TEE) was introduced recently in France. The aim of this study was to review the diagnostic value of this technique after 8 months' use in our cardiology department. A total of 532 TEE studies were carried out between April and December 1988 in 396 patients (average age 54 years, range 17 to 89 years) at Tenon Hospital. The failure rate was 1.8 per cent (N = 10), over half of which occurred at the beginning of the operator's experience. TEE was particularly valuable compared with the standard transthoracic approach in the following instances: the investigation of mitral stenosis, especially before percutaneous valvuloplasty (N = 75). A left atrial thrombus was demonstrated in 5 cases by TEE vs none by standard echocardiography. There was also a much higher diagnostic sensitivity for small interatrial shunts (40 vs 6) resulting from transseptal catheterisation. In the preoperative investigation of severe mitral regurgitation (N = 29). The etiology was accurately diagnosed in 29 vs 26 cases, and the mechanism of the regurgitation was correctly classified especially in cases of ruptured chordae (15 vs 6 cases). In endocarditis (N = 26) by the visualisation of abscess of the aortic ring (7 vs 1) and vegetations (19 vs 8). In prosthetic valve dysfunction (N = 65) by the demonstration of primary degeneration of bioprostheses (7 vs 4), perivalvular leaks (10 vs 4) and non-occlusive thrombi of mechanical prostheses (3 vs 0). In cases of intracardiac tumours, dissection of the thoracic aorta and atrial septal defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational perturbations in retro-analogs of the tBuCO-Ala-Gly-NHiPr dipeptide. Crystal structure of the retro-dipeptide with a reversed Ala-Gly amide bond.

The three retro-analogs of the tBuCO-Ala-Gly-NHiPr dipeptide, in which each amide bond had been successively reversed, were studied in solution by 1H-n.m.r. and i.r. spectroscopy with reference to the conformational properties of their parent dipeptide. Reversal of the Ala-Gly amide bond proved to perturb the folding tendency of the backbone less than the inversion of either of the terminal amide bonds. The crystal structure of the retro-peptide containing a reversed Ala-Gly amide bond was also solved by X-ray diffraction and constitutes the first available data for this retro-peptide series. In contrast to the beta II-folded structure of the parent dipeptide, the retro-peptide molecule adopts an open conformation in the crystal.

Results of percutaneous mitral commissurotomy in 200 patients.

To assess the feasibility and efficacy of percutaneous mitral commissurotomy (PMC), the procedure was attempted in 200 patients with severe mitral stenosis. There were 154 women and 46 men, their mean age was 43 +/- 16 years (range 13 to 79) and 15 were older than 70 years of age. Forty-four had had previous surgical commissurotomy. Forty were in New York Heart Association class II, 152 in class III and 8 in class IV. In regard to valvular anatomy, 67 had calcified valves, 58 had pliable valves and only mild subvalvular disease, and 75 had flexible valves but extensive subvalvular disease. Grade 1+ mitral regurgitation was present in 62 and grade 2+ in 2. In 11 patients the procedure was discontinued because of complications in 3 and technical failure in 8. Six of the 8 technical failures occurred during the first 15 attempts. Effective PMC was performed in 189 patients using 1 balloon in 23 and 2 balloons in 166. After PMC, there was a significant improvement in mean left atrial pressure (21 +/- 7 to 12 +/- 5 mm Hg, p less than 0.0001), mean mitral gradient (16 +/- 6 to 6 +/- 2 mm Hg, p less than 0.0001), cardiac index (2.6 +/- 0.8 to 3.1 +/- 0.8 liters/min/m2, p less than 0.001) and valve area assessed by hemodynamics (1.1 +/- 0.3 to 2.2 +/- 0.5 cm2, p less than 0.0001) and 2-dimensional echocardiography (1 +/- 0.3 to 1.9 +/- 0.4 cm2, p less than 0.0001). No patient died. Embolism occurred in 8 (4%), with no further sequelae. Sixteen (8%) had atrial septal defect detected by oxymetry.(ABSTRACT TRUNCATED AT 250 WORDS)