Hydrostatic pressure effects on a hydrated lipid inverse micellar Fd3m cubic phase.

Over a range of hydration, unsaturated diacylglycerol/phosphatidylcholine mixtures adopt an inverse micellar cubic phase, of crystallographic space group Fd3m. In this study hydrated DOPC:DOG mixtures with a molar ratio close to 1 : 2 were examined as a function of hydrostatic pressure, using synchrotron X-ray diffraction. The small-angle diffraction pattern at atmospheric pressure was used to calculate 2-D sections through the electron density map. Pressure initially has very little effect on the structure of the Fd3m cubic phase, in contrast to its effect on hydrated inverse bicontinuous cubic phases. At close to 2 kbar, a sharp transition occurs from the Fd3m phase to a pair of coexisting phases, an inverse hexagonal H(II) phase plus an (ordered) lamellar phase. Upon increasing the pressure to 3 kbar, a further sharp transition occurs from the H(II) phase to a (fluid) lamellar phase, in coexistence with the ordered lamellar phase. These transitions are fully reversible, but show hysteresis. Remarkably, the lattice parameter of the Fd3m phase is practically independent of pressure. These results show that these two lipids are miscible at low pressure, adopting a single lyotropic phase (Fd3m); they then become immiscible above a critical pressure, phase separating into DOPC-rich and DOG-rich phases.

Time-resolved scattering investigations of brome mosaic virus microcrystals appearance.

The behavior of brome mosaic virus in solution as a function of physico-chemical conditions has already been characterized by Small Angle X-ray Scattering studies. The most striking result was that the precipitates induced by the addition of polyethylene glycol were in fact made of microcrystals. This result was reinvestigated on the ID2 beamline at ESRF (Grenoble, France) to determine whether there was an amorphous state before the organized one, and measure the necessary period of latency for the microcrystals to form. The stopped-flow device associated to the high brilliance of the beamline enabled us to characterize the growth of the diffraction peaks as a function of time.

Spherical plant viruses: interactions in solution, phase diagrams and crystallization of brome mosaic virus.

Brome mosaic virus (BMV) is a small icosahedral plant virus of mean diameter 268 A. Interactions between BMV particles in solution were studied by means of small-angle X-ray scattering in order to find crystallization conditions. The interactions between biomacromolecules as large as these viruses have not yet been systematically studied by this method. As it is known that usually proteins crystallize in, or close to, attractive regimes, the interactions between BMV particles in solution were studied as a function of pH, type of salt and size and concentration of polyethylene glycol. An unexpected result of these studies is that the precipitates obtained upon addition of PEG alone or PEG combined with salt were in fact made of microcrystals, which were all characterized by the same series of diffraction peaks, with positions close to those of a centered cubic space group. A phase diagram of the virus as a function of PEG concentration was established by means of microbatch experiments. From the precipitation zones, conditions for crystallization were tested from 5 to 40 mg ml(-1) virus with 3-10%(w/v) PEG 8000 or PEG 20,000. Small crystals were obtained in several conditions after a few days and continued growing for several weeks.

Crystallographic analysis of freeze-fractured three-dimensionally ordered specimens.

We describe here an original approach for solving the structure of three-dimensionally ordered specimens at low and medium resolutions. It combines freeze-fracture electron microscopy and quantitative image processing and has been first successfully applied to the crystallographic study of different lipid-containing cubic phases. The structure preservation during cryofixation is controlled by recording X-ray diffraction before and after freezing. Well frozen cubic phases show fracture planes which look like well defined cleavage planes of 3-D crystals. These fracture planes (domains) reveal a mosaic of 2D ordered sub-domains which are geometrically related to each other by simple crystallographic operations. The symmetry properties of the images mirror faithfully the symmetry of the space groups. The shifts and rotations observed between adjacent sub-domains are related to this symmetry. Different cubic phases display different fracture behavior, highly characteristic for a given space group. Interpretation of the averaged images of different domains in terms of molecular structure is done by the comparison of the averaged periodic motifs either with the corresponding sections of the electron density map (from X-ray diffraction data) or with the corresponding sections of a 3-D-space filling model. We show here that the same procedure may be applied to other three-dimensionally ordered specimens such as 3-D crystals of membrane proteins or of other proteins, including naturally occurring protein crystals of some secretory organelles. Finally, the same approach could also provide a powerful tool for the study of membrane protein crystallogenesis, particularly for the formation of 3-D crystals.

Crystallographic analysis of freeze-fracture electron micrographs: application to the structure determination of cubic lipid-water phases.

An original approach combining freeze-fracture electron microscopy and quantitative image processing has been developed as an alternative to X-ray analysis. It has been applied to the crystallographic study of different lipid-water cubic phases [bicontinuous or micellar and of type I (oil-in-water) or type II (water-in-oil)] and has enabled significant advances in the study of these phases. Freeze-fracture electron microscopy has revealed that the cubic phases fracture preferentially along a few crystallographic directions which appeared on the images as noisy planar fracture surfaces containing periodic information. The visibility of the corresponding unit cells has been considerably improved by image-filtering techniques based on correlation averaging, allowing a quantitative analysis of the fracture images to be made. This analysis yielded faithful information on the symmetries of the cubic structure (rotation axes and mirror planes) as well as on the structure of the cubic phase itself. Eventually, the different parameters that determine the most favourable fracture pathways within the structures were established. This novel approach constitutes a powerful tool of general interest, complementary to X-ray diffraction, for solving complex ordered macromolecular structures at low resolution.

Freeze fracture electron microscopy of lyotropic lipid systems: quantitative analysis of the inverse micellar cubic phase of space group Fd3m (Q227).

An inverse micellar cubic phase of cubic aspect 15 formed by dioleoylglycerol/dioleoylphosphatidylcholine mixtures has been studied by freeze fracture electron microscopy. The structure was well preserved after freezing samples which had been hydrated either in pure water or in 30 vol% aqueous glycerol solutions. Electron microscopy images of high quality and resolution have been obtained. Four types of fracture planes, perpendicular to the [111], [110], [311] and [100] crystallographic axes, were identified by optical diffraction of the images from selected areas of the replicas. This is the largest number of different fracture planes yet observed in any lipid mesophase by electron microscopy. These planes are also perpendicular to the directions of the lowest order, and most intense reflections in the X-ray patterns from this cubic phase. The images were filtered using correlation averaging techniques, and they revealed the presence of mirror planes, which establishes that the space group is Fd3m (Q227) rather than Fd3. The interpretation of the images was aided by the novel use of standard deviation (s.d.) information obtained from the averaging procedures. The results are easily interpreted with the structure model deduced from X-ray diffraction and consisting of a complex packing of two different sizes of quasi-spherical inverse micelles located at positions (a) and (d) of the Fd3m unit cell. The results also show clearly that the fracture pathways always coincide with the regions of high CH3 concentration, located between the crystallographic planes containing the larger inverse micelles located at positions (a).

DEFPARAM: a program package for aligning elliptical sections of biological objects containing an n-fold symmetry.

A program package has been developed to align automatically images of biological objects containing an n-fold symmetry, and to remove the distortions induced on their circular shape by the microtomy. It uses an original procedure based on correlation techniques and replaces usual manual processing. Examples of direct averaging of transverse sections of biological objects are given to illustrate the program's capabilities.

Freeze-fracture electron microscope study of lipid systems. The cubic phase of space group Pm3n.

The cubic phase Q223 (space group Pm3n) of lipid-water systems has been studied by freeze-fracture electron microscopy. Four types of fracture planes were identified; all display highly ordered two-dimensional domains, each subdivided into subdomains related to each other by displacements and rotations connected to the symmetry of the space group. The images were filtered using cross-correlation averaging techniques and the filtered images were compared with the corresponding planar sections of the electron density map. The similarity of the two distributions was assessed via a mathematical parameter, the matching index, whose values were determined as a function of the apodization of the electron density map and of the position of the plane in the direction normal to the fracture. By definition, the best coincidence corresponds to an extremum of the matching index. Several conclusions were drawn. (1) The structure of the sample is well preserved in the replicas. (2) The "resolution" of the electron microscope experiments is remarkably close to that of the X-ray scattering study. (3) Of the two types of models that have been envisaged for phase Q223, the micellar is found to be in better agreement with the electron micrographs than the cagelike. (4) The symmetry of the space group is faithfully reflected in the electron microscope images; in particular, the presence of mirror planes rules out the non-centric of the two space groups that are compatible with the crystallographic data. (5) The crystallographic orientations of the fracture planes that are most frequently identified coincide with those of the most intense X-ray reflections; this observation was interpreted as an indication that the fracture propagates differently in the hydrocarbon and in the water volumes. (6) The thinner metal deposits in the replicas were found to coincide with the hydrocarbon regions of the fractures. (7) The fracture seems to be planar across the water matrix and the micelles seem to be removed from the replica and replaced by dips.

Cubic phases of lipid-containing systems. Elements of a theory and biological connotations.

It has recently been shown that the structure of two of the six cubic phases so far identified in lipid-containing systems is micellar, one (Q223) of type I, the other (Q227) of type II. The micelles of both phases belong to two distinct classes, those of each class being centred at one of the special positions of the space group. From the chemical viewpoint, phase Q227 seems to require a heterogeneous mixture of water-miscible and water-immiscible lipids, whereas phase Q223 has been observed with chemically pure lipids. Also, the area/volume ratio measured at the polar/apolar interface takes the same value in the two types of micelles of phase Q223, different values in those of phase Q227, in keeping with the notion that the area/molecule ratio is closely related to the chemical activity of the lipid components. The topological properties of the micellar phases are profoundly different from those of the bicontinuous phases. The bicontinuous cubic phases (Q230, Q224, Q229) are often presented as paradigms of the infinite periodic minimal surfaces (IPMS). Some authors have generalized that notion and sought in the IPMS a unified theory underlying the entire field of lipid polymorphism. These analogies entertain some confusion between the mathematical concept of surface and the physical notion of interface. A few electron density maps are presented to document the distance that separates the polar/apolar interfaces from the IPMS. The maps also show that some of the geometric singularities (points, lines, surfaces) of the structures coincide with the locus of the CH3 ends of the chains and with the very centre of the water matrix, i.e. with the regions where the short-range disorder is highest. We introduce the expression chaotic zones to designate these regions. In all the lipid phases the chaotic zones are found to occupy special geometric positions, either related to the symmetry elements or to the IPMS. It thus appears that it is energetically more advantageous to adopt an orderly disposal of the short-range disorder than to minimize the area of the polar/apolar interfaces. Finally, regarding the possible biological significance of lipid polymorphism, the point is stressed that among the phases that are observed in equilibrium with excess water (these phases are also the most likely candidates for a biological role) those with a cubic symmetry deserve special attention.(ABSTRACT TRUNCATED AT 400 WORDS)

Centrosome organization and centriole architecture: their sensitivity to divalent cations.

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved (i) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also observed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A scheme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization.

Cryo-electron microscopy of vitrified specimens: an approach to the study of bulk specimens.

We are using and developing cryo-electron microscopy of vitrified specimens. Our main interests concern the structure of muscle and muscular components. Micrographs which generally contain periodic features are analyzed by numerical image processing methods. To detect artifacts induced by the electron microscopy techniques, we correlate our results to those obtained by X-ray diffraction. In this paper, we describe our approach to the study of bulk specimens. Vitrification of such specimens is assessed by cryo-X-ray diffraction. Microscopy is done on cryo-substituted specimens.

Polymorphism of a lipid extract from Pseudomonas fluorescens: structure analysis of a hexagonal phase and of a novel cubic phase of extinction symbol Fd--.

The phase diagram of the Pseudomonas fluorescens lipid extract is unusual, in the sense that it displays a cubic phase straddled by a hexagonal phase. The hexagonal phase was studied over an extended concentration range, and the reflections were phased on the assumption that the structure contains circular cylinders of known radius. The cubic phase, whose extinction symbol is Fd--, was analyzed by reference to space group No. 227 (Fd3m). The phases of the reflections were determined by using a novel pattern recognition approach, based upon the notion that the average fourth power of the electron density contrast mean value of (delta r)4 is dependent on chemical composition but not on physical structure, provided that the function delta r(r) satisfies the constraints mean value of (delta r) = 0 and mean value of (delta r)2 = 1. As a further constraint, a shape normalization is used, in the form of a Gaussian apodization of the intensities, which has the effect of normalizing the curvature of the autocorrelation function at the origin. We analyzed two cubic samples of different composition: for each of them we generated all the phase combinations compatible with the X-ray scattering data and we searched for those whose mean value of (delta r)4 best agrees with the hexagonal phase. Taking advantage of the favorable properties of the phase diagram, we carefully explored the effects of various parameters; we concluded that the chemical composition of the phases being compared must be identical, that the X-ray scattering data should not be truncated artificially, and that the apodization must be mild so that the curvature takes a value intermediate between those corresponding to the raw data of the two phases. When all these precautions were taken, mean value of (delta r)4 was found to be remarkably invariant; this conclusion is important in view of the possible usefulness of the novel technique in tackling ab initio--and at very low resolution--structural problems of more general interest. The structure of the cubic phase consists of a 3D network of rods joined tetrahedrally 4 by 4 according to a diamond lattice and of a family of quasi-spherical disjointed micelles; the core of the rods and of the micelles is polar, and the interstices are filled by the hydrocarbon chains (structure of type II). All the dimensions (diameter of rods and micelles, area per chain at the polar/apolar interface) are consistent with the chemical properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino acid substitutions in structurally related proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix.

Amino acid substitutions in evolutionarily related proteins have been studied from a structural point of view. We consider here that an amino acid al in a protein p1 has been replaced by the amino acid a2 in the structurally similar protein p2 if, after superposition of the p1 and p2 structures, the a1 and a2 C alpha atoms are no more than 1.2 A apart. Thirty-two proteins, grouped in 11 classes, have been analysed by this method. This produced 2860 amino acid pairs (substitutions), which were analysed by multi-dimensional statistical methods. The main results are as follows: (1) according to the observed exchangeability of amino acid side-chains, only four groups (strong clusters) could be delineated; (i) Ile and Val, (ii) Leu and Met, (iii) Lys, Arg and Gln, and (iv) Tyr and Phe. The other residues could not be classified. (2) The matrix of distances between amino acids, or scoring matrix, determined from this study, is different from any other published matrix. (3) Except for the distance matrices based on the chemical properties of amino acid side-chains, which can be grouped together, all other published matrices are different from one another. (4) The distance matrix determined in this study seems to be very efficient for aligning distantly related protein sequences.

Cubic phases of lipid-containing systems. Structure analysis and biological implications.

Of the six cubic phases identified so far in lipid-containing systems, the structures of only two have been determined unambiguously. We tackle the structure determination of the other four. We use for that purpose a novel pattern recognition approach, which consists of generating all the sets of phase angles (phi-sets) compatible with the observed reflections, and of screening them in a search for the "best" one. Two criteria are used for screening: both involve the parameter [(delta rho)4] (delta rho is a dimensionless function proportional to the Fourier transform of the set of observed structure factors). One is a test of smoothness, based upon the postulate that the "best" phi-set is that whose [(delta rho)4] is minimum; this criterion, equivalent to maximum entropy, is fulfilled when the system is devoid of heavy atoms, and when the polar and the hydrocarbon moieties occupy almost equal volumes. The other criterion is based upon the notion that [(delta rho)4] takes the same (or similar) values in thermodynamic phases with the same (or similar) chemical composition, whatever the structure of the phases. The validity of the two criteria is verified using numerous examples. The six cubic phases are analysed using this approach. The structure of three of them (Q230, Q224, Q229) can be described in terms of two three-dimensional networks of connected rods, mutually intertwined and unconnected: in Q230 the rods are coplanarly joined 3 by 3; in Q224 the rods are tetrahedrally joined 4 by 4; in Q229 the rods are cubically joined 6 by 6. The structures of Q212 and Q227 are related to those of Q230 and Q224, respectively; one of the two networks of rods is preserved, the other is replaced by a lattice of closed micelles. The structure of Q223 appears to consist of a cage-like continuous three-dimensional network of connected globules, coplanarly joined 3 by 3 at one end and 4 by 4 at the other, enclosing a three-dimensional lattice of closed micelles. The analogies of the structures of Q230, Q224, Q229 with the three fundamental cubic infinite periodic minimal surfaces are discussed. More interestingly, the structures of, on the one hand Q230, Q224, Q229 and of Q212, Q227, Q223 on the other, are shown to provide topological generalizations of the two paradigms of lipid organization; namely, the bilayer and the monolayer.(ABSTRACT TRUNCATED AT 400 WORDS)

[Surgery of giant bullae of emphysema in adults]. / La chirurgie des bulles géantes d'emphysème de l'adulte.

Over a 20 years period 50 patients with giant emphysematous bullae (bilateral in 20 cases) were operated upon. Fifteen had been exposed to an occupational risk (coal mining, steel milling, chemicals); all were, or had been, tobacco smokers; 11 were in acute respiratory distress at the time of surgery. Respiratory function tests showed mixed restrictive and obstructive impairment. Ten patients had chronic cor pulmonale. There were 8 post-operative deaths, 4 of them with right-sided heart failure. Mortality was highest in patients with bilateral lesions and in emergency situations. Late complications involved the lungs in 10 cases and the heart in 2 cases. The results of pre- and post-operative lung function tests could be compared in 13 patients.

[Gastroesophageal reflux. Anatomical, physiological and surgical aspects. Apropos of 61 consecutive cases of peptic esophagitis surgically treated with the same procedure]. / Le reflux gastro-oesophagien. Considérations anatomiques, physiologiques et chirurgicales. A propos de 61 cas consécutifs d'oesophagite peptique opérés selon le même protocole.

Based on a literature review and a personal series of cases treated, it is considered that gastroesophageal reflux results mainly from a mechanical process. The importance of the gastric musculature is emphasized, particularly the oblique fibers which form a thick belt encircling the esophagogastric function. Reflux is assumed to be almost exclusively due to loss of fixation of the point of attachment of this belt which, because of its particular texture, allows gastric dilatation to be propagated to the esophagogastric junction. The principal cause is the angular movement of the mobilized cardia. The same result is observed after iatrogenic destruction of this so-called muscular belt. Ulcerated reflux esophagitis is considered to be a serious disease comparable to duodenal ulcer, and the operative technique used aims both at reducing gastric acidity (vagotomy) and the construction of an anti-reflux assembly based on Hill's method. Results of the application of these principles in 61 consecutive cases are discussed.

[Intrathoracic chemodectomas. A report on four observations (author's transl)]. / Les chémodectomes intra-thoraciques. A propos de quatre observations.

Intrathoracic chemodectomas are extremely rare tumours, the number, published up to the present time being less than 50. We wish to report here four personal cases, all resected. The diagnosis was never made before surgery but solely by histopathology. The course was benign in two cases and malignant with slow progression in one case and malignant with rapid progression in the other. On the basis of these four cases, a general review is undertaken of this particular site on the basis of publications in the literature.