Biomedical subjects

J P Bretaudiere

Publications and source records attributed to J P Bretaudiere.

At least 19 recordsLinked to original sources

PubMed1996

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle.

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three-dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z-filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.

Animals↗

PubMed1992

Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis.

Fatty Acid Synthases↗

PubMed1991

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy.

The structure of ornithine decarboxylase (Mr approximately 1.04 x 10(6] from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted +/- 45 degrees. The two edge views were related by a 30 degrees rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-A resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10 degrees with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.

Image Processing, Computer-Assisted↗

PubMed1991

Similar features in Z bands of both skeletal and cardiac muscle revealed by image enhancement.

We have shown previously that the small square (ss) and basket weave (bw) states of the Z band lattice in cardiac and skeletal muscle are related to the contractile state of the muscle. We have used two-dimensional image processing techniques on digitized electron micrographs to enhance the structural features of each projected lattice form in cardiac and skeletal muscle. Four different processing techniques were employed to assess the effect of enhancement artifacts on the resulting Z band images. We observed only slight differences between enhanced images of a particular Z band form produced by the four different techniques. Every enhanced image showed an approximate four-fold symmetry independent of muscle type or Z band lattice form. Each enhanced image showed four cross-connecting Z-filaments which appeared to connect each axial filament to the four nearest axial filaments. In bw images from both cardiac and skeletal muscle, axial filaments had a greater apparent diameter and a greater interaxial filament spacing than in the ss images. In both muscle types, the cross-connecting Z-filaments appeared to overlap half-way between axial filaments in the ss images while the bw images showed no such overlap. These structural features are consistent with a dynamic Z band lattice that participates in muscle contraction.

Algorithms↗

PubMed1991

Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy.

Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis.

Cryopreservation↗

PubMed1989

Electron microscope studies of human alpha 2-macroglobulin-chymotrypsin complex: demonstration that the two structures assigned to native and proteolyzed alpha 2-macroglobulin represent two views of the proteolyzed molecule.

Electron microscope studies of native and protease-bound human alpha 2-macroglobulin have led to two contradictory models for these two structures. One viewpoint maintains that the native structure has the shape of )+(, which contracts on binding of the protease to the shape of ([). An opposing view proposes that the native structure has the shape of a padlock and that )+( and ([) are the side and end views of the proteolyzed molecule. In this investigation, electron microscope studies of the alpha-chymotrypsin-treated alpha 2-macroglobulin utilizing a tilt stage have shown that the two shapes [)+( and ([)] interconvert. This demonstrates that these two shapes represent the side and end views of the proteolyzed alpha 2-macroglobulin which are related by a 90 degree rotation of the prototype molecule.

Chymotrypsin↗

PubMed1988

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Well-preserved structures of native and alpha-chymotrypsin-bound alpha 2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native alpha 2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the alpha 2-macroglobulin-chymotrypsin conjugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of alpha 2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex.

Chymotrypsin↗

PubMed1988

Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli.

Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC. The method is applied to two sets of 204 and 177 images that show the 70S ribosome of Escherichia coli, in the range of overlap views as defined by A. Verschoor and co-workers, and to two sets of 480 and 496 images of the 50S subunit of E. coli depleted of L7/L12 proteins in the well-defined crown view. Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates. These classes appear to be related to different orientations on the specimen grid (in the case of the 70S particle) and to different conformational states (50S subunit).

Escherichia coli↗

PubMed1987

General metabolism in head injury.

Seventy-six patients with closed head injuries alone were studied to define the relation between the severity of the head injury and secondary alterations of general metabolism. The effect of metabolic changes on neurological outcome and the importance of nutritional support on nutritional status and neurological outcome were also evaluated. Using a powerful statistical tool, convergence analysis, it was possible to take into consideration the effects of a number of confounding factors that obviously affected general metabolism. Most of the patients were hypermetabolic for prolonged periods. In addition, many did not receive even basal requirements of calories or protein for many days. Despite this, their outcomes were determined by their initial neurological status and the amounts that they were fed, admittedly relatively modest, did not influence their courses. Despite such feedings, their visceral protein levels, which often dropped initially, rose toward normal levels, indicating effective adaptation. Indeed, it could not be shown that these patients developed complications of malnutrition such as infections. However, it will require a sophisticated randomized clinical trial of vigorous intravenous hyperalimentation to determine whether this complex, dangerous, and expensive therapy is helpful for severely head-injured patients.

Craniocerebral Trauma↗

PubMed1986

Reconstitution of molecule images analysed by correspondence analysis: a tool for structural interpretation.

Correspondence analysis is gaining increasing importance in the analysis of electron micrographs of macromolecules. Partial or complete reconstitution of images from their factorial representations is introduced as a useful tool in interpreting variational patterns and tracing their physical origin. The value of image reconstitution is demonstrated with two examples, one using a set of model images and the other a set of images of a haemocyanin molecule assembly product.

Animals↗

PubMed1985

Comparison of enzyme immunoassay and immunoprecipitation for creatine kinase MB in diagnosis of acute myocardial infarction.

We compared the clinical performance of measuring creatine kinase (EC 2.7.3.2) isoenzyme MB by use of an enzyme immunoassay (Enzygnost CK-MB, Behring Diagnostics) with an immunoprecipitation method (Isomune-CK, Roche Diagnostics) for the diagnosis of acute myocardial infarction. Sera from 80 patients admitted to the coronary care unit because of chest pain were examined: 40 who had this diagnosis of myocardial infarction, and 40 in whom it was ruled out. In addition, sera from 40 apparently healthy individuals were examined. The clinical sensitivity and specificity of these methods were evaluated by use of receiver operating characteristic curves. We conclude that for clinical efficiency, this enzyme immunoassay is slightly superior to the immunoprecipitation assay we used, because of its greater analytical sensitivity and precision for measuring the mass of the isoenzyme.

Adult↗

PubMed1984

Effect of blood contamination on lecithin to sphingomyelin ratio in amniotic fluid by different detection methods.

Amniotic phospholipid detection methods such as cupric acetate measure unsaturated lecithin whereas others such as phosphomolybdate detect both unsaturated and saturated lecithin. Because of the extreme unsaturation in serum and red blood cell lecithin, we compared lecithin (L) and sphingomyelin (S) content of maternal blood as well as the effect of blood contamination on amniotic fluid L/S ratios. L/S ratios were obtained by thin-layer chromatography utilizing both cupric acetate and phosphomolybdate for phospholipid detection. The L/S value (mean +/- SD) of maternal serum obtained by cupric acetate was 1.90 +/- 0.19 and that for phosphomolybdate 1.78 +/- 0.17. The results of increasing serum concentrations in amniotic fluid prior to analysis suggest that as little as 0.5% contamination alter results and by 2% contamination values approach the L/S ratio of actual serum whether the amniotic fluid was initially mature or immature by either method. The serum L/S ratio by cupric acetate equaled its maturity threshold of 2.0 while the serum L/S ratio by phosphomolybdate was below its threshold of 3.0. Whereas both methods would have falsely immature values in the presence of blood only phosphomolybdate would assure against false maturity.

Amniotic Fluid↗

PubMed1984

Removal of a component interfering with phosphatidylglycerol estimation in the "Helena" system for amniotic fluid phospholipids.

A simple " prechromatography " step removes a component of amniotic fluid ("pseudo PG") that otherwise co-chromatographs with phosphatidylglycerol in the system for amniotic fluid analysis developed by Touchstone et al. (Lipids 15: 61-62, 1980) and incorporated into the Helena "Fetal-Tek" kit. The final positions of the major phospholipids are practically unchanged but the faster migrating, more neutral components are swept into a tight band near the solvent front, providing improved general resolution. The resulting change in the ratio of "phosphatidylglycerol" to sphingomyelin ("PG"/S) is as much as 0.5 for some specimens, but no change is seen for others. In 20% of the 73 cases examined, including four of the six fluids associated with respiratory distress syndrome, the PG initially reported was found to be "pseudo PG" and was completely removed by the prechromatography step. This step produces a negligible decrease in the lecithin to sphingomyelin (L/S) ratio in amniotic fluid.

Amniotic Fluid↗

PubMed1983

Influence of phospholipid saturation on classical thin-layer chromatographic detection methods and its effect on amniotic fluid lecithin/sphingomyelin ratio determinations.

We compared seven techniques, commonly used for detection of amniotic fluid phospholipids in thin-layer chromatography, with respect to their sensitivity to saturation of the fatty acid carbon-chain of lecithin. The techniques fell into two classes: sensitive and insensitive; those classed as saturation sensitive were less than or equal to 10% as sensitive to fully saturated lecithin as to lecithin with singly unsaturated acid moieties. Color development increased with the number of carbon-carbon double bonds per molecule but required only a single unsaturated acid ester in either the alpha or the beta position. Mixtures of lecithins with defined saturation, when detected by saturation-sensitive methods, mimicked the uneven coloration of amniotic fluid lecithin bands, supporting the hypothesis that this uneven coloration results from natural saturation heterogeneity. Detection techniques representative of these classes are shown to give widely differing values for the lecithin/sphingomyelin ratio for actual specimens of amniotic fluid.

Amniotic Fluid↗

PubMed1981

Suitability of control materials for determination of alpha-amylase activity.

The suitability of control materials for determination of alpha-amylase activity was assessed in comparison with reference groups of authentic human serum specimens containing alpha-amylase of either pancreatic or salivary origin, specimens from patients with no pancreatic pathology, and normal specimens to which porcine pancreatic alpha-amylase was added. After determination of alpha-amylase activity by 11 commonly used techniques (five different principles), the results were processed by both classical (linear representation, regression) and multivariate (correspondence analysis, principal-components analysis) statistical techniques. Specimens containing porcine pancreatic alpha-amylase did not behave like any of the other groups. We conclude that porcine enzyme should not be used for interlaboratory quality-control surveys or intermethod comparison studies. Determination of human salivary and pancreatic alpha-amylase showed intermethod biases similar to those for authentic patients' specimens. Human salivary alpha-amylase, both because of its behavior and its commercial availability, is a satisfactory source for alpha-amylase activity of quality-control specimens. The nature of the matrix (polyvinylpyrrolidone, albumin, delipidated serum, bovine serum, or human serum) little influenced the behavior of the specimens for any of the methods studied.

Amylases↗

PubMed1981

Measurement of aspartate aminotransferase isoenzymes: six procedures compared.

Six procedures were evaluated for aspartate aminotransferase (EC 2.6.1.1) isoenzyme assay in human serum and tissue homogenates. Results of procedures based on immunochemical precipitation by use of antibodies directed against either the mitochondrial or (with greater precision) soluble isoenzyme correlated well with those by a differential kinetic assay involving both different pH conditions and adipate inhibition. Results with a DEAE-Sephadex ion-exchange chromatographic procedure correlated well with these techniques for specimens containing purified isoenzymes, but showed substantial positive bias for determination of the mitochondrial isoenzyme in human serum. An assay based on the differential effects of pH alone discriminated between the isoenzymes with less bias than did the chromatographic assay. Precision of the two differential pH assays was limited by significant reagent blank activity resulting from destruction of NADH at pH 6.0 or 6.2. An electrophoretic procedure in which diazonium salt is used to make oxalacetate visible was least accurate for measuring samples for which the isoenzyme composition was known.

Aspartate Aminotransferases↗

PubMed1981

Suitability of control materials. General principles and methods of investigation.

We propose methods for characterizing the behavior of quality-control specimens. Candidate quality-control specimens and authentic patients' specimens were analyzed by various methods. Patients' specimens were chosen to be fully representative of those encountered, including subsets from persons who were healthy, had defined disease states, were in therapy, or whose specimens were lipemic, icteric, etc. The analytical methods chosen include those most commonly used as well as reference analytical methods. Procedures for characterizing the behavior of patients' specimens and candidate quality-control specimens are proposed and their applicability is demonstrated. The linear ratio method is a univariate graphical approach in which differences in accuracy among methods for any specimen or group of specimens are each displayed on a linear scale. Correspondence analysis is a descriptive multivariate statistical technique that allows both the specimens and the analytical methods to be characterized. The statistical techniques, in our application, allow the behavior of quality-control specimens to be assessed with respect to authentic patients' specimens without influencing the assessment process. Correspondence analysis provides a graphic representation by projecting both the specimens and the analytical methods on factorial planes. The appropriateness of te behavior of a quality-control specimen may be inferred from its position relative to those of authentic patients' specimens. These statistical techniques also provide some information regarding the specificity of analytical methods.

Blood Chemical Analysis↗

PubMed1981

Measurement of alkaline phosphatase activity: characterization and identification of an inactivator in 2-amino-2-methyl-1-propanol.

An inactivator of alkaline phosphatase (EC 3.1.3.1) in 2-amino-2-methyl-1-propanol is demonstrated and characterized. This time-dependent inactivation results from chelation of enzyme-bound Zn2+; it is reversed by addition of Zn2+ and, to a lesser extent, other divalent metal ions. Cu2+ is an effective spectral indicator and can be used to determine the presence and quantity of inactivator. Data obtained from enzyme inactivation, Cu2+ absorbance spectra, "high-performance" liquid chromatography, thin-layer chromatography, Fourier-transform infrared spectroscopy, and mass spectroscopy indicate that the inactivator is 5-amino-3-aza-2,2,5-trimethylhexanol. This compound, even in trace amounts (less than 0.05% on a molar basis), shown to inactivate alkaline phosphatase.

Alkaline Phosphatase↗