Biomedical subjects

Y Usson

Publications and source records attributed to Y Usson.

At least 55 records · Page 3Linked to original sources

PubMed1995

Application of confocal laser microscopy and three-dimensional Voronoi diagrams for volume and surface estimates of interphase chromosomes.

This study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomal in situ suppression hybridization with chromosome-specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three-dimensional (3-D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were extracted by applying the Delaunay graph, the dual of the Voronoi diagram, which describes the neighbourhood in the Voronoi diagram. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3-D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X- and 7-chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X-territories but not between chromosome 7-territories.

Amniotic Fluid↗

PubMed1994

Internalization of the alpha 5 beta 1 integrin does not depend on "NPXY" signals.

The alpha 5 beta 1 integrin is a constitutively internalized fibronectin receptor. It contains in the cytoplasmic tail of its beta 1 subunit two NPXY sequences which have been proposed to mediate internalization. Indeed a NPXY motif constitutes the internalization signal for the Low Density Lipoprotein (LDL) and insulin receptors. To learn more about the putative role of the two NPXY sequences in internalization of the alpha 5 beta 1 receptor, we have made and expressed mutants of the human beta 1 subunit in Chinese Hamster Ovary (CHO) cells, in which the two tyrosines of the NPXY motifs were replaced by serine residues. A cytoplasmic variant beta 1B which does not contain any NPXY sequence was also analyzed. Our results indicate that the NPXY mutants and the cytoplasmic variant are still internalized. Thus in the alpha 5 beta 1 receptor, the highly conserved NPXY sequences do not function as internalization motifs.

Amino Acid Sequence↗

PubMed1994

Method for the study of the three-dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy.

A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 microns inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.

Cell Nucleus↗

PubMed1994

Improvements in visualisation and localisation of human papillomavirus DNA in CaSki cells by fluorescence in situ hybridization, laser scanning confocal microscopy and three-dimensional image reconstruction.

The visual interpretation and localisation of specific DNA sequences in three dimensions in cell nuclei was investigated by fluorescence in situ hybridization (FISH) and laser scanning confocal microscopy (LSCM) using CaSki cells containing 600 copies per cell of human papillomavirus (HPV) DNA type 16 integrated in cellular DNA. Biotinylated DNA probes were used and DNA-DNA hybrids were revealed by a three-step reaction involving a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. The DNA from cell nuclei was counterstained with propidium iodide. With standard fluorescence microscopy, some dense fluorescent spots were seen in the cell nuclei. Similarly, with LSCM, some hybridization spots were observed in the cell nuclei but they were at different levels of the nuclei as shown by successive nuclear sections taken along the z axis. The visualisation of multiple hybridization spots confirmed the presence of multiple integration sites of HPV 16 DNA in CaSki cells. Association of LSCM with three-dimensional reconstructions lead to spatial images of hybridization spots obtained by stacking (x,y) images from consecutive confocal planes. Rotation of the reconstructed cell nuclei around the y axis makes it possible to distinguish closely adjacent spots. The combination of these techniques improves the detection of hybridization spots and may be of interest to further determine whether the HPV DNA is episomal or integrated in infected cells.

DNA, Viral↗

PubMed1993

Adhesion of CHO cells to fibronectin is mediated by functionally and structurally distinct adhesion plaques.

We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endocytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progressively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cell-substratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.

Animals↗

PubMed1992

Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence.

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.

Algorithms↗

PubMed1992

Eukaryotic DNA replication is a topographically ordered process.

This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd-labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature.

Bromodeoxyuridine↗

PubMed1992

Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.

Autoantigens↗

PubMed1991

A morphometric evaluation of the effects of trichloroethylene and dichloroacetylene on the rat mental nerve. Preliminary results.

Morphometric analysis was used to compare the effects of trichloroethylene (Tri) and dichloroacetylene (Dca) on the fibre parameters of the trigeminal nerve. Treated animals were clearly separated from controls according to a discriminant analysis. Furthermore, in the class of nerve fibres defined by a clustering analysis and corresponding to the largest fibres, myelin thickness was significantly decreased in the Dca group, but less so in the Tri group. In the group of the smallest fibres however, the myelin thickness was significantly increased by the treatments, but especially by Tri. Such a variability in the effects of Tri has already been demonstrated. Mechanisms for this are quite unclear although demyelination could be involved as already suggested. Our results thus show the ability of Tri and Dca to alter nerve parameters but probably with different modes of action depending on the size of the fibre.

Acetylene↗

PubMed1991

Morphometry of human nerve biopsies by means of automated cytometry: assessment with reference to ultrastructural analysis.

The morphometric analysis of myelinated fibres is a tool for diagnosis of neuropathies and for assessing nerve regeneration after reconstructive surgery. In order to obtain reliable information, sampling techniques should be avoided and the measurement of all the fibres within a nerve fascicle requiring the use of automated cytometry is necessary. We have developed a programme on the SAMBA cytological image analyser for the automated measurement of myelinated fibres. Different techniques of segmentation were tested and adaptive grey level thresholding gave the more reliable result. Accuracy and reproducibility were tested using a set of five human superficial peroneal nerve biopsies that were previously analysed by means of semi-automatic ultrastructural morphometry. A maximum difference of 9% in the number of fibres counted was obtained when comparing the automatic and the semi-automatic methods. In all cases the histograms of the morphometrical variables (fibre and axon diameters, myelin sheath thickness) were found to be identical to the reference histograms obtained by the semi-automatic method.

Evaluation Studies as Topic↗

PubMed1990

An automatic method for bone histomorphometry: assessment with reference to usual static and dynamic parameters.

To perform a fast and reproducible analysis in bone histomorphometry, we developed an automatic method for calculating static and dynamic parameters. A color automatic image analyzer (SAMBA 200) was used to obtain the usual parameters of bone histomorphometry: bone volume (Cn-BV%TV), osteoid volume (Cn-OV%BV), and osteoid surface (Cn-OS%BS). A specialized algorithm was designed for calculation of the mineral apposition rate (MAR). Eroded surface (Cn-ES%BS) was read in a semiautomatic mode using a cursor. To validate this program, we input 30 samples from patients with bone disease (20 osteoporosis, 6 renal osteodystrophy, 2 osteomalacia, and 2 hyperparathyroidism) using manual and automatic modes. The results obtained showed a highly significant correlation with the usual manual method for all parameters: OS/BS, r = 0.93; OV/BV, r = 0.98; MAR, r = 0.90. With the automatic method, larger values were found for osteoid parameters and MAR and lower values for BV/TV. There were no statistical differences for OV/BV and MAR when compared to the reference manual method. This study establishes that automatic measurements of osteoid parameters and MAR can be performed by a fast analyzer with as good reproducibility and accuracy as the manual method.

Adolescent↗

PubMed1989

There is no simple adequate sampling scheme for estimating the myelinated fibre size distribution in human peripheral nerve: a statistical ultrastructural study.

Morphometric studies of peripheral nerves (PN) usually involve some sampling of the myelinated fibres (MF). In order to scrutinize the statistical properties of the sampling processes in common use and the reliability of the resulting estimates, a quantitative analysis of human superficial peroneal nerves from 8 different normal subjects was undertaken at the ultrastructural level, both in terms of MF spatial distribution and of their size distribution. This study used sampling rates involving more than 10% of the whole myelinated fibre population observed in each nerve fascicle. However, in nearly all the fascicles evaluated, the sampling fluctuations are so high that neither the number of axons nor their diameter distribution can be assessed with enough accuracy. A systematic study of the myelinated fibres shows that the spatial distribution of their size is not uniform. This marked heterogeneity in the MF size distribution imposes measurement of large enough samples (500 or 600 MFs usually represent about one-half or two-thirds of the whole MF population) in a way to secure a reliable enough estimate of the density and size distributions. However, the practical usefulness of sampling schemes requiring more than one-half of the whole MF population in a nerve fascicle, is questionable.

Adult↗

PubMed1989

Automated morphometric study of human peripheral nerves by image analysis.

In this paper we describe a program using the image analyzer SAMBA, which allows an automatic analysis of silver stained semithin nerve sections. The operator can interactively delimit the contour of the fascicle to be analysed by means of a digitizing tablet connected to the system which generates a mask of the region. Segmentation of the fibre images is conducted as a function of brightness threshold defined by the operator. Fibre clusters are automatically separated using morphological procedures like dilatation. Morphometric parameters such as the external and axonal diameters, myelin sheath thickness and circularity are measured. We are now testing this method on normal and pathological human superficial peroneal nerves. Preliminary results are promising and the development of adequate statistical analysis of morphometric data will provide us with a new tool for the diagnosis of peripheral neuropathies.

Humans↗

PubMed1989

Cellular insertion of primary and secondary myotubes in embryonic rat muscles.

Mammalian muscles develop from two populations of myotubes; primary myotubes appear first and are few in number; secondary myotubes appear later and form most of the muscle fibres. We have made an ultrastructural study to investigate how primary and secondary myotubes in embryonic rat muscles transmit tension during the period of their development. Primary myotubes extend from end to end of the muscle from the earliest times, and attach directly to the tendon. In contrast, newly formed secondary myotubes are short cells which insert solely into the primary myotubes by a series of complex interdigitating folds along which adhering junctions occur. As the secondary myotubes lengthen and mature, their insertion is progressively transferred from the primary myotube to the tendon proper. We suggest that this variable insertion of immature secondary myotubes, combined with complex patterns of innervation and electrical coupling in developing muscle, makes it difficult to predict the overall contribution of secondary myotubes to muscle tension development. This work extends other studies showing the unique relationship between a primary myotube and its associated secondary myotubes, indicating that these may constitute a developmental compartment.

Animals↗

PubMed1989

The origin of secondary myotubes in mammalian skeletal muscles: ultrastructural studies.

The distribution of secondary myotubes and undifferentiated mononucleated cells (presumed to be myoblasts) within foetal IVth lumbrical muscles of the rat was analyzed with serial section electron microscopy. In all myotube clusters for which the innervation zone was located, every secondary myotube overlapped the end-plate region of the primary myotube. No secondary myotubes were ever demonstrated to occur at a distance from the primary myotube innervation zone. This indicates that new secondary myotubes begin to form only in the innervation zone of the muscle. Some young secondary myotubes made direct contact with a nerve terminal, but we cannot say if this is true for all developing secondary myotubes. Myoblasts were not clustered near the innervation zone, but were uniformly distributed throughout the muscle. Myoblasts were frequently interposed between a primary and a secondary myotube, in equally close proximity to both cell membranes. We conclude that specificity in myoblast-myotube fusion does not depend on restrictions in the physical distribution of myoblasts within the muscle, and therefore must reflect more subtle mechanisms for intercellular recognition.

Animals↗

PubMed1988

Schwann cell proliferation in the sciatic nerve of hypothyroid chick embryos studied by autoradiography and image analysis.

The proliferation of Schwann cells in the sciatic nerve of chick was studied from day 11 to day 27 of development in control and thyroid-deficient embryos. Hypothyroidism was induced by tetramethylthiourea injection on days 8 and 19 of incubation. The parameters of the cell cycle were determined using autoradiographs (tritiated thymidine) and by image analysis of Feulgen-stained nuclear smears. The duration of the cell cycle was lengthened and the growth fraction was reduced in hypothyroid animals, at 11 and 15 days of incubation. At later stages (days 21 and 27), these parameters were not significantly different from the controls as if the sensitivity of Schwann cells to thyroid hormones was scheduled to occur during a limited period of development. The total number of axons was the same in control and hypothyroid animals suggesting that the slowing down of Schwann cell proliferation is not a consequence of neuronal cell death. The consequence of that slowing down is a delay in the isolation of promyelin axons and a reduction in the proportion of myelinated axons at all the stages studied.

Animals↗

PubMed1987

A method for automatic classification of large and small myelinated fibre populations in peripheral nerves.

The statistical analysis of morphometric data collected from biopsies of human superficial peroneal nerve is complicated by the heterogeneity of the population of myelinated fibres. In order to make separate statistical analyses of the subpopulations of large and small fibres we have developed a computer program (written in PASCAL) for their automatic separation. The method is based on a dynamic centres clustering algorithm and was applied to the multifactorial space defined by the principal component analysis of the morphometric variables: axonal diameter, myelin sheath thickness, circularity index and g-ratio. The classification technique was applied to measurements obtained from 5 control nerves, and to simulated data, and in each case it gave consistent Gaussian subpopulations with no need for the introduction of supplementary variables.

Algorithms↗

PubMed1987

Morphometric analysis of the cerebellar Purkinje cell in the developing normal and hypothyroid chick.

A morphometric analysis of Purkinje cells in the developing cerebellar cortex of the chick was performed in normal animals and embryos made hypothyroid by one or two spaced injections of tetramethylthiourea. Profiles of 162 Purkinje cells, from Golgi-Cox treated sections were analysed. Soma area, perimeter and circularity index, cumulative length of the dendrites and number of dendritic bifurcations were studied. The results showed significant differences between control and hypothyroid animals. There were no important differences between birds rendered transiently hypothyroid with a single injection and those made chronically hypothyroid with dual injections. This confirms that the Purkinje cell is very dependent on thyroid hormone especially during the early phases of its morphogenesis. The development of the Purkinje cell was the most affected process of cerebellar cortex maturation in the thyroid-deficient chick. The dendritic arborization was particularly hypoplastic. Moreover, a dynamic balance appeared to exist between the development of the dendritic arborization and that of the perikaryon.

Animals↗