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  • Articles  (851)
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  • 1990-1994  (851)
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  • 1
    ISSN: 1059-910X
    Keywords: Three-dimensional light microscopy ; Brain slices ; Neurobiology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large-and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented. © 1994 Wiley-Liss, Inc.
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  • 2
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    Microscopy Research and Technique 29 (1994), S. 279-289 
    ISSN: 1059-910X
    Keywords: Fluorescence microscopy ; Ca channels ; Pyramidal neurons ; CA1 region ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca2+ -sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole-cell patch-clamp electrodes. The combination of imaging and electrophysiology was essential for interpreting the changes in [Ca2+]i. We compared the increases in [Ca2+]i produced by either of two methods-direct depolarization of the cell via the somatic electrode or high-frequency stimulations of synaptic inputs. The increases in [Ca2+]i in the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within seconds in healthy cells. However, the spatial patterns of distal Ca2+ increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of [Ca2+]i transients. We isolated and observed what appeared to be a voltage-independent component of the synaptically mediated [Ca2+]i transients. This work demonstrates that the combination of neurophysiology and simultaneous confocal microscopy is well suited for visualizing and analyzing [Ca2+]i within neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]i changes to structural and functional modifications. © 1994 Wiley-Liss, Inc.
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  • 3
    ISSN: 1059-910X
    Keywords: Sinus afferent pathway ; SP interneurons ; Double immunocytochemistry ; Ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The ultrastructure of substance P-containing nerve terminals synapsing on catecholamine neurons in the rat commissural subnucleus of the nucleus tractus solitarii (NTScom) was studied using a double immunocytochemical labeling technique. Although there were numerous tyrosine hydroxylase-immunoreactive (TH-I) somata present, substance P immunoreactive (SP-I) cell bodies were only occasionally found in the NTScom. At the light microscopic level, many SP-I terminals were seen closely associated with TH-I dendrites and somata. At the electron microscopic level, SP-I terminals synapsing on TH-I structures were also readily encountered. SP-I terminals contained small, clear, and predominantly spherical vesicles (32 ± 4 nm diameter), as well as large dense-cored vesicles approximately 100 nm in diameter. Postsynaptic TH-I dendritic profiles of various calibers and somata were encountered. These postsynaptic TH-I structures often showed postsynaptic densities. The morphological features of the SP-TH synapses in the present study, that is, the size of synaptic vesicles and the presence of postsynaptic densities, are quite different from those of central carotid sinus afferent synapses reported in our previous study [Chen et al. (1992), J. Neurocytol., 21:137-147]. Therefore, most of the SP terminals of the SP-TH synapses in the NTScom appear not to originate from the carotid sinus afferents. SP-I second-order neurons of the carotid sinus afferent pathway [Chen et al. (1991), J. Auton. Nerv. Syst., 33:97-98] may be one of the possible sources of such terminals. © 1994 Wiley-Liss, Inc.
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  • 4
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    Microscopy Research and Technique 29 (1994), S. 310-318 
    ISSN: 1059-910X
    Keywords: Hippocampus ; Dendrites ; 3-D imaging ; Pyramidal cell ; Neurophysiology ; Confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Studies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recording were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio-Rad 600 confocal attachment on an Olympus BH-2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images.Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system. © 1994 Wiley-Liss, Inc.
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  • 5
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    Microscopy Research and Technique 29 (1994), S. 329-343 
    ISSN: 1059-910X
    Keywords: Sensory map ; Neural map ; Mechanosensory afferents ; Database ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We describe the development and analysis of a quantitative database representing the global structural and functional organization of an entire sensory map. The database was derived from measurements of anatomical characteristics of a statistical sample of typical mechanosensory afferents in the cricket cercal sensory system. Anatomical characteristics of the neurons were measured quantitatively in three dimensions using a computer reconstruction system. The reconstructions of all neurons were aligned and scaled to a common standard set of dimensions, according to a highly reproducible set of intrinsic fiducial marks. The database therefore preserves accurate information about spatial relationships between the neurons within the ensemble.Algorithms were implemented to allow the integration of electrophysiological data about the stimulus/response characteristics of the reconstructed neurons into the database. The algorithms essentially map a physiological function onto a “field” representing the continuous distribution of synaptic terminals throughout the neural structure. Subsequent analysis allowed quantitative predictions of several important functional characteristics of the sensory map that emerge from its global organization. First, quantitative and testable predictions were made about ensemble response patterns within the map. The predicted patterns are presented as graphical images, similar to images that might be observed with activity-dependent dyes in the real neural system. Second, the synaptic innervation patterns from the sensory afferent map onto the dendrites of a postsynaptic target interneuron were predicted by calculating the overlap between the interneuron's dendrites with the afferent map. By doing so, several aspects of the stimulus/response properties of the interneuron were accurately predicted. © 1994 Wiley-Liss, Inc.
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  • 6
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    Microscopy Research and Technique 29 (1994), S. 344-349 
    ISSN: 1059-910X
    Keywords: Epithelium ; Eye ; Hyaluronate ; Microscopy ; Rabbit ; Regeneration ; Retina ; Sodium iodate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The distribution of hyaluronate (HA) in regenerating retinal pigment epithelium (RPE) of the rabbit was examined using immunohistochemistry and confocal laser scanning microscopy. The goal was to determine if there is a correlation between differentiation and HA expression, like that seen in developing tissues, where HA accumulates and then disappears as the tissue matures. In normal RPE cells HA is associated mainly with the apical surface. In regenerating RPE (produced by i.v. injection of sodium iodate to damage the epithelium, regeneration arising from spared cells), HA exhibits a patchy distribution among the more immature cells and is especially prominent where they overlap or pile up on each other. Where cells are more mature and form a compact monolayer of cells, HA is expressed mainly on the apical surface, as in normal RPE. The accumulation of HA among the more immature cells in the regenerating epithelial sheet supports the hypothesis that HA influences differentiation by suppressing cell-cell associations until the proper time for their formation. © 1994 Wiley-Liss, Inc.
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  • 7
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    Microscopy Research and Technique 29 (1994), S. C1 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 8
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    Microscopy Research and Technique 27 (1994), S. 125-133 
    ISSN: 1059-910X
    Keywords: Follicle cell ; Cumulus-oocyte-complex ; Transzonal processes ; Tubulin ; Actin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Electron and fluorescence microscopic techniques have been used in a complementary fashion to study the patterns of follicle cell-oocyte interactions within cumulus-oocyte-complexes of various mammals. The principal findings are: (1) two distinct types of transzonal processes exist that are distinguishable on the basis of cytoskeletal composition; (2) in some of the species examined (pig, goat, primate), corkscrew-shaped processes rich in tubulin, traverse the zona pellucida and are invaginated into the oocyte cortex; (3) actin-rich processes either ramify as a network at the outer surface of the zona pellucida or penetrate the zona and make contact with the oolemma in a species specific manner. These results are discussed with respect both to the need to employ complementary optical methods in assessing connectivity patterns within COC and to the possible role that extracellular matrix-cell interactions play in the homeostatic control of oocyte growth and maturation. © 1994 Wiley-Liss, Inc.
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  • 9
    ISSN: 1059-910X
    Keywords: Brain mitochondria ; Microtubules ; Neurofilaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The surface distribution of several proteins (porin, hexokinase, and two proteins associated with microtubules or actin filaments) on the outer membrane of brain mitochondria was analyzed by immunogold labelling of purified mitochondria in vitro. The results suggest the existence of specialized domains for the distribution of porin in the outer mitochondrial membrane. Similarities between the distribution of porin and the distribution of microtubule-associated proteins bound in vitro to mitochondria suggested that mitochondria and microtubules interact by binding microtubule-associated proteins to porin-containing domains of the outer membrane. This hypothesis was supported by biochemical studies on outer mitochondrial proteins involved in in vitro binding of cytoskeleton elements. In vitro interactions between mitochondria and microtubules or neurofilaments were analyzed by electron microscopy. These studies revealed cross-bridging between the outer membrane of mitochondria and the two cytoskeleton elements. Cross-bridging was influenced by ATP hydrolysis and by several proteins associated with the surface of mitochondria or with microtubules. In addition, unidentified proteins which were recognized by antibodies to all intermediate filaments subunits were associated either with the mitochondrial surface or with microtubules. This data suggest the participation of additional cytoplasmic proteins in the interactions between cytoskeleton elements and mitochondria. © 1994 Wiley-Liss, Inc.
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  • 10
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    Microscopy Research and Technique 27 (1994), S. 220-232 
    ISSN: 1059-910X
    Keywords: Mitochondrial DNA ; Mitochondrial nuclear division ; Mitochondriokinesis ; Physarum polycephalum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Our present understanding of mitochondrial division can be summarized as follows:Mitochondria contain a specific genome, synthesize their own DNA, and multiply semi-autonomously. Strands of mitochondrial DNA (mt-DNA) in the in vivo organelles of all eukaryotes are organized to form mitochondrial nuclei (nucleoids) (mt-nuclei) with specific proteins including a histone-like protein and transcription factors at the central region of the mitochondrion. We can easily observe the mt-nucleus in vivo mitochondria in various organisms such as fungi, algae, plants, and animals by using high-resolution epifluorescence microscopy. Therefore, the process of mitochondrial division can be clearly separated into two main events: division of the mt-nuclei and mitochondriokinesis analogous to cytokinesis.Mitochondria undergo binary division which is accompained by the division of the mt-nucleus. A remarkable characteristic of mitochondrial multiplication during the mitochondrial life cycle is that mitochondria can multiply the mt-chromosome by endoduplication until 50-100 copies are present. Mitochondria can then divide without mitochondrial DNA synthesis to eventually contain 1-5 copies of the mt-chromosome. This characteristic phenomenon can be observed during cell differentiation, such as during the formation of plasmodia and sclerotia of Physarum polycephalum and during embryogenesis and the formation of meristematic tissues in plants.The mitochondrial chromosome has a mitochondrial “kinetochore (centromere)” which is A-T rich and contains specific sequences such as topoisomerase binding sites, tandem repeats, and inverted repeats. A bridge of proteins may exist between the kinetochore DNA and membrane systems. Mitochondrial chromosomes can divide according to the growth of a membrane system between the kinetochores.Mitochondriokinesis progresses steadily along with mitochondrial nuclear division. As the membrane at the equatorial region of a mitochondrion contracts, the neck of the cleavage furrow narrows, and eventually the daughter mitochondria are separated. An actin-like protein may power mitochondriokinesis by separating the daughter mitochondria. In general, mitochondriokinesis occurs by contraction rather than by partition of the inner membrane. © 1994 Wiley-Liss, Inc.
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  • 11
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    Microscopy Research and Technique 27 (1994) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 12
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 13
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    Microscopy Research and Technique 27 (1994), S. 294-306 
    ISSN: 1059-910X
    Keywords: Cryo-electron microscopy ; Image analysis ; ATPase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The 1F0 ATP synthase is the large multisubunit complex which uses the proton gradient of energetically active membranes to synthesize ATP. While biochemical and genetic approaches have characterized the composition of the enzyme and elucidated many details of its mechanism and assembly, electron microscopy has been the tool of primary importance in determining the arrangement of the many subunits which comprise the F1F0. The highly cooperative catalytic mechanism is tightly coupled to transmembrane proton translocation in a separate and rather distant sector of the complex. An understanding of this intricate process and its control requires an appreciation of subunit interactions, starting with their locations relative to one another. Electron microscopy has provided most of the available structural information on the F1F0, and recent applications of cryo-electron microscopy have captured different functionally relevant configurations which may finally address longstanding questions about subunit rearrangement during the catalytic cycle. © 1994 Wiley-Liss, Inc.
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  • 14
    ISSN: 1059-910X
    Keywords: Cristae ; 3D structure ; Hepatocytes ; Fibroblasts ; Adrenal cortex ; Brown fat ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Rat adrenal cortex was processed for high resolution scanning electron microscopy (HRSEM) to confirm tubular cristae, reported by transmission electron microscopy to be present in cortex mitochondria. Mitochondria in several other tissue and cell types were also observed and their ultrastructure confirmed by using three-dimensional, stereo, high resolution scanning electron microscopy. The mitochondria in rat and human hepatocytes as well as human skin fibroblasts mitochondria proved to be long, up to 46 micrometers and branching, as compared to those in liver which were spherical in shape. Cold adapted brown fat cells were packed with mitochondria, these containing plate or shelf-like cristae. Branched, rat striated muscle mitochondria were observed to curve around contractile protein filament bundles. The muscle mitochondrial cristae were found to be both tubular and plate-like, within the same mitochondrion. The ratio of tubular cristae to plate-like cristae varied considerably between muscle mitochondria. In order to use ultrastructural changes in mitochondria for differential diagnosis, and because 3D reconstruction of mitochondria based on transmission electron microscopy serial sections is severely limited in resolution, it is imperative to first develop a correct understanding of tissue specific, normal mitochondrial ultrastructure based on three-dimensional, HRSEM methods. © 1994 Wiley-Liss, Inc.
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  • 15
    ISSN: 1059-910X
    Keywords: Matrix ; Membrane ; SEM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The three-dimensional organization of the internal compartments of conventionally fixed and embedded rat-liver mitochondria has been determined by tomographic reconstruction from tilt-series images collected on the Albany high-voltage electron microscope. The results indicate that the inner membranes of these organelles are predominantly tubular in the orthodox (expanded matrix) conformation, as previously suggested by scanning electron microscopy. In the condensed (contracted matrix) conformation, the intracristal space opens up into large irregularly shaped compartments which are connected to each other and to the external (intermembrane) space by tubes with approximately the same diameter (20 nm) as those observed in the orthodox state. These results raise several questions, in particular about the nature of the structural transitions that occur in the cristae during matrix expansion and contraction, and about the influence of inner-membrane shape on the diffusion of ions and metabolites between the intracristal and intermembrane compartments. © 1994 Wiley-Liss, Inc.
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  • 16
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    Microscopy Research and Technique 27 (1994), S. 284-293 
    ISSN: 1059-910X
    Keywords: Mitochondrion ; Contact sites ; Protein translocation ; Ribosomes ; Import intermediates ; Receptor proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established.The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites.In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles.Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites.Based on these ultrastructral data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites. © 1994 Wiley-Liss, Inc.
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  • 17
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    Microscopy Research and Technique 27 (1994), S. 350-354 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 18
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    Microscopy Research and Technique 28 (1994), S. 79-79 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 19
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    Microscopy Research and Technique 28 (1994), S. 141-148 
    ISSN: 1059-910X
    Keywords: X-ray microanalysis ; Respiratory epithelium ; Secretory cells ; Cryofixation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In respiratory epithelium, the mucus is densely packed inside the secretory granules (SG) of secretory cells (SC) before being released by exocytosis in the airway lumen. We have previously shown that the frog palate is a representative model of respiratory epithelium and that rapid cryofixation is a very effective technique in preserving the integrity of the mucus SG. The concentration of phosphorus (P), sulphur (S), and calcium (Ca) were analysed inside the SG of the SC of frog palate after quick freezing, cryosubstitution, and embedding in Lowicryl resin at low temperature. The experiments were carried out using X-ray microanalysis conducted with energy dispersive spectrometry (EDS) at 100 kV. The quantitation was carried out using the continuum method with reference to Agar standards. The cryofixation permitted us to distinguish two types of SG depending on whether they were electron dense (serous cells) or electron-lucent (mucous cells). A significant (P 〈 0.001) difference in the S concentration was observed between the individual serous (239 ± 79 mmol.kg-1) and the mucous SG (161 ± 48 mmol.kg-1). No significant difference could be identified in the Ca concentration between the two SG phenotypes. In the serous SG, the P content was high (41 ± 17 mmol.kg-1) compared with the mucous SG where it was not measurable. The comparison of the three element concentrations in each type of secretory cells showed that significant differences in concentration of S and Ca concentration could be observed from one SC cell to another. A significant correlation (r = 0.76, P 〈 0.01) was observed between the S concentration and the topographical position of the SG inside the SC, the more proximal to the lumen, the higher the S concentration, suggesting that the maturation of the SG involves an increase in the protein content possibly due to a maturation process before the mucus exocytosis. Therefore, these results suggest that the elemental composition of granules varies according to the phenotype of the secretory cells and that changes in the S content from one SG to another or even inside the same cell may reflect a differential state in the functional activity of the secretory cells. © 1994 Wiley-Liss, Inc.
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  • 20
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    Microscopy Research and Technique 27 (1994), S. 359-359 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 21
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    Microscopy Research and Technique 27 (1994), S. 360-375 
    ISSN: 1059-910X
    Keywords: Biological composites ; Structural biocomposites ; Microarchitecture ; Materials design ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Biomimetics is a newly emerging interdisciplinary field in materials science and engineering and biology in which lesson learned from biology form the basis for novel technological materials. It involves investigation of both structures and physical functions of biological composites of engineering interest with the goal of designing and synthesizing new and improved materials. This paper discusses microarchitectural aspects of some structural biocomposites, presents microstructural criteria for future materials design and processing, and identifies areas of future research. © 1994 Wiley-Liss, Inc.
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  • 22
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    Microscopy Research and Technique 27 (1994), S. 376-388 
    ISSN: 1059-910X
    Keywords: Biomineralization ; Crystal nucleation ; Crystal growth ; Crystal engineering ; Biomimetic chemistry ; Molecular recognition ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Effective protocols for controlling crystal structure, size, and morphology attract considerable interest given the requirement for particles of modal size and shape in many areas of materials fabrication and the importance of crystallochemical selectivity in determining the exploitable properties of inorganic solids. For this reason biomineralization merits particular attention since many biominerals are deposited in a highly controlled manner to produce crystals which are uniformly sized and crystallographically unique. Studies of biominerals have revealed that while a complex array of strategies have evolved for regulating their formation, one feature is common to the biological paradigm; interactions between organized biopolymeric assemblies and the nascent inorganic solids play a pivotal role in controlling the crystallization process. In order to gain a better understanding of the molecular interactions which take place at organic-inorganic interface and address the fundamental chemical problems of biomineralization, a crystal chemical approach has been adopted. Organized organic assemblies (phospholipid vesicles, Langmuir monolayers, polypetide templates) of precise molecular design (head group identity, packing conformation, peptide sequence, etc.) were assayed for their effectiveness in controlling the nucleation and growth of inorganic solids. This work has established that through systematic changes in the nature of the organic matrix the size, crystallographic orientation, and growth of the mineral phase can be controlled. Critical to this process was the translation of specific molecular information at the organic-inorganic interface: epitaxial alignment, stereochemical complementarity, and electrostatic interactions were an essential feature of this recognition event. © 1994 Wiley-Liss, Inc.
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  • 23
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    Microscopy Research and Technique 28 (1994), S. 263-276 
    ISSN: 1059-910X
    Keywords: Phagocytosis ; Mac 1 ; Arthritis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The mechanism of human neutrophil clearance of peptidoglycan group A-specific polysaccharide polymers derived from streptococcal cell walls (PG-APS) was investigated by high voltage immunoelectron microscopy (HVEM) in order to determine how neutrophils process this highly inflammatory bacterial debris. Neutrophil monolayers were incubated from 5-30 min with serum-opsonized PG-APS. Cells were lightly fixed with 0.5% glutaraldehyde, and the PG-APS was localized on the neutrophil surface by immunogold using antibodies to N-acetyl-glucosamine and 15 nm colloidal gold coupled to goat anti-rabbit IgG. Neutrophils were viewed unsectioned by stereo HVEM. Patches of PG-APS were distributed randomly on the plasmalemma of well-spread neutrophils within 5 min. In polarized cells, PG-APS was densely localized on the uropod and retraction fibers. Within 15 min, PG-APS was predominantly concentrated into a large aggregate, measuring approximately 1 μm in diameter, near the cell margin or nucleus. The aggregate of PG-APS was engulfed in the vicinity of the indentation of the nucleus (hof). Intact microfilaments were required for aggregation and internalization of PG-APS. Binding of PG-APS was dependent upon complement fixation. Furthermore, PG-APS elicited an increase in density of complement receptor type 3 (CR3, C3bi receptor) on the neutrophil surface as determined by morphometry of immunogold labeled anti-CR3. When cells were stained for both PG-APS and CR3, co-localization was observed, and stereomicroscopy revealed clusters of CR3 in areas associated with phagocytosis. These data suggest that neutrophils use an efficient mechanism for removal of bacterial debris. Unlike whole streptococci which are phagocytosed at multiple sites, these bacterial cell walls are first collected into a large aggregate, or cap, which is then internalized at one site. © 1994 Wiley-Liss, Inc.
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  • 24
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    Microscopy Research and Technique 28 (1994), S. 308-326 
    ISSN: 1059-910X
    Keywords: Glycoproteins ; Spread cells ; GPIIb-IIIa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Exposure of blood platelets to foreign surfaces results in dramatic changes in physical appearance and conversion from a non-sticky to an adhesive state. Membrane glycoproteins and cytoskeletal assembly play a pivotal role in these interactions. Cytochemical techniques commonly applied for demonstration of macromolecules in tissues have been used for the localization of target glycoproteins on spread cells. The present review examines different experimental strategies and immunocytochemical techniques that can be combined to better understand the organization of platelet receptors during surface activation. Glycoprotein IIb-IIIa (GPIIb-IIIa) was localized by immunocytochemical techniques on fixed, surface-activated platelets. The distribution of functional fibrinogen receptors expressed on GPIIb-IIIa was revealed by incubation of fixed platelets with fibrinogen-gold conjugates (Fgn/Au). The movement of receptor complexes was investigated in additional experiments in which surface-activated platelets were interacted with Fgn/Au and then fixed at different periods. The overall impression of these observations suggests that fibrinogen receptors on surface-activated platelets do not redistribute spontaneously and that particulates (gold particles), rather than fibrinogen, may trigger the movement. These results are presented in detail and their significance discussed in the light of current theory. Applications and limitations of such techniques are also discussed. © 1994 Wiley-Liss, Inc.
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  • 25
    ISSN: 1059-910X
    Keywords: Lymphoma ; Splenomegaly ; SEM ; TEM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Peripheral blood mononuclear cells (PBLs) from 14 patients with low grade non-Hodgkin's B-cell lymphomas with predominant splenomegaly were studied by means of scanning (SEM) and transmission electron microscopy (TEM). All patients had peripheral blood and bone marrow involvement, the absence of lymphoadenopathy, and, except in one case, immunophenotypic features of a malignant proliferation of mature spleen B-cells arising from outside the germinal center, but not consistent with CLL or HCL. Several distinctive cytological features were observed in PBLs of the different subgroups. The SEM surface features of PBLs in patients with intermediate differentiation lymphocytic lymphoma (IDL) (five cases), lymphoplasmacytoid immunocytoma (LP-IC) (two cases), and mixed small and large cells malignant lymphoma (one case) were characterized by the presence of numerous well-developed microvilli. Some distinctive TEM ultrastructural features were also seen in the different cases. In the two cases of splenic lymphoma with villous lymphocytes (SLVL), SEM revealed large and elongated surface microvilli generally arising from two or three poles of the cells. This surface morphology, confirmed by TEM analysis, may be pathognomonic of this disease. Four additional cases, tentatively classified as small lymphocytic lymphoma on the basis of immunophenotypic data, were extremely heterogeneous at both SEM and TEM analysis. The ultrastructural features revealed by SEM and TEM may be useful for the more precise characterization of this heterogenous group of diseases, which is generally difficult to define even when immunophenotypic and molecular approaches are used. © 1994 Wiley-Liss, Inc.
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  • 26
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    Microscopy Research and Technique 29 (1994), S. 161-168 
    ISSN: 1059-910X
    Keywords: Heart ; Catecholamine fluorescence ; NADPH diaphorase ; NO synthase ; Nitric oxide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In spite of accumulating evidence for a modulation of sympathetic neurotransmission by endogenously produced nitric oxide (NO), it remains unclear in which parts of the vascular system and at what level this interaction takes place. The aim of the present study was to investigate the distribution of endothelial and neuronal NO synthase (NOS) along the vascular tree of the heart at the light and electron microscopic level using NADPH-diaphorase (NADPH-d) staining as a marker for NOS. In addition, the functional effects of exogenous NO on coronary vascular resistance and cardiac adrenergic nerves was studied using the isolated perfused rat heart as a model. The intraaxonal catecholamine content of adrenergic nerve fibers was visualised and morphometrically assessed by applying glyoxylic acid-induced histofluorescence. The expression of endothelial NOS in the heart was found to depend on the diameter of the blood vessel. Arteries 〉100 μm always showed intense staining, whereas staining in smaller arteries and veins was considerably weaker. Smooth-muscle free vessels were essentially devoid of NADPH-d activity. In atrial and ventricular myocardium, neuronal NOS localised in autonomic nerve fibers along the entire vascular tree. Ultrastructurally, NADPH-d staining revealed adjacent localisation of NOS-positive and -negative axons, suggesting an interaxonal modulation of adjacent autonomic nerve fibers by NO. In isolated perfused rat hearts, the intracoronary application of 10-8 M NO produced a marked decrease of coronary perfusion pressure, which was accompanied by a distinct increase in intraaxonal catecholamine levels of intramural adrenergic nerve fibers. These results suggest that the entire vascular system from arteries to veins is under the influence of NO and implies that two independently operating NO-driven processes are involved in the modulation of blood vessel tone: the well-known pathway of endothelium-derived NO acting directly on smooth muscle, and a second indirect pathway that inhibits noradrenaline release from perivascular nerve endings by endothelially or neuronally produced NO. The uneven distribution of endothelial NOS furthermore suggests that the latter mechanism predominates when the size of the blood vessel decreases. © 1994 Wiley-Liss, Inc.
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  • 27
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    Microscopy Research and Technique 29 (1994), S. 169-176 
    ISSN: 1059-910X
    Keywords: Celiac ganglion ; Chromaffin cells ; Autonomic nervous system ; Ultrastructure ; Guinea pig ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Utilizing electron microscopic observation, several contacts between small, granule-containing cells (SGC) and postganglionic neurons (PGN) in the celiac ganglion of the guinea pig have been observed. A SGC in very close association with a PGN was seen to receive a distinct synaptic contact that contained many vesicles with dense cores. This contact was morphologically unlike cholinergic synapses previously reported on chromaffin cells. Because the SGC and PGN were clearly separated by a thin rim of satellite cell cytoplasm mutual to both cells, it is not known how or if the SGC would possibly exert a synaptic or paracrine effect on the PGN. Also, intraganglion SGC existed as large well-vascularized islands within the celiac ganglion. These intraganlion clusters sometimes contained more than 50 cells and perhaps could be considered to function as localized neuroendocrine components within the ganglion by secreting granule products into the nearby blood vessels for local or distant effects, although this certainly is not known. This work reports a unique synaptic ending upon a single-occurring SGC, which, in turn, closely approximates a ganglion neuron in a soma-somatic relationship. In addition, a very close association (but no actual contact) was observed between granule-containing processes, presumably emanating from the intraganglion clusters, and PGN. Whatever the function of ganglionic SGC may be, the exact relationship between SGC and PGN presumably would be of great interest and potential importance. © 1994 Wiley-Liss, Inc.
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  • 28
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    Microscopy Research and Technique 28 (1994), S. 398-408 
    ISSN: 1059-910X
    Keywords: Aging ; Proteoglycans ; Electron microscopy ; Intervertebral disc ; Hyaline cartilage ; Nucleus pulposus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules. © 1994 Wiley-Liss, Inc.
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  • 29
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    Microscopy Research and Technique 28 (1994), S. 427-429 
    ISSN: 1059-910X
    Keywords: STEM ; Z-contrast microscopy ; Impregnated metal ; Catalysts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: High-angle annular dark-field or Z-contrast microscopy was used to demonstrate that well dispersed metal supported catalysts consist of nanometer sized clusters. Depending upon the impregnated metal, different cluster sizes were observed. Grouping of Pd clusters could also be confirmed by analytical electron microscopy. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 448-451 
    ISSN: 1059-910X
    Keywords: Angular relationship ; Tilting ; Electron microscopy ; Goniometer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A simple formula has been derived for the tilt angle of a specimen in terms of the two tilt angles of a side entry, double tilt holder in a transmission electron microscope. An expression for calculating the direction of the apparent tilt axis in relation to the observed diffraction pattern has also been derived. The accuracy and reproducibility of specimen tilting has been assessed experimentally. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 32
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    Microscopy Research and Technique 28 (1994), S. 455-469 
    ISSN: 1059-910X
    Keywords: Chondrogenesis ; Chondroblasts ; Cartilage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The morphology and fine structure of day 12 rat embryonic mesenchyme from forelimb bud, mandibular arches, and frontonasal prominence is described as the cells undergo chondrogenesis in high density, micromass culture. The cultures began as a multilayered “pavement” of flattened mesenchymal cells, 3-4 deep, with moderate intercellular space but little identifiable electron-dense extracellular matrix. Pre-cartilage condensations, which consisted of aggregates of cells which had rounded up, displaying little or no intercellular space, formed within the first 24 h in limb mesenchyme and after an additional 24 h in mandibular and frontonasal cultures. Gap junctions occur between these cells, indicating a phase of direct cell-cell communication. Chondrogenesis within these aggregates began within the next 24 h in limb cultures but was delayed an additional 24-48 h in the frontonasal and especially in mandibular cultures. The aggregates in both limb and mandibular mesenchyme form discrete nodules bordered by a perichondrium consisting of 2-3 layers of flattened cells. Evidence from late stage mandibular cultures suggests that chondroblasts are added to the nodules from the perichondrium, as occurs in vivo. By contrast, the frontonasal cartilage is initially unbordered and forms anastomosing trabecular arrays. Some of these arrays fuse into larger structures with time, but others become surrounded by proceeds. The sequence of cartilage matrix production, as revealed in long-term facial cultures, appears to occur in three stages, an early phase in which the extracellular matrix consists primarily of proteoglycans, followed by a phase of homogeneous collagen-proteoglycan matrix and a mature, territorial matrix. In all three cultures the cartilage ultimately produced resembles mature rat hyaline cartilage with chondroblasts surrounded by a territorial matrix of type II collagen and proteoglycan granules. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 492-504 
    ISSN: 1059-910X
    Keywords: Calcification ; Hydroxyapatite ; Matrix synthesis ; FT-IR microscopy ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5′-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM β-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells.In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is present. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 35
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    Microscopy Research and Technique 28 (1994), S. 527-534 
    ISSN: 1059-910X
    Keywords: EBIC, a-Si:H ; Generation function ; Electron beam ; Electron range ; Energy loss ; Diode ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The electron beam induced current technique was used to study electron energy loss in amorphous hydrogenated silicon a-Si:H. This study leads to the determination of the electron generation function which is needed when using the variable energy electron beam induced current technique (EBIC) analysis of a-Si:H device. A series of identical n-i-p a-Si:H diodes with a thin aluminium top electodes were fabricated and varying thicknesses of a-Si:H layer were deposited on it. In EBIC measurements, the n-i-p diode was reverse biased at maximum potential. The electron range of a-Si:H was determined directly by measuring the energy at which the electron beam is completely stopped in the top layer and no carrier generation is possible in the n-i-p diode. The generation function is then deduced from EBIC contrast measurements between the aluminium electrode and the top a-Si:H layer. © 1994 Wiley-Liss, Inc.
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  • 36
    ISSN: 1059-910X
    Keywords: Cryofixation ; Electron microscopy ; Extracellular material ; Microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Conventional fixation of the delicate, highly folded rat ciliary body and its iridial extension, as well as of vitreal structures, is associated with the induction of a number of artifacts, thus limiting the reliability of morphological interpretations. Improved ultrastructural preservation may be achieved by microwave heating in combination with osmium tetroxide fixation. This protocol, although simple and cheap, yields results, particularly with respect to the extracellular matrix compartment between inner and outer ciliary epithelial cells, which are not greatly inferior to those obtained by implementing the sophisticated high pressure freezing and freeze substitution technique. The latter affords good to very good ultrastructural preservation of epithelium and stromal components, such as blood vessels, neural elements, smooth muscle cells, fibrocytes, and free cells, up to a depth of 50-100 μm from the tissue surface. Its superiority over osmium tetroxide/microwave fixation is revealed in the cytoplasmic, intraorganellar, and vitreal matrix compartments, which incur no obvious losses. © 1994 Wiley-Liss, Inc.
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  • 37
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    Microscopy Research and Technique 29 (1994), S. 37-46 
    ISSN: 1059-910X
    Keywords: Tritrichomonas foetus ; Freeze-fracture ; Electron microscopy ; Fast-freezing fixation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Tritrichomonas foetus was studied using different physical and chemical fixation methods such as fast-freezing (by high pressure, “slam-freezing,” and jet-propane), freeze-substitution, conventional freeze-fracture and deep-etching, cryoultramicrotomy, and routine preparation for transmission electron microscopy. The use of fast-freezing fixation (FFF) proved to be superior in terms of structural preservation due to the rapidity of this fixation compared to that obtained using conventional chemical fixation. The low temperature techniques used here were useful to confirm data already obtained by conventional freeze-fracture using chemical fixation and cryoprotection, such as the presence of flagellar rosettes and costa structure. Cryoultramicrotomy and slam-freezing also demonstrated the presence of hair-like structures projecting out from the protozoan surface. New aspects of organelles of T. foetus were demonstrated. Published 1994 by Wiley-Liss, Inc.
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  • 38
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    Microscopy Research and Technique 27 (1994), S. 71-79 
    ISSN: 1059-910X
    Keywords: Parotid gland ; Submandibular gland ; Labial gland ; Sodium ; Potassium ; Calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Electron probe X-ray microanalysis (EPXMA) has now been successfully applied to several salivary gland preparations. This paper briefly reviews the principles underlying this technique and the specific sample preparation procedures which permit accurate measurement of elemental concentrations in the various intracellular spaces. Findings from salivary gland studies indicate that cytoplasmic and nuclear spaces of nonstimulated acinar cells have high concentrations of K and P, and low concentrations of Mg, Ca, and S; and that mature secretory granules have high concentrations of Ca and S, and relatively low concentrations of K and P. No consistent differences have been found between the elemental concentrations of mucous and serous secretory granules. In vivo and in vitro EPXMA studies of the elemental changes associated with secretory granule maturation indicate there are at least two stages in this process: an early stage during which granule S concentration increases in parallel with mass density as condensing vacuoles mature into secretory granules, and a late stage during which granule mass density and protein content increase with no further elemental concentration changes. Findings from other in vivo and in vitro studies indicate that secretory granule membranes are permeable to Na, K, and Cl ions because the granular concentrations of these elements are altered by electrochemical gradients. Recent EPXMA results indicate that cells stimulated with parasympathomimetic agonists have decreased K and Cl concentrations, and increased Na concentrations. Furthermore, the magnitude of these changes are quantitatively consistent with changes measured using radio-isotope equilibration and other techniques. In contrast, cells stimulated with the β-adrenergic agonist, isoproterenol, have increased concentrations of Na and Cl, but unchanged K concentrations. In terms of pathology, Na concentration is found to be elevated in secretory granules from labial salivary glands of patients with cystic fibrosis, but the pathophysiological consequences of this change are not known. Thus, studies utilizing EPXMA have obtained detailed and unique information about physiological and pathophysiological changes in elemental concentrations of intracellular organelles in salivary gland cells. © 1994 Wiley-Liss. Inc.
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  • 39
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    Microscopy Research and Technique 27 (1994), S. 355-357 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Topics: Natural Sciences in General
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  • 40
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    Microscopy Research and Technique 29 (1994), S. 103-111 
    ISSN: 1059-910X
    Keywords: Acetylcholinesterase ; NADHd ; NADPHd ; NOSynthase ; Enteric nervous system ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Using acetylcholinesterase (AChE), nicotinamide adenine dinucleotide diaphorase (NADHd), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) enzyme histochemical techniques, the ganglionated plexuses of the porcine enteric nervous system were investigated in small intestine whole-mount preparations. Both AchE and NADHd techniques revealed a majority of the neurons in the ganglia of all three major plexuses. The AchE technique also demonstrated clearly the axodendritic networks of the plexus myentericus. Intraganglionic blank areas revealed the localization of negative cell groups. A very high correlation was found between the activity of both enzymes in one neuron, although this correlation was certainly not linear. Many neurons exhibited a stronger signal for one enzyme. A very small part of the positive nerve cells showed intense staining for both AchE and NADHd. The NADPHd technique demonstrated that the NADPHd-positive neurons fill the negative intraganglionic spaces in the ganglia. Double staining with the two other enzymes showed virtually no colocalization of NADPHd with either NADHd or AchE in the porcine jejunal enteric ganglia. Little negative intraganglionic spaces were seldom found, leaving room for perhaps still more negative enteric neurons. Based upon these results we suggest that the enteric neurons of the porcine small intestine can be subdivided into AchE-NADHd and NADPHd subpopulations. Since the latter colocalizes with the neuronal NO synthase enzyme, we further suggest a subdivision of the enteric nerve cells into AchE-NADHd and NOS-NADHd neurons. © 1994 Wiley-Liss, Inc.
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  • 41
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    Microscopy Research and Technique 29 (1994), S. 120-130 
    ISSN: 1059-910X
    Keywords: Galanin ; Vasoactive intestinal polypeptide ; Tyrosine hydroxylase ; Nerve regeneration ; Immunohistochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 μm, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 μm), whereas VIP occurs mainly in larger neurons (26.1 μm). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 216-225 
    ISSN: 1059-910X
    Keywords: Type IV collagen ; Laminin ; Immunohistochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In this paper, the use of immunohistochemistry for the analysis of basement membrane components and related extracellular matrix proteins in human cancer is reviewed. Basement membranes in cancer are dynamic structures that are constantly degraded but also deposited, in close collaboration between tumor cells and stromal cells. Basement membrane immunohistochemistry, using antibodies against type IV collagen and laminin, appears to be a useful tool in the analysis of lesions on the borderline between premalignant and malignant. Basement membrane interruptions, however, cannot be used as the only criterion for the diagnosis of malignancy. Type VII collagen is often degraded prior to type IV collagen and laminin in early invasion. This protein also tends to be expressed in carcinomas when it is not found in the corresponding normal tissue. Tenascin seems to play a complex role in the development of human tumors, including promotion of cell growth and differentiation, cell migration during invasion, and tissue remodeling during the development of primary and metastatic lesions. Further systemic exploration of extracellular matrix molecules in neoplasms should yield new information relevant for cancer biologists and useful in cancer diagnosis. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 193-203 
    ISSN: 1059-910X
    Keywords: Extracellular matrix ; Radial glia ; Glia limitans ; Pial-glial barrier ; Wound repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Although remarkable progress has been made during the last two decades concerning the biosynthesis, expression and assembly of extracellular matrix (ECM) macromolecules in nonneural cells, we are still far from a complete understanding of the role and function of the ECM and the basement membrane (BM) in the central nervous system (CNS). With the aid of correlative light and electron microscopic, Golgi and immunohistochemical studies of the developing neural tube of both early human fetus and mouse, we have shown that the establishment of the pial-glial barrier (PGB) is one of the earliest histogenetic events in neurogenesis. This is accomplished by coordinated interaction among the processes of radial glia, various ECM components, and mesenchymal cells at the pial surface, with the formation of a BM that tightly abuts the glia limitans. The PGB and the BM appear to be critical to the migration and final positioning of neurons and to the differentiation of the laminar cortical pattern within the developing neopallium. This hypothesis is further supported by our study of the brain of a human newborn infant in whom multiple sites of disruption of the BM and PGB resulted in abnormal neuronal migration and massive ectopia of neurons within the subarachnoid space, with abnormal cortical lamination. Finally, studies of the experimental cryogenic injury to the neonatal rat cerebrum have shown that the final positioning of neurons within the developing cortical plate appears to depend largely on the reconstitution of the BM and PGB, which presumably provide crucial positional signals for migrating neurons. Also, one of the essential reparative features seen following cryogenic injury to the adult rat cerebrum is an orderly and dynamic interaction between various ECM components and neural cells, resulting in the formation of the BM. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 204-215 
    ISSN: 1059-910X
    Keywords: Heparan sulfate proteoglycan ; Laminin ; Collagen ; Amyloid P component ; β-Amyloid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Alzheimer's disease (AD) is one of several systemic and cerebral diseases that involve the abnormal deposition of fibrillar proteins called amyloids. All amyloids share conformational and staining characteristics, as well as an association with resident tissue macrophages and two extracellular matrix components [heparan sulfate proteoglycan (HSPG) and amyloid P component]. Vascular, glomerular, and Schwann cell basement membrane pathologies have been documented in many forms of amyloidosis, and often amyloid fibrils fuse to and project from the basement membrane in these diseases. The present report demonstrates the vascular basement membrane (VBM) alterations in AD autopsy samples, and details the methodologies used.Electron microscopy reveals the fusion of amyloid fibrils with the VBM and the alteration of the VBM in the absence of amyloid accumulation. Double-labelling and pre-embed immuno-electron microscopy techniques demonstrate the colocalization of amyloid P component and VBM components with amyloid, and also reveal that amyloid P component is not localized to the cerebral VBM. Finally, a novel correlative light/electron microscopy technique demonstrates the association between amyloid P component and cerebral resident tissue macrophages, the microglia. Taken together, these data suggest that the physicochemical processes of amyloid formation, rather than amyloid deposition, may be responsible for VBM pathology. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 254-258 
    ISSN: 1059-910X
    Keywords: X-ray microanalysis ; Freeze-substitution ; Diffusible ions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A comparative study of some aspects of freeze-substitution as a preparative method for X-ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X-ray microanalysis of thin sections of mouse pancreas were used. Freeze-substitution for 3 weeks in diethy ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze-substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze-substitution in acetone resulted in noticeable loss and redistribution of ions and freeze-substitution in methanol gave very poor results. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 243-253 
    ISSN: 1059-910X
    Keywords: Senescence ; Lipofuscin ; Secretion and content of saliva ; Protein synthesis ; Amylase mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Various salivary glands in senescent humans and other animals have been examined extensively to characterize the structural and functional changes that occur during aging. Although a wide range of different structural changes, involving both the parenchymal and stromal tissues, have been described, it is unclear how any of these changes affects the function of the salivary glands. One major change in structure is the reduction in the volume of acini with a concomitant increase in the ductal volume. Despite this loss of functional acini, the salivary output and the contents seem to be unaltered, or minimally altered, due to aging. One consistent change observed in many salivary glands of aged animals is the decline in the rate of synthesis of proteins and their messenger RNA (mRNA). However, the salivary acinar cells from aged animals can synthesize secretory proteins at an elevated rate just as effectively as those from their younger counterparts in response to external stimuli, which are known to enhance the rate of protein synthesis. Thus, it appears that the salivary acinar cells, which remain structurally intact during aging, seem to retain their functional efficiency. Furthermore, these acinar cells, although reduced in number, are sufficient in quantity to carry out most of the salivary gland functions. © 1994 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Microscopy Research and Technique 27 (1994), S. 420-428 
    ISSN: 1059-910X
    Keywords: Self-assemblies ; Chitin ; Collagen ; Polarizing microscopy ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Microscopic studies at different scales have shown that in biological tissues the three-dimensional arrangement of chitin-protein or of collagen fibrillar networks can follow the same spatial distributions as those described in certain liquid crystals. The present work reviews the structural analogies established between the dense fibrillar organic matrix found in two materials: crab cuticles and compact bones. In both systems mobile fringes are described in polarizing microscopy, periodic cleavage aspects in scanning electron microscopy (SEM), and arced patterns in transmission electron microscopy (TEM). In parallel to these structural data, results obtained in vitro are recalled which corroborate the relationship established between ordered arrays of biopolymers and liquid crystalline assembly principles, highly concentrated solutions of purified collagen molecules spontaneously form ordered assemblies characterized in polarizing microscopy as cholesteric phases. This particular state of matter joins both fluidity and order and could correspond to a transient state of collagen or chitin secretion, before the stiffening of these skeletal structures, bone or cuticle, by molecular cross-links and crystalline deposition. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994), S. 469-469 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994), S. 451-458 
    ISSN: 1059-910X
    Keywords: Lipid monolayers ; Lipid bilayers ; Electron diffraction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Lipid monolayer is a half leaflet model for lipid bilayer, which forms the basis of biological membranes. Within a certain range of surface area per molecule of phospholipid monolayers at the air-water interface, where the compressibility was nearly infinite, two phases with different molecular packings were observable by fluorscence microscopy. Mixed-phase monolayers of L-1,2-dipalmitoyl-N-monomethyl-3-phosphatidylethanolamine [DP(Me)PE] or L-1,2-dipalmitoyl-N-dimethyl-3-phosphatidyl-ethanolamine [DP(Me)2PE] were deposited on marker grids coated with Formvar films. The molecular organization in the dark and bright fluorescent areas on the grids was investigated by low dose, selected area electron diffraction. Sharp reflection arcs, at a spacing of 4.2Å and arranged in a hexagon pattern, were detected from dark domains of both lipids. A diffuse reflection ring at a spacing of 4.6Å was derived from the bright background areas. Diffraction patterns were obtained from neighboring areas along selected dark domains of both lipids. The orientations of diffraction patterns from areas along smooth and curving boundries of DP(Me)2PE domains were found to turn with the boundaries. In the branching domains of DP(Me)PE, the orientations of diffraction patterns indicated that the branches were formed by twinning. Electron diffraction thus provides an unique way to sample the local molecular packing order and orientation within individual domains in phospholipid monolayers. © 1994 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Microscopy Research and Technique 29 (1994), S. 368-380 
    ISSN: 1059-910X
    Keywords: EM tomography ; Dendritic spines ; Axonal arborizations ; Reconstruction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: High voltage electron microscopy (HVEM) and HVEM tomography of selectively stained cell processes in the neostriatum have offered an alternative to serial thin section reconstruction for accurate 3-D visualization and measurement of axons, dendrites, and dendritic spines. Tissue preparation is simple and rapid, allowing examination of large numbers of specimens required for quantitation of neuronal morphology. The resolution of the images exceeds that available from any light microscopic technique and is appropriate for measurement of the finest axons and dendritic spine necks. HVEM tomography allows the direct measurement of dendritic surface area, required for computational modeling of synaptic integration. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 357-367 
    ISSN: 1059-910X
    Keywords: Neuron ; Astrocyte ; High voltage electron microscopy ; Three dimensional ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Neurons and glia cells in the mammalian central nervous system have many complicated processes. They are too fine for light microscopic study and too complicated and widely spread for thin section electron microscopy. High-voltage electron microscopic (HVEM) stereo observation of thick Golgi preparation provides detailed 3-D images of their processes. Three-dimensional fine structures of astocytic processes in the neuropile and on the surface of neuronal somata, and those of the ruffed cell axon initial segment and thorny excrescences of CA3 pyramidal cell dendrites, are elucidated with the aid of HVEM stereoscopy of thick Golgi preparations. In addition, some results obtained by 3-D morphometrical analysis of dendritic spines using HVEM stereo images are shown.The examples presented here clearly show the usefulness of high-voltage electron microscope stereo observation of thick specimens for detailed morphological and morphometric study of the central nervous system. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 507-515 
    ISSN: 1059-910X
    Keywords: Cerebral endothelial cells ; Intracellular signaling ; Messenger molecules ; Brain oedema ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Recent advances in our knowledge of the blood-brain barrier have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro blood-brain barrier model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained mainly from this in vitro approach. Different elements of the intracellular signaling messenger systems have been detected in the course of our studies in the cerebral endothelial cells. It has been shown that the synthesizing enzymes of and substrate proteins for the second messenger molecules are present in the cerebral endothelial cells, and their activity and/or amount can change in pathological circumstances, i.e., during the formation of brain oedema. Pharmacological treatments interfering with the second messenger systems proved to be effective in the prevention of brain oedema formation. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 516-527 
    ISSN: 1059-910X
    Keywords: Glucose transporter ; Interendothelial junctions ; Mitochondrial density ; Endothelial vesicles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This paper describes a quantitative approach to evaluating the ultrastructural features of brain capillaries that relate to the low non-specific permeability of the blood-brain barrier (BBB). Critical features in this approach include examination of large numbers of tissue samples and consistent, objective means of measuring features of interest. Junctional clefts, i.e., continuous channels between tight junctional regions correlate well with the know vascular permeability, being low in normal adult blood-brain barrier, high in fetal brain, and high in tumours, both human and rat. Endothelial vesicles do not always correlate with vascular permeability. They have a low density in normal adult BBB, but are also low in fetal BBB and low in some intracranial tumour vessels. However, they have a high density in muscle capillaries, and others have shown that they increase in BBB vessels damaged by hypertension. Fenestrations are consistently high in leaky vessels, but not all leaky vessels have fenestrations. The density of mitochondria in endothelial cells is high in BBB vessels of some species but not in others. Glut-1, the glucose transporter of the BBB is asymmetrically distributed between the luminal and abluminal membranes of BBB capillaries, being almost four times as numerous on the abluminal face. A large intracellular pool of glucose transporter may provide a means for rapid upregulation of the surface transporters. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 452-453 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 28 (1994), S. 470-482 
    ISSN: 1059-910X
    Keywords: Cartilage ; Type II collagen ; Aggrecan ; Chondrogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Cartilage is an important tissue in skeletogenesis, in the growth of long bones, and as a flexible component of the mature skeleton. The extracellular matrix proteins type II collagen and aggrecan comprise 90% of the matrix and are characteristic of cartilage. Type II collagen provides structural integrity to the tissue, while aggrecan confers resiliency. The quantity of type II procollagen is controlled at the level of transcription of mRNA from the COL2A1 gene. In addition, type II procollagen can be expressed in two isoforms by differential splicing of the primary gene transcript, a post-transcript, a post-transcriptional control mechanism. The two mRNAs either include exon 2 (type IIA) or exclude exon 2 (type IIB) which encodes the major portion of the amino (NH2)-propeptide [Ryan and Sandell (1990), J. Biol. Chem., 265:10334-10339]. The aggrecan gene also encodes alternative splice forms that may be developmentally expressed. The regulation of aggrecan splicing or transcription has not been studied in detail. To determine the spatial and temporal patterns of expression of extracellular matrix in the development of cartilage, we have examined the expression of type II collagen and aggrecan during chondrogenesis in the vertebral column and during elongation of a newborn growth plate. Our results indicate that there is a developmental sequence of type II collagen splice form expression during chondrogenesis with type IIA expressed in prechondrocytes and type IIB expressed in chondrocytes. During elongation of the growth plate, mature chondrocytes express type IIB procollagen and then differentiate into hypertrophic chondrocytes and initiate expression of type X collagen. In all cases, aggrecan was coordinately expressed with type IIB procollagen. As cartilage-like proteins have been observed in more primitive structures such as notochord, the expression of type II collagen mRNAs was also examined in the notochordal remnants of the vertebral column. In the notochord, the predominant collagen expressed was the type IIA collagen prechondrocyte isoform. Notochordal cells also expressed mRNAs more characteristic of fibroblasts such as versican and decorin: low expression of type I collagen, type IIB collagen, and aggrecan were observed. © 1994 Wiley-Liss, IncThis Article is a US Government work and, as such, is in the public domain in the United States of America..
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    Microscopy Research and Technique 28 (1994), S. 483-491 
    ISSN: 1059-910X
    Keywords: Cartilage cells ; Bone formation ; Ostenectin ; Osteopontin ; Matrix vesicles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin. A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of ostenection (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17-18 sternum (US cells) and grown in monolayer in standard serum-containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen-positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3-week-old cultures with RA (10-100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA-treated cultures. Electron microscopy confirmed the formation of large matrix-associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells). In these cells, RA failed to induce high levels of APase activity, ON and OP gene expression, and mineralization; instead, it greatly promoted cell proliferation. Thus RA appears to have major, stage-specific effects on the maturation program of chondrocytes. The retinoid rapidly induces expression of late maturation genes and activates mineralization of the cartilage matrix. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 505-519 
    ISSN: 1059-910X
    Keywords: Hypertrophy ; IGF-I ; Growth hormone ; Thyroxine ; Growth factors ; Cartilage ; Proteoglycans ; Collagens ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Growth plate chondrocytes play a pivotal role in promoting longitudinal bone growth. The current review represents a brief survey of the phenomena involved in this process at the cellular level; it delineates the contributions made by various activities during the course of the chondrocyte life cycle, notably proliferation and hypertrophy, and illustrates how the relative contributions may be modulated according to the particular needs of an organism at critical phases of growth. The cellular mechanisms by which a few well characterized growth-promoting substances exert their influences are discussed in the light of recent findings pertaining to epiphyseal plate chondrocytes in vivo. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 520-526 
    ISSN: 1059-910X
    Keywords: Cartilage ; Human chondrocyte ; Proteoglycans ; Agarose culture ; In vitro studies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Iliac crest biopsies are important in the detection of human skeletal dysplasias. Therefore, culture of these cells may serve as a valuable method for studying proteoglycan metabolism in chondrocytes of individuals with skeletal abnormalities. Morphological and biochemical studies were performed on human iliac crest chondrocytes grown in monolayer and in agarose gels. Two proteoglycan populations of different hydrodynamic size and glycosaminoglycan composition were synthesized by cells grown in monolayer. Chondrocytes cultured in an agarose gel for 2 weeks synthesized proteoglycans identical to those of the native tissue with respect to hydrodynamic size and glycosaminoglycan chain length. However, the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate was higher than in the native tissue. This ratio was not influenced by different sulfate concentrations in the medium. Moreover, treatment with ascorbic acid did not influence proteoglycan synthesis; however, there was a pericellular accumulation of proteoglycans. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 1-10 
    ISSN: 1059-910X
    Keywords: Aldehyde/UA fixation ; Preservation of cytoskeleton ; Nuclear skeleton ; Perinuclear shell ; Immunogold and lectin-gold labelling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Conventional treatment of tissues for sectioning and transmission electron microscopy uses aldehyde fixation and osmium tetroxide postfixation. Although the result is aesthetically pleasing, osmication destroys some cell components and reduces the chemical activity of others, such as reactions with antibodies and lectins. We have found that aldehyde fixation followed by uranyl acetate preserves and contrasts most structures and visualizes some that are not easily seen after osmication. Aldehyde/UA treated tissues have enough contrast to be observed without section staining while retaining some of the chemical activity that is lost through osmication. Sections of tissues with good preservation and contrast can be used for immunogold and lectin-gold labelling of at least some molecules. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 23-28 
    ISSN: 1059-910X
    Keywords: Morphology ; Immunolabeling ; Negative contrast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Ultra-thin frozen sections are ideal substrates for immunolabeling in high resolution electron microscopy. However, visualization of subcellular structures is inferior to that obtained with corresponding plastic sections. Although negative staining is generally effective and even superior to positive staining, the accumulated stain is often too heavy, obscuring morphology and markers used for immunocytochemical localization of antigens. This paper describes the development of a modified negative contrast staining technique in which a high concentration of uranyl acetate is mixed with methyl cellulose at a low pH. Application of this stain to cryosections of cells and tissue resulted in improved visualization of morphological structures characterized by negative images of membranes and cell organelles. Use of this stain is advantageous for morphological and immunocytochemical studies involving ultra-thin frozen sections. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 29-36 
    ISSN: 1059-910X
    Keywords: Enamel ; Ameloblast ; Maturation ; Alkaline phosphatase ; Smooth ended ameloblast ; Ruffle-ended ameloblast ; Digital backscattered electron imaging ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The different functional conditions of pre-ameloblasts, secretory ameloblasts, and maturation ameloblasts in 9 day rat incisors were recognized using high resolution light microscopic alkaline phosphatase histochemistry: Digital backscattered electron imaging was performed using the block surfaces from which thin sections were taken for histochemical study. It was possible to correlate exact locations in histochemical sections with positions in the block face at all stages of enamel mineralization from early secretion through late maturation. The first steep increase in the rate of mineralization of completed enamel matrix occurs after the first transition from smooth ended ameloblasts to ruffle ended ameloblasts. In the 9 day rat incisors used for this purpose, there are only two smooth to ruffle cyclical transitions, and the width of successional smooth ended bands of ameloblasts in the maturation cycling process is always narrow. Nevertheless, there seems to be a good correlation between mineralization increase and the acquisition of the high alkaline phosphatase activity in the deeply enfolded distal cytoplasm of the ruffle-ended maturation stage ameloblasts. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 47-53 
    ISSN: 1059-910X
    Keywords: Scanning electron microscope ; Dislocations ; Monte Carlo simulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A simple method for the fabrication of a Au/p-Si Schottky barrier suitable for electron beam induced current (EBIC) study has been developed. The mechanical and electrical properties of the fabricated Au/p-Si Schottky barriers have been tested, and EBIC measurements of the dislocation contrast have been conducted using the fabricated Schottky barriers. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 54-61 
    ISSN: 1059-910X
    Keywords: Nuclear envelope ; Nuclear pore complex ; Percoll purification ; Field emission scanning electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 62-68 
    ISSN: 1059-910X
    Keywords: Three-dimensional organization ; Potassium ethoxide ; Extracellular matrix ; Aorta ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A new method is described which enables the visualization of hidden cellular surfaces of biological tissues. The method does not depend on the kind of fixative solution, the duration of the fixation, or the quantity of extracellular matrix. The samples are chemically dissociated with potassium ethoxide until the first individual cells appear in the solution. This initial process is followed by phase contrast microscopy, and remaining tissue is investigated with scanning electron microscopy. The method has proved more effective than conventional enzymatic techniques and is a simple, quick, and effective way to investigate the three-dimensional organization of different normal and pathological tissues. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 29 (1994), S. 262-265 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 29 (1994), S. 267-267 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 68
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    Microscopy Research and Technique 29 (1994), S. C1 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 69
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    Microscopy Research and Technique 29 (1994), S. 290-296 
    ISSN: 1059-910X
    Keywords: Neural plasticity ; Image processing ; Deconvolution ; Three-dimensional reconstruction ; Volume rendering ; Fluorescent tracers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi-like staining of small numbers of cells and processes that can be resolved clearly using confocal microscoopy; viability of stained cells is established by exclusion of the fluorescent DNA-binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high-resolution images are combined in an automated procedure to generate three-dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 97-107 
    ISSN: 1059-910X
    Keywords: Cellular interaction ; Granulosa cells ; Theca cells ; Morphology ; Proliferation ; Ovarian function ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The term paracrine control has recently become very fashionable in the field of endocrinology: however, no study has yet clarified directly the role of paracrine activity in the functional and morphological development of endocrine organs. We developed a culture system in which two types of cells were allowed to attach to opposite sides of a collagen membrane in order to observe cellular communication directly and used it to explore the role of granulosa and theca cell interactions in ovarian structure and function.In the first series of the study, we explored the interaction between theca and granulosa cells by investigating the morphology, proliferation, and steroidogenesis of the cells. Granulosa cells cultured alone were flattened and polygonal in shape and formed a monolayer sheet. Granulosa cells cocultured with theca cells formed multilayer sheets. The apical surface of each cell appeared convex. Numerous filopodia spread over the cellular surfaces to connect individual cells. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cocultured with granulosa cells were also spindle-shaped; however, the apical surface appeared convex. The cell numbers of both granulosa and theca cells in the cocultured group increased approximately twofold compared to control cells cultured alone. The progesterone producing activity of granulosa cells in the cocultured group was reduced to 40% of that of cells in the control group. In contrast, the androstenedione producing activity of theca cells in the cocultured group increased approximately threefold compared to that of control group cells.In the second series of experiments, effects of cellular communication on cellular response to gonadotropins were investigated. When granulosa cells were cultured with theca cells, FSH treatment (1 μg/ml) promoted cellular growth: however, LH treatment (1 μg/ml) suppressed cellular growth and augmented their progesterone production. In contrast, such effects of gonadotropins were not detected when granulosa cells were cultured alone. LH induced estradiol production by granulosa cells both cultured alone and cocultured with theca cells: however, FSH-induced estradiol production was not detected in this experimental condition. LH treatment increased androstenedione production of theca cells cocultured with granulosa cells, but no increase was observed for theca cells cultured alone.These results demonstrate that communication between these two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function and that the actions of gonadotropins on target cells in the follicular wall can also be modified by the communication between these cells. In contrast to granulosa and theca cells cultured alone, cells in coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Therefore, we speculate that interactions between these two types of follicular cells is essential in the maintenance of original structure and function of the bovine follicular wall. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 194-195 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994), S. 165-193 
    ISSN: 1059-910X
    Keywords: Cryopreservation ; Mammalian oocyte ; Cytogenetics ; Fertilization ; Embryogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This study examined the effects of cryopreservation on cellular organization, chromosomal complement, and developmental potential of immature and mature mouse and human oocytes. Chromosomal analyses were performed by DNA fluorescence microscopy and karyotyping on the same metaphase II-stage oocytes before and after freezing. Cellular analyses involved electron microscopy, time-lapse video recording, and fluorescent-probe microscopy of cortical granules. The findings demonstrate that while profound cytoplasmic, nuclear, and nucleolar alterations occur in the immature oocyte during cryopreservation, an apparently normal nucleus and cytoplasm is re-established progressively after thawing and culture. The resulting oocytes mature at high frequency and for the mouse, are fertilizable and capable of normal preimplantation of embryogenesis. Cryopreservation of mature mouse and human oocytes is not accompanied by a significant increase in the frequency of aneuploidy. However, cryopreserved human oocytes, while fertilizable, arrest development during the early cleavage stages and display aberrant patterns of cytokinesis. The possible etiologies of developmental failure in the human embryo that may be related to oocyte cryopreservation, as well as the potential benefits of cryopreservation of the immature oocyte, are discussed with respect to clinical and commercial applications. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 197-197 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994), S. 198-219 
    ISSN: 1059-910X
    Keywords: Mitochondria ; Organelles ; Microtubules ; Fluorochromes ; Fission ; Fusion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Mitochondria are semi-autonomous organelles which are endowed with the ability to change their shape (e.g., by elongation, shortening, branching, buckling, swelling) and their location inside a living cell. In addition they may fuse or divide. These dynamics are discussed. Dislocation of mitochondria may result from their interaction with elements of the cytoskeleton, with microtubules in particular, and from processes intrinsic to the mitochondria themselves. Morphological criteria and differences in the fate of some mitochondria argue for the presence of more than one mitochondrial population in some animal cells. Whether these reflect genetic differences remains obscure. Emphasis is laid on the methods for visualizing mitochondria in cells and following their behaviour. Fluorescence methods provide unique possibilities because of their high resolving power and because some of the mitochondria-specific fluorochromes can be used to reveal the membrane potential. Fusion and fission often occur in short time intervals within the same group of mitochondria. At sites of fusion of two mitochondria material of the inner membrane, the matrix compartment seems to accumulate. The original arrangement of the fusion partners is maintained for some minutes. Fission is a dynamic event which, like fusion, in most cases observed in vertebrate cell cultures is not a straightforward process but rather requires several “trials” until the division finally occurs. Regarding fusion and fission hitherto unpublished phase contrast micrographs, and electron micrographs have been included. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 262-267 
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    Keywords: Thick films ; Low-angle ion milling ; Differential sputtering rate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A new preparation method permits the production of large-area, electron-transparent, transmission electron microscopy (TEM) specimens in cross section of free-standing, thick, multilayered structures. Such production often has been difficult in the past because of large chemical differences between the component layers in the multilayer. This difference usually results in a large difference in thinning rates between the layers. A unique combination of electroplating, lapping, dimpling, and low-angle ion milling is a successful and reproducible technique for producing high-quality TEM specimens of these complex materials. Procedures and results presented here are for a 304 stainless-steel/copper multilayer having a repeat period of 20 nm and a total thickness of 20 μm. © 1994 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Microscopy Research and Technique 27 (1994), S. 61-70 
    ISSN: 1059-910X
    Keywords: Salivary gland ; Diabetes ; Insulin ; Electron microscopy ; Streptozotocin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Effects of experimental diabetes on rat submandibular glands have been documented, but earlier reports suggested that diabetes caused an extensive cellular degeneration and a replacement of the parenchymal cells by fibrous connective tissue. Such observations, however, are difficult to reconcile with the relatively normal physiological responsiveness of the gland (Anderson and Suleiman, 1989). This study, therefore, reexamined the histological, histochemical and ultrastructural effects of streptozotocin-induced diabetes on rat submandibular glands. The tissues were examined at 3 weeks, and 3 and 6 months after the induction of diabetes, and compared with glands from age-matched controls by both light and electron microscopy. Light microscopically, the proportional volumes of the acini and granular ducts remained constant in control rats at about 48% and 38% respectively. In diabetic animals the volume density of the acini increased progressively to 62%, whereas that of the granular ducts decreased to 20%. The diameter and number of granular ducts were reduced in diabetic animals, but acinar cell profile area was only affected 6 months after the induction of diabetes. Ultrastructurally, there was an accumulation of lipid in the acinar cells and, with increasing duration of diabetes, the number of autophagic structures in both the acini and the granular ducts increased. Although there was evidence of some cellular degeneration it was never excessive. Morphometry showed that the volume density of secretory granules within the acinar cells was unaffected, but there was a significant reduction in the volume density of secretory granules within the granular ducts. Thus, in the rat submandibular gland the greatest effect of streptozotocin-induced diabetes was to cause hypotrophic changes in the cells of the granular ducts. The relative contributions of a direct effect of insulin insufficiency and the hypogonadal effects of diabetes, however, are not known. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 81-82 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 27 (1994), S. 108-124 
    ISSN: 1059-910X
    Keywords: Cytoskeleton ; Steroidogenesis ; Mitochondrial development ; Gap junctions ; Haras Oncogene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Granulosa cells, which nurse the oocyte and serve as a major source for estradiol and progesterone production, undergo major morphological changes which correlate very well with modulation of their steroidogenic capacity. These include changes in intercellular contacts and communication, in cell membrane receptors, and in the development and organization of organelles associated with steroidogenesis (i.e., mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes). These biochemical and morphological changes can also be obtained in primary cultures as well as in oncogene transformed granulosa cell lines established recently in our laboratory. A growing body of evidence suggests that plasticity of the cytoskeleton plays a major role in the biochemical and morphological differentiation of granulosa cells as well as in other steroidogenic cells. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 27 (1994), S. 83-96 
    ISSN: 1059-910X
    Keywords: Growth factor ; Inhibin ; Microscopy ; Immunohistochemistry ; In situ hybridization ; Morphology ; Ovary ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Follicular development in the mammalian ovary is a complex process regulated by an orchestrated action of the pituitary gonadotropins, e.g., follicle stimulating hormone (FSH) and luteinizing hormone (LH), and local ovarian factors, such as peptide growth factors and steroids. The mechanism of endocrine/paracrine/autocrine regulation of folliculogenesis (i.e., cell proliferation and functions) has been addressed largely by biochemical means. However, the availability of immunological and molecular tools now enables us to undertake critical microscopic studies revealing the ovarian cell-type specific synthesis and/or accumulation of many of these local peptide modulators, their roles in the proliferation and differentiation of follicular cells, and their regulation by gonadotropins and local factors. The objective of this review is to provide a comprehensive yet complete picture of the endocrine/autocrine regulation of mammalian folliculogenesis as revealed by microscopic studies. Efforts have been made to include adequate research information relevant to update our understanding of the process of follicular development; however, to maintain the brevity, many equally important studies could not be included. This review confirms that FSH and LH are still the primary stimuli for follicular development. However, it is clear that the actions of these hormones at the cell level involve a host of peptide factors which are produced locally by different follicular cell types and which are powerful modulators of gonadotropin actions. The temporal and spatial expression of the genes of these modulators, the synthesis of active factors, their interactions, and the dynamics of their receptors on the follicular cell surface may be the ultimate determinants of cellular events which are crucial to coordinated growth and differentiation of follicular cells leading to folliculogenesis and ovulation. © 1994 Wiley-Liss, Inc.
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  • 81
    ISSN: 1059-910X
    Keywords: Rat ; Albumin ; Fibrinogen ; Immunohistochemistry ; Electromagnetic fields ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Biological effects of electromagnetic fields (EMF) on the blood-brain barrier (BBB) can be studied in sensitive and specific models. In a previous investigation of the permeability of the blood-brain barrier after exposure to the various EMF-components of proton magnetic resonance imaging (MRI), we found that the exposure to MRI induced leakage of Evans Blue labeled proteins normally not passing the BBB of rats [Salford et al. (1992), in: Resonance Phenomena in Biology, Oxford University Press, pp. 87-91].In the present investigation we exposed male and female Fischer 344 rats in a transverse electromagnetic transmission line chamber to microwaves of 915 MHz as continuous wave (CW) and pulse-modulated with repetition rates of 8, 16, 50, and 200 s-1. The specific energy absorption rate (SAR) varied between 0.016 and 5 W/kg.The rats were not anesthetized during the 2-hour exposure. All animals were sacrificed by perfusion-fixation of the brains under chloral hydrate anesthesia about 1 hour after the exposure. The brains were perfused with saline for 3-4 minutes, and thereafter fixed in 4% formaldehyde for 5-6 minutes. Central coronal sections of the brains were dehydrated and embedded in paraffin and sectioned at 5 μm. Albumin and fibrinogen were demonstrated immunohistochemically.The results show albumin leakage in 5 of 62 of the controls and in 56 of 184 of the animals exposed to 915 MHz microwaves. Continuous wave resulted in 14 positive findings of 35, which differ significantly from the controls (P = 0.002). With pulsed 915 MHz microwaves with repetition rates of 200, 50, 16, and 8 s-1, 42 of 149 were positive, which is highly significant at the P = 0.001 level. This reveals that both CW and pulsed 915 MHz microwaves have the potential to open up the BBB for albumin passage. However, there is no significant difference between continuous and pulsed 915 MHz microwaves in this respect.The frequency of occurrence of extravasates (26%) was found to be independent of SAR for SAR 〈 2.5 W/kg, but rose significantly for the higher SAR values (to 43%).The question of whether the opening of the blood-brain barrier constitutes a health hazard demands further investigation. © 1994 Wiley-Liss, Inc.
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  • 82
    ISSN: 1059-910X
    Keywords: Bioelectromagnetics ; Static field effects ; rf bioeffects ; ELF bioeffects ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We have previously reported that exposure of rats to low-field (0.15 T) magnetic resonance imaging (MRI) increases blood-brain barrier (BBB) permeability. However, a number of investigators have failed to observe this effect when high-field MRI (1.5 T or higher) is used. Therefore, we investigated whether or not we would observe changes using our technique at these higher fields.Adult male Sprague-Dawley rats were anaesthetised and then exposed to a 22.5 min imaging or sham procedure. Immediately following exposure, rats were injected with 1 MBq of 153Gd-DTPA intracardially and then immediately re-exposed for an additional 22.5 min. The rats were killed 1h following the second MRI exposure, at which time the brain was resected and 3ml of venous blood collected. The ratio of radioactivity per gram of brain to radioactivity per per milliliter of blood, known as the brain-blood partition coefficient, was determined and used as a measure of BBB permeability. Groups of animals had different exposures. Group 1 (n = 9) was exposed to a clinically relevant MRI procedure. Group 2 (n = 20) was exposed to the same procedure except the rf specific absorption rate (SAR) was reduced to 25% and the animals were positioned 15 cm from imager centre to increase the time-varying magnetic field from 0.4 to 2.8 T/s. For the sham exposures (n = 21), the animals were placed in the imager with the static field ramped down to zero and exposed to a sound recording simulating a MRI examination. In a third group (n = 11), exposure to only the static 1.5 T was investigated, while in a fourth group (n = 15), the exposure field was only to a static 1.89 T. Sham controls for groups 3 (n = 12) and 4 (n = 19) were silent.Group 1 showed an increase in BBB permeability (5.7 ± 0.5, mean ± SEM, P 〈 0.04, n = 9); however, group 2 showed a decrease (3.9 ± 0.2, P 〈 0.002, n = 20). Static field exposure of 1.5 T increased permeability (5.7 ± 0.5, P 〈 0.05, n = 11) as did 1.89 T (4.7 ± 0.4, P 〈 0.02, n = 15). Although there was no difference between sound and silent shams, shams done at night (4.8 ± 0.2, n = 32) were significantly greater (P 〈 0.0004) than those done during the day (3.7 ± 0.2, n = 19). Our results suggest changes in the rf and/or gradient field exposure can produce opposite effects on BBB permeability. Further, experiments involving only rf and only gradient field exposure are needed.The estimation of changes in BBB permeability by measuring the partition coefficient of compounds normally excluded by the BBB offers an objective quantitative technique which can detect changes of the order of 25%. This sensitivity is needed to explore the important, albeit subtle, effects of magnetic fields. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 28 (1994), S. 1-1 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 28 (1994), S. 48-59 
    ISSN: 1059-910X
    Keywords: Lamina lucida ; Basement membrane ; Lamina densa ; Pars fibroreticularis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In tissues prepared with chemical fixation followed by conventional dehydration, basement membranes have been observed to be laminated structures composed of a lamina lucida and lamina densa as well as a poorly limited transitional zone referred to as the pars fibroreticularis. Scattered attempts in the application of new techniques of tissue preparation such as cryofixation or freeze substitution for the study of the basement membrane structure have been made in recent years. From these studies, the possibility has arisen in which basement membranes are composed of only the lamina densa without a lamina lucida. In recent studies in this laboratory, the attempt was made to determine whether or not this lamina lucida is an artefact, and if so, which step in the conventional method of tissue preparation is responsible for its formation. Basement membranes from diverse sources in the mouse and rat including the testis, ductus epididymis, eye, thyroid, kidney, and skin, were observed after either cryofixation by slam freezing followed by freeze substitution, or aldehyde fixation followed by freeze substitution. The basement membranes after preservation with either of these two methods were composed of only the lamina densa with no lamina lucida. It indicates that an artefactual formation of the lamina lucida occurs during dehydration in conventional tissue preparation rather than during chemical fixation. In view of the well known superiority of freeze substitution over conventional dehydration, the lamina lucida of the basement membrane is likely to be an artefact. Therefore, it is concluded that the lamina lucida is an artefact formed during conventional tissue preparation, and in its original condition in the living state, the basement membrane is composed of a single layer made up of lamina densa material. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 368-369 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 28 (1994), S. 356-367 
    ISSN: 1059-910X
    Keywords: Hairy cell leukemia ; Immunogold labelling ; B-ly7 antibody ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Two cases of hairy cell leukemia have been studied by immuno-TEM and immuno-SEM after immunogold labelling of the cell surface antigen recognized by the B-ly7 monoclonal antibody.Most hairy cells appeared significantly labeled, although the density of the expression of the antigen, as demonstrated by immunogold labelling, seems variable from cell to cell. Moreover, some cells with the morphology of hairy cells and which could not be identified as monocytes were not labeled. Labelling for the antigen identified by the B-ly7 mAb does not seem to correlate with the presence of ribosome lamellae complexes which were present only in one of the two cases studied. Rare lymphocytes of unidentified lineage were labeled. Monocytes were significantly absent from the samples of peripheral blood of the two patients studied. In one normal control sample, monocytes were observed unlabelled.The results are discussed in reference to the pathogenesis of hairy cell leukemia, its surprisingly low mitotic rate, and its distinct response to chemotherapy. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 372-377 
    ISSN: 1059-910X
    Keywords: Chondrocyte ; Intermediate filaments ; Actin ; Tubulin ; Differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The cytoskeleton of chondrocytes consists of microfilaments made of actin, microtubules made of tubulin, and intermediate filaments made of a variety of subunits. Actin filaments are not prominent in vivo but may form in vitro. In culture, changes in filament polymerisation are important in determining cell shape, initiating chondrogenesis, and maintaining the chondrogenic phenotype. Microtubules, besides their role in cell division, organise the distribution of organelles and are involved in secretory transport mechanisms in collagen and proteoglycan synthesis. A variety of intermediate filaments may be present, frequently forming large whorled aggregates. The filaments include vimentin, cytokeratins, and glial fibrillary acidic protein. These may occur at different depths in articular cartilage. Vimentin accumulates during development of some fibrocartilages with increased mechanical loading. Together with other elements of the cytoskeleton, intermediate filaments could form part of a mechanotransduction system by which cells respond to external forces and sense changes in their external environment. © 1994 Wiley-Liss, Inc.
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    Microscopy Research and Technique 28 (1994), S. 378-384 
    ISSN: 1059-910X
    Keywords: Cartilage ; Collagen ; Chondrocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Collagens are the major proteinaceous constituents of cartilage. Three collagen types participate in the formation of striated fibrils of cartilage, collagens II, IX, and XI. Collagen II and XI belong to the subgroup of fibrillar collagens and are structurally closely related, differing mainly in their N-propeptides. Collagen IX has a very different structure but is nevertheless an essential constituent of the striated fibrils. Two other collagen types are also found in cartilage but form distinct structures. Collagen VI, found mainly in the periphery of the chondrocytes, forms beaded filaments. These filaments are probably formed by interaction of collagen VI with hyaluronan. Collagen X is expressed by hypertrophic chondrocytes. It has been shown to form in vitro hexagonal lattices and in vivo to be associated either with striated fibrils or with mats which may correspond to the lattices. The functional role of the collagen diversity in cartilage is discussed. © 1994 Wiley-Liss, Inc.
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  • 90
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    Microscopy Research and Technique 28 (1994), S. 76-76 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 91
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    Microscopy Research and Technique 28 (1994), S. 77-77 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 92
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    Microscopy Research and Technique 28 (1994), S. 67-74 
    ISSN: 1059-910X
    Keywords: Al coating ; Frozen hydrated specimens ; Gels ; Longitudinal sections ; Microanalysis ; Morphometric analysis ; Plant tissues ; SEM ; Serial sections ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully-hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at -80°C in cryo-ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (-90°C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo-preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al-coated planed face is the increased accuracy of energy-dispersive X-ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face. © 1994 Wiley-Liss, Inc.
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  • 93
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    Microscopy Research and Technique 28 (1994), S. 78-78 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 94
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    Microscopy Research and Technique 28 (1994), S. 81-94 
    ISSN: 1059-910X
    Keywords: Heparan sulfate proteoglycans ; Nephrogenesis ; Glomerular basement membranes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In vivo labeling of infant rat and mouse glomerular basement membranes (GBMs) with polyclonal anti-laminin IgGs results in binding across the full widths of GBMs at all stages of development. These stages include the pre-fusion, double basement membranes found beneath endothelial cells and podocytes in early glomeruli, and the subepithelial matrix outpockets where newly synthesized GBM is spliced into fused basement membrane during glomerular maturation. Identical binding results are obtained either with peroxidase or post-embedding immunogold techniques. Although injected cationized ferritin also binds abundantly to all developing GBMs, it quickly disappears and, 24 hours after injection, is generally absent from GBMs but remains within mesangial matrices. Injection of newborn mice with monoclonal anti-laminin IgGs results in dense labeling of pre-fusion GBMs but post-fusion GBMs and subepithelial outpockets are weak-negative. Although masking can not be excluded, these results indicate that laminin epitopes are removed during GBM fusion and splicing, either by isoform substitution or proteolytic processing. The loss of bound cationized ferritin is believed to occur mainly through rapid turnover of GBM proteoglycans. © 1994 Wiley-Liss, Inc.
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  • 95
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    Microscopy Research and Technique 28 (1994), S. 95-124 
    ISSN: 1059-910X
    Keywords: Basement membrane ; Lamina densa ; Lamina lucida ; Lamina fibroreticularis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The aim of this contribution is to summarize our knowledge of the morphology of the basement membrane (BM). The first step in this direction is the attempt to define this term. The BM is composed of the Lamina lucida, densa, and fibroreticularis. Subsequently, the historical development of this term is discussed. Our main interest is, of course, focused on the description of the BM-structure up to the macromolecular level and the special forms of this structure. This is supplemented by discussing its chemical composition and establishing a relationship between morphology and biochemistry. The obtained findings yielded some indications as to the molecular composition of the BM which may serve for the construction of “models.” The composition of the Lamina lucida (L.l.) and the Lamina or Pars fibroreticularis (L.f.) must be discussed separately, since, if present, they show a different and strongly varying structure (L.f.). An important aspect is the function of this extracellular layer which comprises mechanical tasks up to inductive effects. Finally, the concepts of the formation of the BM, especially of the Lamina densa (L.d.), are summarized. It obviously consists of a sequence of individual steps which starts with expression and secretion of the L.d.-components and is followed by an induction of integrin expression. © 1994 Wiley-Liss, Inc.
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  • 96
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    Microscopy Research and Technique 28 (1994), S. 125-140 
    ISSN: 1059-910X
    Keywords: Glycosaminoglycans ; Heparan sulfate ; Dermatan sulfate ; Cuprolinic blue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The application of cationic probes for the ultrastructural detection of proteoglycans in basement membranes is reviewed. Proteoglycans are highly negatively charged macromolecules due to their glycosaminoglycan side chains. The interaction of cationic probes with proteoglycans is of an electrostatic nature. Methods are discussed to increase the specificity of probes for proteoglycans. The use of phthalocyanin-like dyes such as Cuprolinic blue, according to the critical electrolyte concentration method, results in a selective staining of proteoglycans. Enzymatic or chemical digestions, however, should be done to validate the proteoglycan nature of the dye-positive granules/filaments, and to establish the class of proteoglycan. The value of cationic probes in basement membrane research on development and pathology is discussed. The potential for deducting molecular information from the ultrastructural appearance of stained proteoglycans is indicated. © 1994 Wiley-Liss, Inc.
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  • 97
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    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 28 (1994) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
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  • 98
    ISSN: 1059-910X
    Keywords: Time-resolved fluorescence ; Fluorescence microscopy ; Europium chelates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time-resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross-linking fixation with glutaraldehyde was used and the signal-to-background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long-term stability for the samples with glutaraldehyde fixation and permanent mounting. © 1994 Wiley-Liss, Inc.
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  • 99
    ISSN: 1059-910X
    Keywords: Alveolar macrophages ; OsO4 fixation ; Elements ; ESI ; EELS ; Energy filtering microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique. © 1994 Wiley-Liss, Inc.
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  • 100
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    Microscopy Research and Technique 28 (1994), S. 165-177 
    ISSN: 1059-910X
    Keywords: Diabetic nephropathy ; Diabetic retinopathy ; Mesangial matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Basement membranes (BMs) were first described in the mid-19th century, but they were not isolated and prepared for compositional studies until nearly 100 years later. Early methods of isolation were carried out on renal glomeruli, which were first sub-fractionated from kidney tissues by sieving. BMs were then isolated from the glomeruli by ultrasonic disruption, which, following low speed centrifugation, yielded “purified” but highly fragmented BM material. In an effort to obviate the mechanical damage to BMs produced by ultrasound, a sequential detergent solubilization technique was introduced that resulted in morphologically intact BMs from a variety of tissue sub-fractions. This was highly advantageous because “acellular” BMs produced by the procedure could be examined critically by light and electron microscopic methods. Subsequently, this procedure has been utilized to demonstrate the substructural heterogeneity of vascular and non-vascular BMs from a wide variety of animal species. The current review describes the results of scanning and transmission electron microscopic studies of acellular BMs prepared from renal glomeruli and from the retinal microvessels of the eye. These BMs are of particular interest to basic scientists and clinicians because they are altered in several disease states, most notably diabetes mellitus. An effort is made to point out the implications of glomerular and retinal vessel BM changes to the pathogenesis of diabetic kidney and retinal vessel BM disease. © 1994 Wiley-Liss, Inc.
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