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  • Cell & Developmental Biology  (1,675)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 106-117 
    ISSN: 1059-910X
    Keywords: Acinar cells ; Duct cells ; Differentiation ; Immunogold ; Amylase ; Proline-rich proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Chronic treatment of mice and rats with isoproterenol (IPR) causes marked hypertrophy and hyperplasia of the salivary glands, and alters the expression of several secretory proteins. We used quantitative postembedding immunogold labeling to study the cellular responses in the rat parotid gland during daily (up to 10 days) injections of IPR and during recovery (up to 14 days) after cessation of IPR treatment. Labeling densities of acinar cell secretory granules with antibodies to amylase and protein SMG-B1 (cross-reactive with the rat homologue of Parotid Secretory Protein, PSP) fell to 10% of control levels after 8-10 IPR injections, then increased during recovery, paralleling previous biochemical determinations of changes in protein and mRNA levels. With antibodies to proline-rich proteins (PRP), labeling densities initially fell, then subsequently showed considerable variability, but never exceeded control levels. These results contrast with biochemical determinations showing a marked induction of PRP synthesis, and may have both immunological and structural explanations.Occasional intercalated duct cells located close to the acini underwent differentiation toward an acinar-like phenotype as a result of IPR treatment. After 1-2 IPR injections, the secretory granules of these cells labeled with antibodies to amylase and PRP. Subsequently, the granules appeared electron-lucent and were increased in size and number. These observations support earlier work, suggesting that intercalated duct cells may differentiate into other gland cell types.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 118-127 
    ISSN: 1059-910X
    Keywords: In vivo ; Tubular epithelium ; Kidney ; Endocytosis ; Cationic albumin ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A-gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde-fixed, osmiumpostfixed, and Epon-embedded kidneys. Cationic BSA-DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium. © 1995 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 128-140 
    ISSN: 1059-910X
    Keywords: Actin ; Phospholipids ; Tight junctions ; Pancreas ; Testis ; Immunocytochemistry ; Fracture-label ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a doublelabeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex. © 1995 Wiley-Liss, Inc.
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  • 4
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    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 141-158 
    ISSN: 1059-910X
    Keywords: Immunocytochemistry ; Lipopolysaccharide ; Pneumocyte ; Macrophage ; Microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gramnegative bacteria that act on numerous cellular functions through the processes of cell activation and damage. The molecular mechanisms involved in the “endotoxic phenomenon” are not defined yet, although extensive studies have been carried out. Immunogold and electron microscopy (EM) have contributed to identify the primary target cells of endotoxins and the subcellular systems that receive the direct action of these bacterial agents. Here, we review our studies on immunogold detection of endotoxins in cellular and subcellular systems. The analysis of the interaction between endotoxins and cells was focussed on the following aspects: (1) morphological characteristics of the LPS aqueous suspensions used in experimental work; (2) binding of endotoxins to the plasma membrane of type II pneumocytes and alveolar macrophages (two of their cellular targets), and influence of the state of aggregation of the LPS; (3) movement and distribution of endotoxins inside the cell, from the plasma membrane to the nucleoplasm; and (4) interaction of LPS with microtubules and its effects on the integrity of the microtubular network. These approaches provide information at the molecular level as well as data for the establishment of physiological models of endotoxicity. © 1995 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995) 
    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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  • 6
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    Microscopy Research and Technique 31 (1995), S. 159-173 
    ISSN: 1059-910X
    Keywords: Immunogold ; Electron microscopy (EM) ; Oncogene ; Mos ; Met ; Ski ; Muc1 ; Mucin ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Immunogold labeling electron microscopy technique has been used to study the ultrastructural localization of oncogenic proteins: Mos, Met, Ski, and the tumor-associated protein, Muc1, as well as their relationship with other tumor-related proteins. By pre- and postembedding immunogold labeling electron microscopy techniques, we showed that the Mos protein pp39mos colocalized with microtubule bundles, suggesting that microtubulin or microtubule-associated protein(s) may be the substrate of Mos. Met protein was labeled at the microvilli of the lumen that are formed in cultured T47D cells, implying its potential involvement in lumen formation. Ski localization experiments revealed a unique globular structure “Ski body” that is present inside the nucleus of interphase chicken embryo fibroblast infected with Ski cDNA FB29 and FB2-29. Ski bodies were also found scattered in the cytoplasm of metaphase FB29 and FB2-29 Ski expressing chicken embryo fibroblasts. In T47D cells, tumor-associated protein Muc1 was associated with both the plasma membrane and the membranes of secretory vesicles in the cytoplasm. In MUC1 infected NIH3T3 cells, however, labeling showed that in addition to the plasma membrane and the membranes of secretory vesicles, some Muc1 gold spheres were seen inside the secretory vesicles, suggesting that the subcellular localization of the protein may vary in different cell types. © 1995 Wiley-Liss, Inc.
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  • 7
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    Microscopy Research and Technique 31 (1995), S. 183-183 
    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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    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 174-181 
    ISSN: 1059-910X
    Keywords: Ultramicrotomy ; Transmission electron microscopy ; Uranium in soils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Uranium-contaminated soils from the U.S. Department of Energy (DOE) Fernald Site, Ohio, have been examined by a combination of backscattered electron imaging (BSE) and analytical electron microscopy with electron diffraction (AEM). The inhomogeneous distribution of particulate uranium phases in the soil required the development of a method for using ultramicrotomy to prepare transmission electron microscopy (TEM) thin sections from the SEM mounts. A water-miscible resin was selected that allowed comparison between SEM and TEM images, permitting representative sampling of the soil. Uranium was found in iron oxides, silicates (soddyite), phosphates (autunites), and uraninite (UO2+x). No uranium was detected in association with phyllosilicates in the soil. © 1995 Wiley-Liss, Inc.
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  • 9
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    Microscopy Research and Technique 31 (1995), S. 184-192 
    ISSN: 1059-910X
    Keywords: DNA ; In situ hybridisation ; Monoclonal antibodies ; Protein ; RNA ; Western blots ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The molecular cell sciences have had a great impact in the analysis of the genetic and epigenetic events of esophageal and gastric tumorigenesis. In other regions of the alimentary tract such as the colon, the serial identification of the molecular events in the corresponding morphological lesions is perhaps most advanced. This is, in part, due to the relative ease of the histological characterisation of the premalignant lesions. In this regard the analysis of morphological and molecular adaptation in the alimentary tract is inextricable. This review aims, therefore, to judiciously assess the relative applications of contemporary techniques in investigative histopathology. © 1995 Wiley-Liss, Inc.
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  • 10
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    Microscopy Research and Technique 31 (1995), S. 4-21 
    ISSN: 1059-910X
    Keywords: DNA ; RNA ; Cell nucleus ; Immunogold techniques ; Nuclear organization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the present review, we report on recent results obtained by in situ transferaseimmunogold techniques as to the ultrastructural distribution of DNA and RNA within the cell nucleus. Special emphasis is placed on the various nucleolar components and the various enigmatic structures of the extranucleolar region: interchromatin granules, coiled bodies, and simple nuclear bodies. These data are discussed in the light of our current understanding of the functional organization of the cell nucleus. © 1995 Wiley-Liss, Inc.
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  • 11
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    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 31 (1995), S. 44-62 
    ISSN: 1059-910X
    Keywords: Mineralization ; Bone ; Cartilage ; Cementum ; Dentin ; Enamel ; Osteopontin ; Osteocalcin ; Bone sialoprotein ; Amelogenin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Immunocytochemistry is a powerful tool for investigating protein secretion, extracellular matrix assembly, and cell-matrix and matrix-matrix/mineral relationships. When applied to the tissues of bones (bone and calcified cartilage) and teeth (dentin, cementum, and enamel), where calcium phosphate-containing extracellular matrices are the predominant structural component related to their weight-bearing and masticatory roles, respectively, data from immunocytochemical studies have been prominent in advancing our understanding of mineralized tissue modeling and remodeling. The present review on the application of postembedding, colloidal-gold immunocytochemistry to mineralized tissues focuses on the advantages of this approach and relates them to conceptual, theoretical, and experimental data currently available discussing matrix-mineral interactions and extracellular matrix formation and turnover in these tissues. More specifically, data are summarized regarding the distribution and role of noncollagenous proteins in different mineralized tissues, particularly in the context of how they interface with mineral, and how this relationship might be affected by the various tissue-processing steps and immunocytochemical strategies commonly implemented to examine the distribution and function of tissue proteins. Furthermore, a technical discussion is presented that outlines several different possibilities for epitope exposure in mineralized tissues during preparation of thin sections for transmission electron microscopy. Cell biological concepts of protein secretion by cells of the mineralized tissues, and subsequent extracellular matrix assembly and organization, are illustrated by examples of high-resolution, colloidal-gold immunolabeling for osteopontin, bone sialoprotein, and osteocalcin in the collagen-based mineralized tissues and for enamel protein (amelogenin) in enamel. © 1995 Wiley-Liss, Inc.
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  • 12
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    Microscopy Research and Technique 31 (1995), S. 79-92 
    ISSN: 1059-910X
    Keywords: Immunogold technique ; Protein A-gold ; Peroxisome ; Subcompartment ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A-gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research. © 1995 Wiley-Liss, Inc.
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  • 13
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    Microscopy Research and Technique 31 (1995), S. 63-78 
    ISSN: 1059-910X
    Keywords: Chitinase ; β-1,3-glucanase ; β-fructosidase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: During the past few years, cyto- and immunocytochemical techniques have been developed and widely used for locating and identifying various molecules in plant cell compartments. The last decade has witnessed tremendous improvements in molecular cytology, thus allowing an accurate in situ detection of various components thought to play important biological functions in the plant metabolism. The use of immunocytochemistry to investigate resistance mechanisms of plants upon pathogen attack has provided key information on the defense strategy that plants elaborate during a host-pathogen interaction. Of the various proteins induced in response to infection, chitinases and β-1,3-glucanases have been the focus of particular attention due to their believed antimicrobial activity through the hydrolysis of the main fungal wall components, chitin and β-1,3-glucans. Attention has also been paid to β-fructosidase, the enzyme that hydrolyzes sucrose into glucose and fructoside. The marked accumulation of this enzyme upon pathogen infection has led to the consideration that infection may greatly infleunce the metabolic activity of colonized tissues by creating alterations of source-sink relationships. Another facet of the plant's defense strategy that has been the focus of considerable interest is related to the accumulation of structural compounds, such as hydroxyproline-rich glycoproteins and callose, to reinforce the wall architecture, thus decreasing vulnerability to microbial enzymes. A number of alternatives designed to improve plant protection towards pathogen invasion have been suggested. Among these, the production of transgenic plants expressing constitutively a foreign resistance gene and the pretreatment of plants with elicitors of defense reactions have been the subject of intensive studies at the molecular, biochemical, and cytological levels. Results of such studies clearly demonstrate the important contribution that cyto- and immunocytochemical approaches can make to our knowledge of how plants defend themselves and how plant disease resistance can be directly enhanced. These approaches will undoubtedly be active areas for future research in the development of biological control alternatives in which the mode of action of the product used is of key importance. © 1995 Wiley-Liss, Inc.
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  • 14
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    Microscopy Research and Technique 31 (1995) 
    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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  • 15
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    Microscopy Research and Technique 30 (1995), S. 208-217 
    ISSN: 1059-910X
    Keywords: Superconductors ; Electron energy loss spectrometry ; Transmission electron microscope ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Electron energy loss spectrometry (EELS) with a cold field emission gun (cFEG) transmission electron microscope (TEM) is implemented to analyze the evolution of the electronic structure and dielectric function of oxide superconductors. The O-K core loss spectra of p-type doped oxide superconductors are analyzed in terms of holes formation on oxygen sites, while low loss spectra are analyzed for free carrier plasmas, other spectral excitations, and their crystallographic confinement.It is illustrated that the transmission EELS with a cFEG TEM very much complement soft X-ray absorption spectroscopy and optical spectroscopy, with the added advantages of high spatial resolution (∼1-100 nm), and is compatible with other analytical, diffraction, and imaging techniques, which are readily available in a cFEG TEM. © 1995 Wiley-Liss, Inc.
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  • 16
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    Microscopy Research and Technique 30 (1995), S. 246-251 
    ISSN: 1059-910X
    Keywords: Bodonidae ; Cell surface ; Chemical fixation ; Cryofixation ; Quick freeze ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Freeze-fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free-living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows. © 1995 Wiley-Liss, Inc.
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  • 17
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    Microscopy Research and Technique 30 (1995), S. 252-257 
    ISSN: 1059-910X
    Keywords: Permeability tracer ; Endocytosis ; Transcytosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Bovine aortic endothelial cells (BAEC) were exposed to glucosylated albumin-gold complexes (GgA), and the distribution of the tracers was compared after cryofixation and after glutaraldehyde fixation. Morphometric analysis revealed differences in the GgA distribution depending upon the method of fixation used. In BAEC monolayers cryofixed after 3 min of incubation with GgA, tracer was observed in predominately apically located vesicular elements. After 16 min of incubation, all vesicular elements were labelled, and multivesicular bodies were the prominent labelled structure. In contrast, chemically fixed monolayers exhibited a heterogeneous distribution of GgA within vesicular profiles after 3 min and 16 min of GgA incubation. The differences in tracer distribution depending upon the fixation method must be resolved before the mechanism of vesiclemediated endothelial cell transport function is defined and universally accepted. © 1995 Wiley-Liss, Inc.
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  • 18
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    Microscopy Research and Technique 30 (1995), S. 265-267 
    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
    ISSN: 1059-910X
    Keywords: Microstructure ; High-Tc Bi(Pb)-2212 superconductor ; Sol-gel method ; SEM ; TEM ; Powder XRD ; EPMA ; Magnetization ; Crack ; Amorphous substance ; Modulated structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Microstructures of Bi(Pb)-family 2212 superconductors, which were prepared by a sol-gel method with three different compositions, were examined mainly by scanning and transmission electron microscopy. The magnetization of the specimens strongly depends on the ratio between Bi and Pb content, while Tc is almost constant. In specimen 1, prepared with the nominal composition of Bi/Pb = 9/1, small grains of 2212 phase are formed with a minor fraction of some impurity phases. In specimen 2, with Bi/Pb = 17/3, which is optimum from the viewpoint of magnetization, large grains of the 2212 phase are formed during heating at 800°C, also with the impurity phases. In specimen 3, with Bi/Pb = 8/2, the 2212 grains are divided by layers of (Bi0.86, Pb0.14) (Ca0.7, Sr0.3)Ox. Moreover, plate-like 2212 crystals are severely bent so that small cracks appear often with an inclusion of amorphous substance being rich in Ca and Pb. These layers and cracks must degrade the magnetization. A modulated structure of Bi-type is formed in the 2212 grains of specimens 1 and 2, while not only Bi-type but also Pb-type are formed in specimen 3. The wavelength of Bi-type is different for each specimen. © 1995 Wiley-Liss, Inc.
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  • 20
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    Microscopy Research and Technique 30 (1995) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 21
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    Microscopy Research and Technique 30 (1995), S. 269-270 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 22
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    Microscopy Research and Technique 30 (1995), S. 218-245 
    ISSN: 1059-910X
    Keywords: (Bi,Pb)2Sr2Can-1CunO4+2n+δ ; HR-TEM ; Crystal structure ; Microstructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This paper summarizes results obtained by high-resolution transmission electron microscopy on the crystal structure and microstructure of the (Bi,Pb)2Sr2Can-1CunO4+2n+δ high-Tc superconducting oxides. The experimental basis for the work presented here are high-resolution structure images obtained at ultra-thin (3 nm) areas of carefully prepared transmission electron microscope (TEM) samples. The analysis was carried out on a 400 kV TEM equipped with a pole piece yielding 0.17 nm point-to-point resolution. From the images obtained the projected crystal potential of the cations can be extracted directly, as confirmed by detailed image simulation. Structural analysis of the oxygen sublattice remains an unsolved problem by high-resolution TEM (HRTEM), mainly because of the small scattering factors, and thus the contribution of the oxygen sublattice to the image contrast is small. The (Bi,Pb)2Sr2Can-1CunO4+2n+δ phases are modulated structures that can be understood as an average structure plus a superimposed displacement field. The crystal structure consists of BiO double layers and perovskite-type cuboids (containing Sr, Ca, Cu, and O), which are sandwiched between the BiO double layers. The displacement field can be directly analyzed by HRTEM, and the largest displacement amplitudes of 70 pm were determined for the Bi atoms in the n = 1 compound. The chemical composition of the n = 2 and n = 3 compounds was determined by EDX in the TEM for the cation sublattice. A significant (Ca + Sr) deficiency (approximately 10%) with respect to Cu was found. The (Sr + Ca)/Cu mole fraction ratio was 1.31 for the Bi-2212 phase and 1.14 for the Bi(Pb)-2223 phase. The oxygen content cannot be determined by EDX in the TEM with the accuracy necessary for a correlation with electrical and superconducting properties. The defect structure present in these materials, that is, intergrown lamellae with different crystal structures and equal or different chemical compositions, stacking faults, and grain boundaries, is summarized. The importance of grain boundaries for understanding and improving superconducting properties is emphasized. © 1995 Wiley-Liss, Inc.
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  • 23
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    Microscopy Research and Technique 31 (1995), S. 408-419 
    ISSN: 1059-910X
    Keywords: Neuronal mosaic ; Coupling ; Network ; Dopamine ; Nitric oxide ; Connexin ; Development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The vertebrate retina is a highly laminated assemblage of specialized neuronal types, many of which are coupled by gap junctions. With one interesting exception, gap junctions are not directly responsible for the ‘vertical’ transmission of visual information from photoreceptors through bipolar and ganglion cells to the brain. Instead, they mediate ‘lateral’ connections, coupling neurons of a single type or subtype into an extended, regular array or mosaic in the plane of the retina. Such mosaics have been studied by several microscopic techniques, but new evidence for their coupled nature has recently been obtained by intracellular injection of biotinylated tracers, which can pass through gap junctional assemblies that do not pass Lucifer Yellow. This evidence adds momentum to an existing paradigm shift towards a population-based view of the retina, which can now be envisaged both as an array of semi-autonomous vertical processing modules, each extending right through the retina, and as a multi-layered stack of interacting planar mosaics, bearing some resemblance to a set of interleaved neural networks. Junctional conductance across mosaics of horizontal cells is known to be controlled dynamically with a circadian rhythm, and other dynamically-regulated conductance changes are also likely to make important contributions to signal processing. The retina is an excellent system in which to study such changes because many aspects of its structure and function are already well understood. In this review, we summarize the microscopic appearance, coupling properties and functions of gap junctions for each cell type of the neural retina, the regulatory properties that could be provided by selective expression of different connexin proteins, and the evidence for gap junctional coupling in retinal development. © 1995 Wiley-Liss, Inc.
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    ISSN: 1059-910X
    Keywords: Gap junctions ; Connexin40 ; Connexin43 ; Conduction system ; Mammals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 337-356 
    ISSN: 1059-910X
    Keywords: Olfactory signal transduction ; G proteins ; Adenylyl cyclase ; Rapid freezing ; Lowicryl ; Cilia ; Microvilli ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Freeze-fracturing and deep-etching are a well-suited set of methods to study membrane and cytoplasmic features. Various approaches are available. Possible variables include tissue preparation, fracturing only or fracturing followed by etching, modes and materials of replication, and various ways of combining freeze-fracturing and/or deep-etching with (immuno)cytochemistry. Freeze-substitution, in particular combined with embedding in methacrylate resins such as the Lowicryls, is becoming rather widely accepted for purposes of ultrastructural (immuno)cytochemistry. Most investigators active in this field agree that this combination yields superior results compared to (immuno)cytochemistry combined with more conventional means of thin section transmission electron microscopy. Yet relatively little information is available on the variations that can occur with different approaches of freeze-substitution immunocytochemistry. This review deals with some of the variations in freeze-fracturing, freeze-etching, and freeze-substitution as applied to olfactory epithelial structures and with the effectiveness of observations obtained by application of the above sets of methods in relating the special morphology of olfactory epithelial cellular structures with those obtained by other approaches. Indeed, the data obtained continue to provide an integral image in which that morphology can be related to the special biochemistry, cell and molecular biology, and electrophysiology of olfactory epithelial structures. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 319-332 
    ISSN: 1059-910X
    Keywords: Rat ; Prostate ; Epithelium ; Stroma ; Cytodifferentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 342-350 
    ISSN: 1059-910X
    Keywords: Apoptosis ; BPH ; Growth regulatory factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Homeostasis in the prostate is recognized to be maintained by a complex interplay between the opposing actions of cell proliferation and Cell death. Growth regulatory factors that promote or inhibit cell proliferation and promote cellular death have been identified in the prostate. The integration of these forces involves cellular cooperation between the prostatic stroma and epithelium. Hormone-regulated production of growth regulatory factors by one cell type may determine growth stimulation, inhibition, or cell death in a reciprocal cell partner. Imbalance between net cell proliferation and net cell death rates may result in abnormal growth leading to BPH. Additional study of the growth regulatory factors associated with distal vs. proximal epithelial cells and stroma and comparison of growth factor expression by the neonatal, postnatal growing, adult quiescent, and aging prostates will likely provide further insight into the regulation of prostate cell division and death. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 351-352 
    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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  • 29
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    Microscopy Research and Technique 32 (1995), S. 13-28 
    ISSN: 1059-910X
    Keywords: Ciliated and secretory cells as related to menstrual cycle ; LM ; SEM ; TEM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Oviductal epithelium of the baboon, Papio cynocephalus, was studied utilizing light, scanning, and transmission electron microscopy. Results of counts made of nonciliated, ciliated, and ciliogenic cells were analyzed statistically. The percentages of nonciliated cells of the fimbria and ampulla during the early proliferative and late secretory stages of the menstrual cycle were significantly greater than those during the mid-proliferative and late proliferative-early secretory stages, due to deciliation. This paper emphasizes previously unreported apical surface morphology as viewed by scanning and transmission electron microscopy. The microvillar pattern of the fimbrial secretory cells differs from that of the ampullar and isthmic cells in that the microvilli originate from thick apical protrusions and vary greatly in length and number as related to the cycle. A ridge demarcating the apical intercellular junction is composed of rows of microvilli during the early proliferative and late secretory stages. During the early proliferative and late secretory stages an increased degree of invagination of the basal and lateral plasma membranes occurs as the height and width of the cells decreases. The general numbers and distribution of the organelles of the various types of oviductal cells agree with that described for the ampulla and isthmus by Verhage et al. [(1990) Am. J. Anat., 187:81-90]; however, fimbrial epithelium was not included in that study. Other cyclic ultrastructural changes not examined previously include variation in the number of lipid droplets and their location, and in the number and relationships of glycogen particles to other structures. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 75-76 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 31
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    Microscopy Research and Technique 32 (1995), S. 77-78 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 32
    ISSN: 1059-910X
    Keywords: Chelonia, Crocodilia ; Rhynchocephalia ; Squamata ; Parathyroid hormone ; Development ; Seasonal variation ; Parathyroidectomy ; Topography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Adult reptiles possess one or two pairs of parathyroid glands that have been shown in many species to derive from the third and fourth pharyngeal pouches, respectively. Up to five pairs may develop during early embryonic life. Excess glands may involute during late embryogenesis. The location of the parathyroid glands differs in the various species. As a general rule, they lie just anterior to the heart, the anterior pair (parathyroid III) being associated with the carotid artery, the posterior pair (parathyroid IV) with the aortic arch. In snakes, however, the anterior pair (parathyroid III) is associated with the carotid artery near the angle of the jaw. As shown by light microscopy and, to a lesser extent, by electron microscopy, the parathyroid parenchyma comprises secretory cells which may form dark and light variants, occasional oxyphil cells, and stellate cells. They are arrangend in cords separated by connective tissue containing a capillary network. Parathyroid secretory cells often form follicles which might be the result of degeneration. Degeneration may occur as a form of involution during winter in species undergoing seasonal changes. The product of parathyroid cells, the parathyroid hormone, is responsible for the maintenance of blood calcium concentration. The sites of action - bones, kidneys, intestine, endolymphatics, and dermal skeleton - are not well understood or not investigated. In some turtles, parathyroid hormone is not the (main) factor for the regulation of calcium homeostasis. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 390-407 
    ISSN: 1059-910X
    Keywords: Development ; Deefferentation ; Muscle cell lineages ; Spindle origin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This review concerns the pattern of expression and regulation of myosin heavy chain (MHC) isoforms in intrafusal fibres of rat muscle spindles detected by immunocytochemistry. The three types of intrafusal fibres - nuclear bag1, nuclear bag2, and nuclear chain fibres - are unique in co-expressing several MHCs including special isoforms such as slow tonic and α cardiac-like MHC and isoforms typical of muscle development, such as embryonic and neonatal MHC. The distinct intrafusal fibre types appear sequentially during rat hind limb development, the nuclear bag2 precursors being first identifiable at 17-18 days in utero as the only primary myotubes expressing slow tonic MHC. Sensory innervation is required for the expression of “spindle-specific” MHC isoforms. Motor innervation contributes to the diversity in distribution of the different MHCs along the length of the nuclear bag fibres. It is suggested that unique populations of myoblasts are destined to become intrafusal fibres during development in the rat hind limb muscles and that the regional heterogeneity in MHC expression is related both to sensory and motor innervation and to the properties of the myoblast lineages. These distinct features make intrafusal fibres an attractive in situ model for investigating myogenesis, myofibrillogenesis, and the mechanisms regulating MHC expression. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 366-380 
    ISSN: 1059-910X
    Keywords: Myogenesis ; Myosin ; MyoD ; Myogenin ; PDGF ; FGF ; Transferrin ; Chicken ; Rat ; C2 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The myogenic precursor cells of postnatal and adult skeletal muscle are situated underneath the basement membrane of the myofibers. It is because of their unique positions that these precursor cells are often referred to as satellite cells. Such defined satellite cells can first be detected following the formation of a distinct basement membrane around the fiber, which takes place in late stages of embryogenesis. Like myoblasts found during development, satellite cells can proliferate, differentiate, and fuse into myofibers. However, in the normal, uninjured adult muscle, satellite cells are mitotically quiescent. In recent years several important questions concerning the biology of satellite cells have been asked. One aspect has been the relationship between satellite cells and myoblasts found in the developing muscle: are these myogenic populations identiacal or different? Another aspect has been the physiological cues that control the quiescent, proliferative, and differentiative states of these myogenic precursors: what are the growth regulators and how do they function? These issues are discussed, referring to previous work by others and further emphasizing our own studies on avian and rodent satellite cells. Collectively, the studies presented indicate that satellite cells represent a distinct myogenic population that becomes dominant in late stages of embryogenesis. Moreover, although satellite cells are already destined to be myogenic precursors, they do not express any of the four known myogenic regulatory genes unless their activation is induced in the animal or in culture. Furthermore, multiple growth factors are important regulators of satellite cell proliferation and differentiation. Our work on the role of one of these growth factors [platelet-derived growth factor (PDGF)] during proliferation of adult myoblasts is further discussed with greater detail and the possibility that PDGF is involved in the transition from fetal to adult myoblasts in late embryogenesis is brought forward. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 408-418 
    ISSN: 1059-910X
    Keywords: Mitosis ; Chromosomes ; Lung cells ; HeLa S3 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphas plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A - specific and groups D - and G - specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 37
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    Microscopy Research and Technique 30 (1995), S. 419-426 
    ISSN: 1059-910X
    Keywords: Sublimation drying ; Peldri II ; Tert-butyl alcohol ; Cell morphology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The increasing importance of in vitro models has presented new challenges in SEM processing techniques. The present study has evaluated the quality of preservation of cultured human lens epithelial cells processed by critical point, Peldri II, and tert-butyl alcohol drying. Specimens processed by critical point drying produced specimens with severe cracking of cell processes and microcracks across cell membrane surfaces. Peldri II and tert-butyl alcohol drying eliminated breakage of the filopodia and lamellipodia as well as eliminating the microcracks across the apical membrane surface. The morphology of lens epithelial cells grown on Cytodex 3 beads appeared rounded with convoluted membrane surfaces. These morphological features were present for cells processed by all three methods. Cytodex 3 beads were subsequently shown to shrink 52% in diameter during dehydration, which results in an 89% reduction in volume for the bead. Cells grown on Biosilon beads, which do not shrink, had a morphology similar to the cells grown on a flat substrate. These results indicate that Peldri II and tert-butyl alcohol drying offer an attractive alternative to critical point drying when preparing cultured cells for SEM. Interpretation of cultured cell morphology must consider shrinkage of the substrate material as a possible contributor to artifact. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 531-532 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Topics: Natural Sciences in General
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  • 39
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    Microscopy Research and Technique 32 (1995), S. 1-12 
    ISSN: 1059-910X
    Keywords: Spermatozoon ; Fertilization ; Embryo ; Growth factors ; Insulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Studies on embryonic development in vitro as well as observations in vivo, suggested that two aspects of oviduct physiology are important for early development. On one side has to be considered the oviduct “environment”: temperature, pH, osmotic pressure, nutrients, oxygen tension, free radical scavengers, etc. On the other, the oviduct “active components”: stimulatory and/or regulatory molecules, supposed to finely regulate the fertilisation process and the first differentiative steps.While the physical environment of the oviduct has been under investigation for some decades, studies on oviduct-specific molecules and their functions have only been developed much more recently. The amount of information on this topic, however, has rapidly reached the size that demands a summary.In this review the descriptive literature on oviduct specific proteins will be examined as a basis for illustrating the possible functions of these molecules. In particular their role in fertilisation and early embryonic cleavages will be analysed in some details. Finally a section is devoted to the presence and physiological significance of growth factors in oviduct fluid. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 29-49 
    ISSN: 1059-910X
    Keywords: Mucus ; Mucous surfaces ; Glycocalyx ; Glycoproteins ; Estradiol ; Fallopian tube ; Oviduct ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The mucous surfaces of the oviducts of mammals, especially humans, are dependant on estradiol. The mucus glycoproteins and glycocalyceal glycoproteins have, however, barely been studied. Biochemical analyses have focussed on the relatively low molecular weight glycoproteins likely to be found in more serous-type granules of the ampulla and not on the very high MW glycoproteins typical of mucus and represented in the isthmus by morphological evidence of mucus secretion. Quantitatively, secretion from the ampulla is likely to predominate, because of its huge surface area compared with the isthmus. But functional closure of the isthmus under the influence of estradiol in the absence of progesterone means that it is the isthmus where luminal secretions accumulate - and where mucus glycoproteins will exert their most important effects on spermatozoa ascending the reproductive tract, and then on fertilized ova en route to the uterus. Further study of the extracellular, intraluminal, carbohydrate-rich environment of the oviductal isthmus, especially in humans, is likely to prove rewarding. Sampling of these secretions is now feasible using transvaginal, transuterine fallopian tube catheters that are in clinical use. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 57-69 
    ISSN: 1059-910X
    Keywords: Oviduct ; Glycoprotein ; Antibody ; cDNA ; Human ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The major objective of this study was to examine the hormonal regulation of a human oviduct-specific glycoprotein (huOGP) throughout the menstrual cycle and in all regions of the human oviduct. Regulation of synthesis and secretion was examined at both the protein (Western immunoblots and immunocytochemistry) and mRNA (Northern and slot blots) levels and correlated with changes in the morphological features of the oviductal epithelial cells throughout the cycle. Immunoblot analysis of oviductal fluid and explant culture media from all regions of the oviduct demonstrated that huOGP is primarily found during the follicular stage of the cycle and is not present in serum, follicular fluid, or uterine endometrium. Moreover, two-dimensional (2-D) immunoblots showed that all major isoelectric variants of huOGP observed on 2-D fluorographs are immunologically related. Light microscopic immunocytochemistry localized huOGP to oviductal secretory cells in both ampulla and isthmic regions, with the most intense immunoperoxidase staining seen in midcycle samples. Using an indirect immunogold technique at the electron microscopic level, huOGP was specifically localized to secretory granules of the ampullary and isthmic nonciliated epithelial cells. The ultrastructural characteristics of these secretory cells during the mid to late follicular phase of the cycle suggested elevated protein synthetic activity. In addition, mRNA expression for huOGP was elevated in all regions of the oviduct in midcycle specimens. Collectively, these data indicate that huOGP is a major tissue-specific, stage-specific secretory product of the human oviduct during the periovulatory stage of the cycle and support the hypothesis that huOGP synthesis and secretion may be regulated by fluctuations in the levels of estrogen and progesterone. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 120-128 
    ISSN: 1059-910X
    Keywords: Parathyroid cell variants ; Fixation ; Membrane disintegration ; Secretory activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Fixation with aldehydes is achieved either by immersion or perfusion. The parenchyma of parathyroid glands fixed by immersion consists of dark cells containing a lot of membranes of those organelles which are concerned with hormone secretion, light cells which are poor in these organelles, intermediate forms between the two, and multinuclear syncytial cells. They have been attributed to represent different functional stages of secretory activity, the dark cell being in an active form, the light cell in a resting form. Studies of the parathyroids of mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, and horses employing various fixation protocols clearly demonstrate that light cell variants and multinuclear syncytial cells are formed during improper immersion fixation as a result of membrane disintegration. Parathyroids fixed by perfusion or by immersion in an appropriate fixation medium comprise only one cell type which correspond to the dark chief cell. Parathyroid cells are polar cells bearing some of the rough endoplasmic reticulum in the basal pole, the rest of it, the Golgi complex, and secretory granules in the apical pole. The secretory product is released by exocytosis at the apicolateral domain of the plasma membrane into the intercellular space. Secretory activity can be altered experimentally, leading to drastic changes in the amount of cell membrane related to hormone synthesis, intracellular transport, exocytic release, and secretion coupled membrane retrieval. The sensitive reaction of parathyroid cells to both the mode of fixation and to fixation media demands careful evaluation of the fixation protocol. This and the polarity of parathyroid ceils have to be borne in mind for estimating secretory activity on the basis of morphological criteria. © 1995 Wiley-Liss, Inc.
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    ISSN: 1059-910X
    Keywords: Lactose repressor ; Mitochondrial ATPase ; F1 ; Freeze-etching method ; 45° unidirectional Pt-C replication of globular proteins ; 45° and vertical replication of gold ball standards on indirect carbon films ; Transmission electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45° angle on an ≈ -100°C surface in a 10 -6 torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 ± 0.25 nm, 2.0 ± 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The “geometric assumption” that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45° and 20° angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45° angle with current high resolution conditions at a substrate temperature of -185°C, and the results of these replicas were compared to the results reported here at ˜-100°C. © 1995 Wiley-Liss, Inc.
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  • 44
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    Microscopy Research and Technique 32 (1995), S. 437-448 
    ISSN: 1059-910X
    Keywords: Sexual differentiation ; Organ culture ; Sertoli cell ; Leydig cell ; Mesonephros ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The influence of mesonephric tissues and the extracellular matrix on mouse gonadal differentiation was examined in vitro. Gonadal ridges, with or without the adjacent mesonephric region, were removed from mouse embryos on day 12 post coitum (p.c.), and cultured in the presence or absence of reconstituted basement membrane (matrigel) for 5 days. Culturing control undifferentiated testes with mesonephric tissues induced normal testicular differentiation. When testes without mesonephric tissues were cultured in the absence of matrigel, testicular cord formation was not observed in the explants. Sertoli cells were irregularly arranged in the testicular parenchyma, and no continuous basal lamina was formed around the Sertoli cells. However, when testes without mesonephric tissues were embedded in matrigel and cultured for 5 days, the Sertoli cells were organized into testicular cord-like structures. The Sertoli cells positioned at the base of the cord-like structures were closely connected to the matrigel at their basal surface, and showed a polarized distribution of vimentin filaments in their basal cytoplasm. Leydig cells, on the other hand, were differentiated in all testicular explants. In all ovarian explants, germ cells normally entered meiotic prophase. Therefore, these findings indicate that the extracellular matrix permits testicular differentiation in the absence of the mesonephros, and that removal of mesonephric tissues leads to developmental failure of cord formation because the components of the extracellular matrix around pre-Sertoli cells are incomplete. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 46
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    Microscopy Research and Technique 31 (1995), S. 293-299 
    ISSN: 1059-910X
    Keywords: Sample preparation for ATEM ; PEELS imaging ; Zn-Fe intermetallics characterization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The formability of galvanneal steel sheets used in the automotive industry is influenced by the presence and distribution of brittle and difficult to distinguish Zn-Fe intermetallics in the coating. Characterization of these intermetallics requires a high spatial resolution technique such as analytical transmission electron microscopy (ATEM). Sample preparation by ion milling is impossible due to iron redeposition, and traditional ultramicrotomy using water affects the coating chemistry. A technique based on dry ultramicrotomy has therefore been developed.To optimize the technique, different parameters (knife angle, cutting medium, thickness setting on the ultramicrotome, cutting speed) have been investigated for the preparation of galvanneal coatings and pure Al sections. Results show that dry cutting does not affect the coating chemistry but shortens the life of the knife. Knife quality (cleanliness, sharpness and absence of defects) is a major factor to obtain good dry sections. The best results for the more ductile pure Al are obtained with a 35° knife whilst for the harder galvanneal coating it is recommended to use a 55° knife. These results suggest that the sectioning mechanism for the harder material involves more a cleavage-fracture mechanism whilst a greater amount of shear is involved when sectioning relatively ductile Al. The optimum parameters for sectioning galvanneal coatings are established and results obtained by parallel electron energy loss spectrum imaging and energy dispersive X-ray spectrometry in the TEM are given. This study shows that with a good control of all the sectioning parameters it is possible to obtain good sections repeatedly and rapidly. © 1995 Wiley-Liss, Inc.
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  • 47
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    Microscopy Research and Technique 31 (1995), S. 285-292 
    ISSN: 1059-910X
    Keywords: Oxide film ; Barrier film ; Ultramicrotomy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A recent advance in metallurgical technology has been the application of rapid solidification techniques to Al alloy production. FVS0812 is the designation given to a microcrystalline Al-based alloy consisting of 8 wt% Fe, 1 wt% V and 2 wt% Si. It is a two-phase alloy, consisting of ca. 27 vol percent of approximately spherical Fe-V-Si-rich dispersoids in an essentially pure Al matrix. The high strength, low density properties of this advanced material, and other related alloys, have not yet been realized, however, due, in part, to the inability of the alloy to form a thick, adherent, abrasion-resistant outer surface oxide film, a feature readily achieved at conventional Al alloys by normal anodizing methods. The present research has involved an electro-chemical study of oxide film growth at the 812 alloy, with the specific goals being to seek an understanding of the origin of the oxide film growth problem and ultimately to propose alternative approaches to the formation of a thick, stable oxide film at this material. The techniques used in this research have included electrochemical methodologies such as cyclic voltammetry and electrochemical impedance spectroscopy. Crucial information has been obtained through transmission electron microscopy (TEM) of ultramicrotomed specimens. Experiments were carried out initially in neutral borate solutions to characterize the compact barrier oxide film formed in this environment and expected to be present beneath the porous oxide film formed in the normal sulfuric acid anodizing medium. In borate solutions, the electrochemical results implied oxide film thicknesses which were less than seen subsequently by TEM work, suggesting either that the barrier film at the 812 alloy can be penetrated by solution in very fine pores (not resolvable by conventional TEM) at its outer surface or that dispersoids trapped in the oxide film cause differential oxide film thicknesses to develop across the alloy surface. In sulfuric acid solutions, dissolution of Fe and V occurs from the 812 alloy during anodization. Both impedance and TEM studies reveal the absence of a barrier film at the 812 alloy surface. Also, the thick oxide overlayer has a tortuous and more open pore structure than formed at Al and the oxide film is also substantially thinner than it should be. It is suggested that the absence of a barrier oxide film indicates that the sulfuric acid anodizing medium is too aggressive for oxide film formation at the 812 alloy, resulting in excessive dissolution and poor oxide film qualities. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 308-310 
    ISSN: 1059-910X
    Keywords: Thin films ; TEM specimen preparation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Ultramicrotomy has been used to prepare TEM cross-sections of typical hard dielectric, semiconductor, and metal coatings, providing a critical capability in the study of structure-property relationships of thin films. Ultramicrotomy of thin film coatings requires meticulous attention to technique and handling. The sample to be microtomed must be very small, well bonded to the epoxy embedding medium, and precisely oriented. In this article we report the ability to microtome TEM cross-sections of diamond and cubic boron-nitride (cBN) coatings. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 357-363 
    ISSN: 1059-910X
    Keywords: Gap junctions ; Connexins ; Immunofluorescence ; In situ hybridization ; Arrhythmias ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Electrical activation of the heart requires intercellular transfer of current through gap junctions connecting individual cardiac myocytes. Using a combination of light and electron microscopic techniques and molecular approaches, we have characterized the number, size, and spatial distribution of intercellular connections at gap junctions in cardiac myocytes and have also cloned, sequenced, and elucidated the subcellular distribution of three physiologically distinct gap junction channel proteins. In this review, we present evidence to suggest that the spatial distribution of myocyte interconnections and the molecular composition of gap junction channels may confer distinct conduction properties on specific tissues of the mammalian heart such as atrial and ventricular myocardium, and the nodes and bundles of the cardiac conduction system. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 375-386 
    ISSN: 1059-910X
    Keywords: Gap junctions ; Myocardium ; Ischemia ; Hypoxia ; Freeze fracture ; Immunohistochemistry ; Connexin43 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Ischemia causes an increase in myocardial resistivity and a decrease in conduction velocity, thereby enhancing cardiac contractile dysfunction and arrhythmic tendency. Myocardial gap junctions, as principal determinants of conduction velocity, may, therefore, be expected to be deranged in ischemia. Despite a lack of consensus, attempts at correlating gap junction ultrastructural morphology with functional state have revealed the component connexons of gap junctions in freeze-fractured myocardium to be in multiple small hexagonal arrays, tending to become randomly distributed and compacted under uncoupling conditions. Further hypoxic uncoupling causes ultrastructural damage and a reduction in gap-junctional surface area. Immunohistochemical detection of connexin43 gap junctions in chronically ischemic non-infarcted human myocardium demonstrates a reduction in junctional surface area within a normal number of intercalated disks per myocyte, and with a normal distribution of junction sizes. In healed canine infarction there are smaller and fewer gap junctions in the fibrotic myocardium adjacent to infarcts, with reductions in overall gap-junctional content and the proportion of side-to-side vs. end-to-end intercellular connections. Immunohistochemical examination of intact human ventricular myocardium shows the myocytes immediately abutting healed infarcts to hve connexin43 gap junctions spread longitudinally over the cell surfaces, and not in discrete transversely orienated intercalated disks as in normal myocardium. Early after canine infarction, and before fibrotic healing, the connexin43 gap junctions in myocytes abutting the infarct show disorganization similar to that described in healed human infarcts, suggesting that this disturbance is an early pathophysiological cellular response, and not simply due to later fibrotic distortion. Such changes in gap-junctional organization in myocardial ischemia and infarction may be implicated in the elusive link between subcellular structure, contractile dysfunction and arrhythmogenesis. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 396-407 
    ISSN: 1059-910X
    Keywords: Gap junction ; Connexin ; Blood ; Haemopoiesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: More than ten research groups have now reported the presence of gap junctions in blood-forming tissue or cultured cells. It is time to accept that these cell-coupling structures are present in this tissue. To find out what they are doing here we need to develop appropriate experimental techniques. This review covers the particular problems of investigating direct cell-cell communication by gap or other junction in undisturbed haemopoietic tissue. It then describes and assesses the published reports of haemopoietic gap junctions. Recently, in the author's laboratory, three means of increasing the number of gap junctions 50- to 100-fold in mouse marrow have been described, as well as techniques for doing so in culture. There is a complete report of this work here. At present it is quite unclear what function gap junctions serve in blood-formation, perhaps it is some consolation that 30 years after their ultramicroscopic discovery it is also true for all other unexcitable tissues. Possibly the ability to up-regulate their expression in haemopoietic tissue will help us find out what their role is in blood formation. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 437-445 
    ISSN: 1059-910X
    Keywords: Intramembrane particle plaques ; Surface distribution ; Experimental manipulation ; Junction neogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Gap junctions provide the basis for the formation of elaborate networks of communication between cells in animal tissues. Electron microscopic examination of thin sections of plastic embedded gap junctions has provided valuable information on the anatomy and function of these remarkable structures. Freeze-fracture electron microscopy, however, has made available unique vistas of gap junction-bearing intramembrane surface-surface previously inaccessible to the researcher's eyes. Data on population density, distribution, size, geometry of intramembrane particle packing, and structural responses of gap junction components to experimental manipulation are simply and easily obtained with freeze fracture. Recent developments of sophisticated protocols of immunocytochemistry as applied to freeze-fracture replicas further serve to reinforce the notion that freeze-fracture is a powerful tool for study of gap junctions. Molecular techniques of gap junction gene transfection promise to add a truly unique dimension to investigations of the broad spectrum of functional roles of gap junctions. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 446-451 
    ISSN: 1059-910X
    Keywords: Freeze-fracture ; Confocal microscopy ; Gap junction channels ; Mathematical analytical model ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the heart, gap junctions electrically couple myocytes together. Electron- and light-microscope-based analyses have revealed that cardiac gap junctions show a variety of organizational patterns. At the level of gap-junctional channel aggregates, freeze fracture has demonstrated diverse channel packing arrangements in the membranes of different myocardial tissues. Ultrastructural and immunohistochemical studies have shown variation and specialization in the 3-dimensional spatial distribution of gap junctional contacts between different types of myocardial cells. Here, we estimate the access resistance of various configurations of gap junctions using physical principles and explore how certain of these specializations in gap-junctional organization may influence access resistance, a potentially important determinant of electrical conductance between coupled myocardial cells. © 1995 Wiley-Liss, Inc.
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  • 54
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    Microscopy Research and Technique 30 (1995) 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 55
    ISSN: 1059-910X
    Keywords: Spermatozoon ; Principal cell ; Sperm maturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747-766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron-lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis.Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda.These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 37-53 
    ISSN: 1059-910X
    Keywords: Tight junctions ; Epididymis ; Vas deferens ; Freeze-fracture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The present study used the freeze-fracture technique and vascular infusion of horseradish peroxidase (HRP) as a junction permeability tracer, visible in thin sections, to investigate potential seasonal variations in the mink epididymis and vas deferens cell junctions. The junctions were studied in kits during the neonatal period, during and after puberty, and during adulthood monthly throughout the annual reproductive cycle. Results showed the existence, at the time of birth, of a junctional complex joining ductal cells that reached the lumen of the epididymis and vas deferens. The lumenal impermeable segment of the junctional complex was characterized by the presence of an occluding zonule made up of continuous tight junctional ridges extending around the perimeter of the cell. The basal permeable segment of the junctional complex contained mainly discontinuous ridges with frequent forming gap and tight junctions next to adhering junctions. Receding annular junctions were present in the apical and lateral cytoplasm of principal and clear cells. The membrane domain apical to the occluding zonule bore 30-35 nm exo/endocytosis blebs and 40-60 crenations associated with deforming tight and gap junctions made up of randomly scattered particles. Patterns of junctional strands varied greatly from one principal and/or clear cell to another. However, cell junctions did not significantly vary from the neonatal period to adulthood nor from breeding to testicular regression. Anatomical subdivisions of the epididymis with fewer junctional strands per zonule and high diversity in their patterns exhibit no permeability differences to HRP when compared with subdivisions containing more numerous strands. The junctions were impermeable during the neonatal period and throughout the annual reproductive cycle. The following conclusions were reached: (1) a competent occluding zonule developed in the mink epididymis and vas deferens before it did in the testis; (2) the number of strands and the diversity of patterns did not correlate with permeability differences; (3) after the development of a lumen in the testicular excurrent duct, neither receding cellular changes caused by testicular regression nor seasonal passage of a bolus of sperm through the duct modified the occluding zonules; and (4) in the testicular excurrent duct, junction modulation (i.e., formation and deformation) paralleled that in the testis and followed the direction of the synthesis-transport-secretion process. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 1-23 
    ISSN: 1059-910X
    Keywords: Testis ; Epididymis ; Immunocytochemistry ; Glutathione S-transferase ; Subunits (Yf, Yo, Ya, Yc, Yb1, Yb2) ; Detoxify ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Glutathione S-transferases (GSTs) are dimeric proteins that come from a multigene family. They can be grouped into five classes (alpha, mu, pi, sigma, theta) based on the degree of amino acid homology of their subunits. These GST isozymes are able to catalyze the conjugation of glutathione with a wide variety of electrophiles, thereby protecting important cellular constituents from electrophilic attack. In the present study, the distribution of the Ya and Yc subunits from the alpha family, as well as the Yb1 and Yb2 subunits from the mu gene family was examined using immunocytochemistry in the adult rat testis and epididymis. The results of these four GST subunits were also compared with two other subunits, the Yf and Yo proteins, which have already been investigated in our laboratory [Veri et al. (1993), J. Androl., 14:23-44; Veri et al. (in press), J. Androl.]. In the testis, Leydig cells were intensely stained for all six subunits. Within the seminiferous epithelium, Sertoli cells were reactive only for antibodies raised against the Ya, Yb1 and Yf subunits. Among germ cells, all spermatogonia, spermatocytes and step 1-15 spermatids were virtually unreactive for each of the six GSTs. However, moderate to intense staining was seen over steps 16-19 spermatids with the anti-Yo and anti-Ya antibodies, and intense staining over step 19 spermatids with the anti-Yb1, and anti-Yb2 antibodies. In the rete testis, Yf, Yo, Yb1, and Yb2 subunits were intensely reactive over the epithelial cells with weak staining for Yc and no staining for Ya antibodies. Interestingly, in the efferent ducts the Yc, Yb1, and Yf proteins were intensely reactive over ciliated cells, whereas only the Yc protein was intensely reactive over nonciliated cells. In the epididymis, immunoreactivity varied among the principal and basal cells of a given epididymal region for each GST antibody. In the case of principal cells, several of the GSTs showed a similar immunostaining pattern along the tubule. Although not identical in intensity of reaction, the Yc, Yb1, Ya and Yo proteins showed an increase in staining intensity from the proximal to distal segments of the epididymis. In contrast, the Yb2 protein was intensely expressed only in the distal caput with weak levels throughout the rest of the epididymis. The Yf reactivity was strongest from the distal initial segment to the distal caput and unreactive in the corpus and proximal cauda epididymidis. In the case of the Yb2 subunit, reactivity was more intense over the nucleus of principal cells than the cytoplasm in the proximal cauda epididymidis. Similar results were seen for Yb1 protein over principal cells from the middle region of the initial segment to the proximal caput. In the distal caput, principal cells displayed a checkerboard-like staining pattern with the Yb1, and Yf proteins. A population of apically located cells of the proximal initial segment was intensely reactive with the Yb1, and Yo proteins and of the middle initial segment with the Yf protein. Intense staining was also noted for several GSTs in basal cells. The Yf and Yc proteins were stained in basal cells along the entire length of the epididymis. The Yb1, Ya and Yb2 proteins were intensely reactive over basal cells of the corpus epididymidis with weak to no staining in other regions. The Yo protein was negative in basal cells. Clear cells were always unreactive. These results indicate that expression of GST proteins varies considerably along the length of the epididymis. Changes in levels of GST expression in principal and basal cells along the length of the epididymis may result from the need to protect spermatozoa from a changing environment of harmful electrophiles. The results also suggest that expression of the various GSTs is complex and under the control of different regulatory factors. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 30 (1995), S. 67-81 
    ISSN: 1059-910X
    Keywords: Transport ; Proteins ; Ions ; Micropuncture ; Tight junctions ; Permeability ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: To understand the process of sperm maturation, an understanding of interactions between the spermatozoa with the luminal fluid microenvironment and with the epididymal epithelium is necessary. The composition of epididymal luminal fluid of several species is well documented but the manner by which the epididymis contributes to the formation of this specialized milieu is not so well understood. A major role played by the epididymis is to finely regulate the movement of molecules into and out of the lumen. This ensures that as spermatozoa progress along the duct they are exposed to a continually changing, but optimal environment necessary for their maturation and survival. This review focusses on our current understanding of the contributions of the epididymal epithelium to the formation of a specialized luminal fluid microenvironment. The role of the blood-epididymis barrier, the composition of the epididymal luminal fluid, the permeability properties of the epididymal epithelium, and recent studies on a number of luminal fluid proteins and expression of the genes which encode these proteins are discussed. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 82-100 
    ISSN: 1059-910X
    Keywords: Vasectomy ; Epididymis ; Vas deferens ; Hydrostatic pressure ; Antisperm antibodies ; Spermatic granulomas ; Inflammation ; Lysosmes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Common principles can be discerned in the response of the epididymis to vasectomy, despite species differences. Increases in the size and number of lysosomes are the most frequent changes in the epididymal epithelium. The presence or absence of additional alterations such as changes in the height of the epithelium may be related to variations in distensibility of the vas deferens and epididymis. Direct measurements by micropuncture of epididymal and seminiferous tubule hydrostatic pressure indicate that, contrary to dogma, increased pressure in the distal epididymis after vasectomy is not generally transmitted to the seminiferous tubules. The epididymal interstitium shows microscopic changes indicative of chronic inflammation, with infiltration of macrophages, lymphocytes, and plasma cells, and rats with these lesions have higher antisperm antibody levels than animals lacking epididymal changes. Macrophages and neutrophils may enter the duct through the epididymal epithelium, at sites of rupture of the duct, and in the efferent ductules. Cyst-like spermatic granulomas occur in virtually all species where the epididymis or vas deferens ruptures with escape of spermatozoa. The sites and timing of granuloma formation may depend on the mechanical properties of the tract in different species, and they are probably important in the immune response to vasectomy. Postvasectomy sera in Lewis rats recognize a consensus repertoire of dominant autoantigens that closely resembles the antigens bound by sera from rats immunized with isologous spermatozoa. There are multiple routes for disposal of the sperm that continue to be produced after vasectomy. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 101-101 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 30 (1995), S. 138-154 
    ISSN: 1059-910X
    Keywords: Copper oxycarbonates ; Layered structures ; Tc superconductors ; Life and Medical Sciences ; Cell & Developmental Biology
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    Topics: Natural Sciences in General
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    Microscopy Research and Technique 30 (1995), S. 123-137 
    ISSN: 1059-910X
    Keywords: Sr-Cu-O system ; High-Tc superconductors ; Infinite-layer structure ; Planar defects ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The crystal chemistry of the alkaline-earth cupric oxides stabilized at high pressure was studied by high-resolution electron microscopy. Two families of superconductors, (Ca1-ySry)1-xCuO2 and Srn+1Cun O2n+1+δ (n = 1, 2), and one series of semiconductors, Srn-1Cun+1 O2n, are reported. Their structural characteristics are systematically interpreted on the basis of the simple infinite-layer structure seen in SrCuO2. Microstructures and their relevance to high-Tc superconductivity are discussed, particularly in the alkaline-earth-deficient infinite-layer phase. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 102-122 
    ISSN: 1059-910X
    Keywords: High Tc ; Superconductors ; Deformation modulations ; Oxygen ordering ; Superstructures ; YBCO ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A short overview is given of the possibilities of electron microscopy in the determination of the local, atomic scale structure of high Tc superconducting materials. Examples include the detection of weak oxygen ordering, description and characterization of deformation modulations in layered superconductors, and analysis of very long period superstructures. The ordering principles for tetrahedral chains in Ga-, Co-, or Al-substituted YBCO are discussed and their complex defect structures are described. The incommensurate modulation in YBCO-based materials containing SO4-tetrahedra, centered on the Cu(1) sites of the CuO-chain plane is attributed to the ordering of b-oriented SO4-rich chains in the Cu(1)-S-O layer; the structure is described in terms of an SO4-concentration wave. As examples of the new mercury-based superconducting family we discuss Y0.6Ca0.4Ba2Hg1-xMxCu2O6+y, which crystallizes in the space group P4/mmm with a = 0.3870(1) nm, c = 1.2537(1) nm. This cuprate belongs to the 1212 series; susceptibility measurements show a Tc (onset) of 90K, with a diamagnetic volume fraction of 27% at 4.2K to be reached. A second example is related to the compound Tl2HgBa4Cu2O10+y, in which ordering between single Hg layers and double Tl layers is observed. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 155-166 
    ISSN: 1059-910X
    Keywords: High-resolution transmission electron microscopy (HRTEM) ; Oxycarbonate superconductor ; Superstructure ; Modulation structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Arrangements of CO3 groups in various types of oxycarbonate superconductors are examined by high-resolution transmission electron microscopy (HRTEM). Every other B-site of basic perovskite structure is replaced with CO3 groups in the first carbonate superconductor, (Ba0.56Sr0.44)2Cu1.1(CO3)0.9Oy. The 123-related oxycarbonate superconductor obtained in a Y-Ca-Sr-Cu-O system, (Y0.475Ca0.475Sr0.05)Sr2Cu2.4(CO3)0.6Oy, has a superstructure with 2α periodicity due to ordered replacements of Cu-site with CO3 groups. The non-superconducting counterpart with 123-related structure, (Y0.84Sr0.16)2Sr2Cu2.6(CO3)0.4Oy, on the other hand, shows more disordered arrangements of CO3 groups with nearly 3α periodicity. Similar superstructures, due to ordered replacements of Cu sites with CO3 groups, are also observed in the 223-related oxycarbonate superconductors, (Ln,Ce)2Sr2Cu2.5(CO3)0.5Oy (Ln = Ho, Dy). Homologous series of compounds, (CaSr)n+1Cun(CO3)Oy (n = 1-5), consist of alternate stacking of Sr2Cu(CO3)Oy and SrCuO2 (infinite-layer) types of blocks. They become superconductive by additionally doping the BO3 group. Another homologous series of Bi-based oxycarbonate superconductors, (Bi,Pb)2Sr2n+2Cun+1 (CO3)nOy (n = 1-3), contain alternate CuO2 and CO3 layers in between the two (BiO)2 layers. Both mercury (Hg)-and thallium (TI)-based oxycarbonate superconductors, MBa2Sr2Cu2(CO3)Oy (M = Hg or TI) show quite unique modulation structures, where both HgO (or TlO) and CO3 layers repeat in the same plane, along [110] in the Hg compound and [100] in the Tl compound, to form longperiod superstructures with wavy distortion of atom planes. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 167-180 
    ISSN: 1059-910X
    Keywords: Cleaving ; Ion beam thinning ; Ion milling ; Ultramicrotomy ; Jet polishing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We have investigated a wide variety of oxide superconductors and report here on a number of techniques that can be effectively used to prepare transmission electron microscopy (TEM) specimens from these materials. Crushing, cleaving, ion milling, ultramicrotomy, and jet polishing all were successfully utilized, and details of each technique, as well as equipment used, are described. Selection among these methods depends both on the starting form of the material and the information required. Ion milling and crushing generally give the best results and have the widest applicability in our particular work, while crushing and cleaving involve the least equipment cost. In some cases, particularly with ion milling and jet polishing, small variations in the details of preparation have a dramatic effect on the success rate. We have found it to be a great advantage that the same techniques can be applied in a similar manner to a whole range of oxide materials, even (with some refinements and special precautions) to those that are extremely oxygen or moisture sensitive. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 30 (1995), S. 193-207 
    ISSN: 1059-910X
    Keywords: Superconductivity ; n-type superconductors ; Nonstoichiometry ; Superconducting oxides ; T, T′ ; T* structures ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: After reviewing microstructural studies on superconducting materials showing T, T′, and T* structural types, results are presented on the microstructure of some n-type superconductors and related materials prepared with accurate control of the oxygen stoichiometry. Electron microscopy is used to describe the ordering of interstitial oxygen defects in T-type La2NiO4+δ leading to the formation of the n = 2 term of a homologous series with the general formula La8nNi4nO16n+1. Structural transitions and superstructure formation in the Pr2-x-yCexSryCuO4-δ system are reported, where T, T′:;, and T* phases are isolated as a function of both Ce and Sr content. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 30 (1995), S. 181-192 
    ISSN: 1059-910X
    Keywords: Electron Ronchigrams ; Numerical reconstruction ; Lens aberrations ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In a dedicated STEM instrument equipped with a field emission gun, shadow images are easily obtained and have many uses. They are very sensitive to misalignment of the instrument and astigmatism, and therefore can be used for rapid and accurate alignment of the microscope. For crystalline materials, the shadow image contains both the bright-field and dark-field images. It is a summation of the transmitted and diffracted beams, and is basically a kind of Gabor's in-line hologram. Under small or medium defocus, shadow images of a thin, well-orientated crystalline specimen take the characteristic form of Ronchigrams, which offer a unique means to calibrate the microscope operation parameters, such as the spherical aberration coefficient Cs and defocus settings of the objective lens, with high accuracy. With the calibrated values of Cs and δ, a transfer function of the objective lens may be generated. In the stage of numerical reconstruction, by adapting this transfer function to the experimentally recorded hologram the lens aberration introduced in forming the hologram may be corrected and an improved resolution may be achieved for electron microscope images. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 234-247 
    ISSN: 1059-910X
    Keywords: Immunolocalisation ; Electron microscopy ; Ultrastructure ; hSP ; pS2 ; TFG α ; EGFR ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF α) takes its name from one of its alternative ligands, epidermal growth factor and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF α and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF α is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF α but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF α, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF α, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 265-266 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 31 (1995), S. 248-256 
    ISSN: 1059-910X
    Keywords: Esophageal reflux ; Morphometry ; Distinctive cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In Barrett's esophagus, metaplastic columnar epithelium replaces the normal squamous epithelium. The importance of this lesion lies in the increased incidence of adenocarcinoma of the esophagus occuring in patients with Barrett's esophagus. We characterized the surface epithelial cells of Barrett's esophagus using quantitative scanning electron microscopy. Three distinct surface cell types, in addition to the goblet cell, were recognized in Barrett's epithelium: the gastric-like cell and the intestinal-like cell, both of which were similar to normal gastric and small intestinal surface cells, respectively, by quantitative scanning electron microscopy, and the variant cell which had a range of surface features. In four biopsy specimens from the squamo-Barrett's junction in three patients, we found the distinctive cell that had features intermediate between those of squamous and columnar epithelium. On the distinctive cell's surface there are two disparate structures not normally present on the same cell in the gastrointestinal tract: microvilli (a scanning electron microscopy feature of glandular epithelium) and intercellular ridges (a scanning electron microscopy feature of squamous epithelium). The surface characteristics of this cell were almost identical to those of cells found in the transformation zone of the uterine cervix, an area in which squamous epithelium physiologically replaces columnar epithelium. Scanning electron microscopy of Barrett's esophagus has increased our understanding of this precancerous lesion by showing striking cellular heterogeneity. It has also identified the distinctive cell which may represent an intermediate step in the development of Barrett's epithelium during which the surface characteristics of two different cell types, columnar and squamous, coexist in the same cell. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 257-264 
    ISSN: 1059-910X
    Keywords: Cell count ; Cell division ; Computer-assisted image processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The development of computer-assisted image analysis has provided the technology to rapidly determine the population size of cultured cell monolayers in situ. We have adapted this technology to determine the population growth rate of cultured fibroblasts for use in a highreplicate format. Human lung fibroblasts were seeded into 1/2 A 96-well plates that had one-half the culture area of standard 96-well plates. The cells were cultured in medium supplemented with different concentrations of FBS and on days 0, 1, 2, 3, 5, and 7, and their nuclei were stained with propidium iodide. A microscopic field representing one-quarter of a well of fluorescent nuclear images was captured onto a Macintosh computer, and the number of nuclei were counted using an image analysis software program. There were no significant differences between the number of nuclei counted manually and the number counted using computer-assisted software, until day 7 where the cells were multilayered (P 〈 0.05). This image analysis method was compared to other assays typically used to estimate cell proliferation or population size, namely hemocytometer counting, a rapid colorimetric staining assay using naphthol blue-black, and [3H]-thymidine incorporation. The growth rates derived using image analysis were in close agreement with results derived from hemocytometer counts and [3H]-thymidine incorporation. However, the growth rates of cells grown in high concentrations of FBS as determined using naphthol blue-black were substantially lower than results from image analysis. We conclude that this adaptation of computer-assisted image analysis provides a method to derive accurate growth curves by directly counting the number of cells in a large number of replicates. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 267-274 
    ISSN: 1059-910X
    Keywords: Preparation technique ; Artefacts ; Sectioning-induced texture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The methodology and one of the first attempts to produce transmission electron microscopic (TEM) specimens of nanocrystalline metals, alloys and ceramics by ultramicrotomy are presented. Samples of the pure elements Co, Pd; alloys of Y-12 at.% Fe, Al-7 at.% Ag and W-30 at.% Ga; and ZnO ceramic, were found to section successfully to varying degrees. Advantages of sections prepared through ultramicrotomy over ion beam methods include extensive electron-transparent regions of uniform thickness and absence of ion beam damage. Typical artefacts were observed (knife marks, tearing, pull-out, shear lamellae, section curling, and anodic dissolution) but did not impede TEM analysis significantly. A potentially important effect observed was that of a texture development upon sectioning of the Co and Pd samples. It is thought that this unusual phenomenon results from the extremely fine scale of the microstructure and the purity of the Co and Pd samples, and may be enhanced by frictional heating effects and the state of the knife edge. © 1995 Government of Canada.Exclusive worldwide publication rights in the article have been transferred to Wiley-Liss, Inc., in perpetuity.
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    Microscopy Research and Technique 31 (1995), S. 275-284 
    ISSN: 1059-910X
    Keywords: Ultramicrotomy ; Cross-sections ; Epilayers ; Semiconductors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Ultramicrotomy has found novel application in materials research using transmission electron microscopy, for both analytical and high resolution work. Specimens 20-50 nm in thickness with lateral dimensions of up to 100 microns may routinely be prepared, with the additional advantage of precise area location for cross-sectional samples from real devices (e.g., VLSI structures). Much of this work is possible through the availability of diamond knives with various included angles and lengths at reasonable cost. This paper reports on various applications of ultramicrotomy, in particular, lattice imaging of surfaces and interface regions from epilayers of II-VI compound semiconductors and related materials. Ultramicrotomed cross-sections have enabled modern electron beam imaging, diffraction and analytical techniques to be brought to bear near surfaces and across interfaces of multilayer structures, yielding high spatial resolution information on crystallography, defect structure and composition. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 255-262 
    ISSN: 1059-910X
    Keywords: Cavalieri's principle ; Length density ; Morphometry ; Stereology ; Surface density ; Vertical sections ; Vertical slices ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We compare the effectiveness of morphometric methods for extimating lung parameters. Various stereological methods are applied on human lungs and described in detail. The lung volume was estimated by Cavalieri's principle and by fluid displacement. But methods are reliable, but Cavalieri's principle is superior when systematic sections are needed or when volumes of parts of the lung are wanted. Point counting demonstrated that 87.5% of the lung is parenchyma, 5.4% is vessel volume, and 7.1% is bronchia volume. Alveolar surface was estimated on vertical and isotropic uniform random tissue (IUR) sections. The capillary length and length density was estimated on projected images of vertical slices (Gokhale method) and on IUR sections. Only minute differences were found whether IUR sections or vertical sections were used. Of the total variation, approximately 2% was due to the stereological variation and approximately 98% was due to the biological variation on IUR sections and vertical sections. Estimates for volumes, surfaces, and lengths coming from model-based and design-based methods gave similar results for human lungs. In our hands, the design-based methods were easier to use and required less time. However, only the design-based methods offer the guarantee of an unbiased estimate. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 263-263 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 32 (1995), S. 267-285 
    ISSN: 1059-910X
    Keywords: MHC ; Class I ; Class II ; Brain ; Spinal cord ; CNS ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: MHC-restricted T cells are thought to contribute to clinical demyelination in MS and other circumstances. The step-by-step mechanisms involved and ways of controlling them are still being defined. Identification of the MHC+ cells in the CNS in situ has been controversial. This chapter reviews MHC expression in neural tissue, including normal, pathological, experimental, and developing tissue in situ and isolated cells in vitro. A basic pattern is defined, in which MHC expression is limited to nonneural cells and strongest class I and II expression are on different cell types. Variations from the basic pattern are reviewed. Ways of reconciling divergent findings are discussed, including the use of “mock tissue” to help choose between technical and biological bases for divergent findings, the potential contribution of internal antigen to the in situ staining patterns, and the possibility that class I upregulation is actively suppressed in situ. Functional implications of the observed patterns of MHC expression and ways of confirming the function of each MHC+ cell type in situ are described. It is suggested that modulating MHC expression in different cell types at different times or in different directions might be desirable. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 467-468 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 31 (1995), S. 469-469 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Microscopy Research and Technique 31 (1995), S. 488-496 
    ISSN: 1059-910X
    Keywords: Lectins ; Ampulla ; Isthmus ; Rabbit ; HCG ; Estradiol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Localization of individual glycosidic residues and sialic acid acceptor sugars was investigated by conjugated lectins in the rabbit oviduct under physiological hormonal conditions and human chorionic gonadotropin (HCG) administration. Ampulla and isthmus were found to exhibit lectin binding profiles typical of each hormonal stage. Two different sialylated glycomolecules were identified within the epithelial lining; in particular, sialoglycoconjugates characterized by the terminal sequence sialic acid-galactose were visualized in the secretory cells and the sialic acid-N-acetylgalactosamine terminal disaccharides were localized on both ciliated and secretory cells of the entire oviduct. Surface and cytoplasmic sialoglycoconjugates were also found to exhibit a differential behaviour inside the two oviduct tracts examined. Present findings further supported the idea that in ampulla and isthmus, the greatest modifications consequent to hormone treatment take place at different times. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 31 (1995), S. 507-518 
    ISSN: 1059-910X
    Keywords: Matrigel ; In vitro differentiation ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This report describes an easy method of isolation and cell culture of the epithelial cells of cow oviduct. Incubation of cow oviduct with 0.1 mg/ml collagenase in the lumen for 90 minutes helped to dislodge large numbers of ciliated and secretory epithelial cells. The isolated cells, when seeded on plastic, proliferated very quickly and became confluent in 8-10 days in 35 mm Petri dishes. The isolated ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The cultured epithelial cells were keretin positive. The isolated bovine oviduct epithelial cells, when cultured on plastic precoated with 10 mg/ml matrigel, organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. The epithelial cells seeded on 2 mg/ml matrigel became subconfluent in 15-20 days after seeding. The histoarchitecture of the secretory cells growing in vitro on matrigel resembled that of intact oviduct secretory epithelial cells. Occasional ciliated cells containing large number of mitochondria were observed in the monolayer cultured on 2 mg/ml matrigel substratum but possessed few cilia. The oviduct epithelial cells cultured on 2 mg/ml matrigel incorporated 35S-methionine linearily into protein up to 8 hours in the presence of estradiol or progesterone. The fluorograph of the newly synthesized proteins indicated the presence of an additional 60 kd protein in the cell extract of epithelial cells incubated with estradiol. © 1995 Wiley-Liss, Inc.
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    Microscopy Research and Technique 32 (1995), S. 449-454 
    ISSN: 1059-910X
    Keywords: Calibration sample ; Transmission electron microscope ; Magnification steps ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A calibration sample for transmission electron microscopy (TEM) has been developed that performs the three major instrument calibrations for a transmission electron microscope: the image magnification calibration for measurements of images, the camera constant calibration for indexing diffraction patterns, and the image/diffraction pattern rotation calibration for relating crystal directions to features in the image. This offers an improvement over commercially available calibration standards, where up to five different samples are required to perform these three calibrations. The new calibration sample consists of an electron-transparent cross-sectional TEM sample made from a molecular beam epitaxy (MBE)-grown, single-crystal semiconductor wafer. When the calibration structure is viewed in a TEM, it appears as a series of light and dark layers where the layer thicknesses are very accurately known. The calibrated thickness measurements of these light (silicon) and dark (SiGe alloy) layers are based on careful TEM measurements of the {111} lattice spacing of silicon which is visible on the calibration sample itself, and are supported by X-ray diffraction measurements. Furthermore, the layer thickness variation across the entire silicon wafer has been verified to be less than 1%, allowing all samples prepared from the same wafer to have errors in the given layer thickness values of less than 1%. As the sample is a single crystal of silicon, the calibrations requiring electron diffraction information such as the camera constant calibration and the image/diffraction pattern rotation calibration can also be performed easily and unambiguously. One single calibration sample can therefore be used to provide all three of the major TEM instrument calibrations at all magnifications and all camera lengths. © 1995 Government of Canada.Exclusive worldwide publication rights in the article have been transferred to Wiley-Liss, Inc., in perpetuity.
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  • 85
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    Microscopy Research and Technique 32 (1995), S. 520-532 
    ISSN: 1059-910X
    Keywords: Perinuclear ; theca proteins ; Spermiogenesis ; Acrosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The perinuclear theca (PT) is a unique cytoskeletal element that encapsulates the mammalian sperm nucleus. It is divided into subacrosomal and postacrosomal regions. The objective of this study was to analyze and stage the intracellular distribution of several promiment bull PT proteins during spermatogenesis. For this purpose, polyclonal antibodies raised and affinitypurified against the 15.5-, 25-, 28-, 32-, 36-, and 60-kDa bull PT polypeptides were used as probes on sections of aldehyde-fixed testes. Immunoperoxidase staining revealed that the PT polypeptides first appeared early in spermiogenesis, concomitant with early steps of development of the acrosomic system. Immunogold labeling further showed that these polypeptides were peripherally associated with the entire acrosomal membrane, before and during the attachment of the acrosomic vesicle onto the spermatid's nucleus. Once the acrosome had capped the nucleus the labeling resided mainly in the subacrosomal region of the spermatid, between the inner acrosomal membrane and nuclear envelope. Later, during the elongation of the spermatid's nucleus, the labeling with all antibodies except the anti-15.5-kDa antibody extended caudally over the assembling postacrosomal sheath. This study suggests that the perinuclear theca proteins play an instrumental role in the attachment, spreading, and binding of the acrosome onto the nucleus of spermatids. © 1995 Wiley-Liss, Inc.
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  • 86
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    Microscopy Research and Technique 32 (1995), S. 459-497 
    ISSN: 1059-910X
    Keywords: Anamniotes ; Spermatocysts ; Amniotes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Spermatogenesis appears to be a fairly conserved process throughout the vertebrate series. Thus, spermatogonia develop into spermatocytes that undergo meiosis to produce spermatids which enter spermiogenesis where they undergo a morphological transformation into spermatozoa. There is, however, variation amongst the vertebrates in how germ cell development and maturation is accomplished. This difference can be broadly divided into two distinct patterns, one present in anamniotes (fish, amphibia) and the other in amniotes (reptiles, birds, mammals). For anamniotes, spermatogenesis occurs in spermatocysts (cysts) which for most species develop within seminiferous lobules. Cysts are produced when a Sertoli cell becomes associated with a primary spermatogonium. Mitotic divisions of the primary spermatogonium produce a cohort of secondary spermatogonia that are enclosed by the Sertoli cell which forms the wall of the cyst. With spermatogenic progression a clone of isogeneic spermatozoa is produced which are released, by rupture of the cyst, into the lumen of the seminiferous lobule. Following spermiation, the Sertoli cell degenerates. For anamniotes, therefore, there is no permanent germinal epithelium since spermatocysts have to be replaced during successive breeding seasons. By contrast, spermatogenesis in amniotes does not occur in cysts but in seminiferous tubules that possess a permanent population of Sertoli cells and spermatogonia which act as a germ cell reservoir for succeeding bouts of spermatogenic activity. There is, in general, a greater variation in the organization of the testis and pattern of spermatogenesis in the anamniotes compared to amniotes. This is primarily due to the fact there is more reproductive diversity in anamniotes ranging from a relatively unspecialized condition where gametes are simply released into the aqueous environment to highly specialized strategies involving internal fertilization. These differences are obviously reflected in the mode of spermatogenesis and this is particularly true of the stage of spermiogenesis where the morphology of the species-specific spermatozoon is determined. Moreover, unlike amniotes, many anamniotes display a spermatogenic wave manifest, depending upon the species, either at the level of the cyst or seminiferous lobule.This variation in the organization of the testis makes certain anamniotes perfect models for investigating germ cell development and maturation. For instance, the presence of a spermatogenic wave provides an opportunity to manually isolate discrete germ cell stages for analysis of specific Sertoli/germ cell interactions. Furthermore, for many anamniotes, germ cells mature in association with a morphologically poorly developed Sertoli cell. This seeming independence of Sertoli cell regulation allows the in vitro culture of isolated germ cells of some species of anamniotes through several developmental stages. Thus, due either to the anatomical organization of the testis, or structural simplicity of the germinal units, nonmammalian vertebrates can provide excellent experimental animal models for investigating many basic problems of male reproduction. © 1995 Wiley-Liss, Inc.
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  • 87
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    Microscopy Research and Technique 32 (1995), S. 148-163 
    ISSN: 1059-910X
    Keywords: Secretory granules ; Calcium ; Parathyroid hormone storage and release ; Hydrolysis ; Trans Golgi network ; Vacuolar bodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Both prosecretory and storage granules are concomitantly formed at the trans Golgi network including the innermost Golgi cisterna. Prosecretory granules develop into small secretory granules that release their contents by exocytosis finely regulated by a complex mechanism for maintaining calcium homeostasis. In the rat parathyroid cells, storage granules are large secretory granules storing parathyroid hormone for an emergency supply. The hormone is rapidly discharged by exocytosis when serum calcium concentration is decreased. The granules are constantly produced even under conditions of low serum calcium concentration in the regions of 8 mg/dl. The granule content is constantly hydrolyzed when not discharged, leading to a decreased core and finally to the formation of vacuolar bodies. The fate of the vacuolar bodies is unknown. Hypercalcemic conditions accelerate hydrolysis. The threshold value of calcium concentration required for the release of storage granule contents is between 8.0 and 7.5 mg/dl and that of calcium concentration for accelerating degradation of storage granules is about 11.5 mg/dl. Sympathetic stimulation causes storage granules to be discharged regardless of hypercalcemia or hypocalcemia. Parasympathetic stimulation accelerates hydrolysis. The degradation of storage granules seems to be closely associated with an intracellular regulatory mechanism for parathyroid hormone secretion. © 1995 Wiley-Liss, Inc.
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  • 88
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    Microscopy Research and Technique 32 (1995), S. 164-179 
    ISSN: 1059-910X
    Keywords: Parathyroid gland ; Hyperparathyroidism ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Parathyroid glands (n = 271) removed from 130 patients were examined by light and electron microscopy. A standardized method of tissue processing was employed and morphometry was performed. The aim of the paper is to provide a description of the human parathyroid chief cell ultrastructure in health and disease, with quantitative evaluation of structures involved in secretion of parathyroid hormone in a large case series, and to discuss their role in current diagnostic histopathology. The patients were euparathyroid (n = 10), or affected by primary (n = 97), secondary (n = 8), or tertiary (n = 15) hyperparathyroidism. In normal glands, solid parenchyma was composed of chief cells, large clear cells, transitional-oxyphil cells, and oxyphil cells. Chief cell hyperplasia, pseudo-adenomatous hyperplasia, adenoma, water-clear cell hyperplasia, and carcinoma were the most usual forms of parathyroid disease responsible for primary hyperparathyroidism. In chief cell hyperplasia, all the parathyroid glands were enlarged and the chief cells were in an active state of hormone secretion, with a large Golgi complex, abundant rough endoplasmic reticulum (RER), small lipid droplets, and tortuous plasma membrane. In pseudo-adenomatous hyperplasia, one gland was enlarged and the others displayed a normal size; however, electron microscopic examination and morphometric analysis showed that all the glands had active cells. Adenomas displayed a pattern similar to those of pseudo-adenomatous hyperplasia, with one gland enlarged and the others of normal size. However, ultrastructural examination and morphometry showed that the normal-size glands were hypo-active. Water-clear cell hyperplasia showed cells filled with cytoplasmic vacuoles. In these cells, structures with intermediate features between secretory granules and vacuoles were visible. Nucleo-cytoplasmic atypias were frequently visible in parathyroid carcinoma cells. In secondary and tertiary hyperplasia, active chief cells were regularly mixed with oxyphil or transitional-oxyphil cells. The tertiary hyperplasia was characterized by RER-associated structures that were not found in the normal or other pathologic conditions. These results demonstrate that electron microscopy and morphometry represent useful tools in parathyroid histopathology. © 1995 Wiley-Liss, Inc.
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  • 89
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    Microscopy Research and Technique 32 (1995), S. 204-214 
    ISSN: 1059-910X
    Keywords: Central and peripheral nervous systems ; Demyelination ; Remyelination ; Oligodendrocytes ; Schwann cells ; Globoid cell leukodystrophy ; Krabbe disease ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Twitcher mouse is an authentic murine model of human genetic demyelinating disease, globoid cell leukodystrophy (GLD), or Krabbe disease. Since its discovery at the Jackson Laboratory (Bar Harbor, ME) this model has been used extensively for the morphological, biochemical-enzymatic studies to clarify pathogenesis and also for therapeutic manipulation of genetic demyelinating disease in humans. As a result of these studies, now we know that (1) GLD is caused by a deficiency of lysosomal enzyme galactosylceramidase, and a toxic metabolite, psychosine, accumulates in the tissue, including the nervous system, damaging myelin forming cells and resulting in secondary demyelination; (2) morphological features of demyelination and associated cellular reactions in demyelination in this mutant are similar to those seen in autoimmune or toxic demyelination; and (3) with enzyme supplementation provided by bone marrow transplantation, remyelination occurs to some extent in demyelinated fibers in both central and peripheral nervous systems of twitcher mouse. © 1995 Wiley-Liss, Inc.
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  • 90
    ISSN: 1059-910X
    Keywords: EAE ; TMEV ; Demyelination ; Remyelination ; Multiple sclerosis ; Oligodendrocyte ; Schwann cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Theiler's murine encephalomyelitis virus (TMEV) infection and experimental allergic encephalomyelitis (EAE) are considered among the best models of human multiple sclerosis (MS). In both models, clinical disease is characterized by paralysis, while pathological changes consist of inflammatory demyelination. In both models there is a genetic influence on susceptibility/resistance to the development of disease. This has been thoroughly studied in TMEV infection, and it has been found to depend on both major histocompatibility complex (MHC) and non-MHC genes. At least four genes have been so far identified. Because of this genetic influence, some strains of mice are more susceptible to both clinical and pathological changes than others, and susceptibility appears to best correlate with the ability of a certain murine strain to develop a delayed-type hypersensitivity (DTH) response to viral antigens. We have also observed that even among mice which are equally susceptible clinically, striking differences may be seen under pathological examination. These consist of different gradients of severity of inflammation, particularly in regards to the macrophage component. There is an inverse relationship between the number of macrophages, and their length of stay in the CNS, and the ability of mice to remyelinate their lesions. The most severe lesions are in SJL/J mice, and remyelination in this strain is extremely poor. The least severe lesions in terms of macrophage invasion are in strains such as NZW and RIIIS/J, and these are able to remyelinate lesions very successfully. Murine chronic relapsing EAE (CR-EAE) shows pathological changes in many ways similar to those in TMEV-infected SJL/J mice, although less severe in terms of degrees of macrophage infiltration and tissue destruction. Mice with CR-EAE have a correspondingly limited ability to remyelinate their lesions. In both models the pathology appears to be mediated through a DTH response. However, while in EAE the DTH response is clearly against neuroantigens, the response in TMEV infection is against the virus itself. The end result in both models would be that of myelin destruction through a lymphotoxincytokine-mediated mechanism. The importance of the DTH response in both models is well illustrated by the effects of tolerance induction in EAE and TMEV infection to neuroantigens and virus, respectively. These are important models of human MS, since the current hypothesis is that a viral infection early in life, on the appropriate genetic background, may trigger a secondary misdirected immune response which could be directed either against myelin antigens and/or possible persistent virus(es). © 1995 Wiley-Liss, Inc.
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  • 91
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    Microscopy Research and Technique 32 (1995), S. 264-265 
    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 32 (1995), S. 286-294 
    ISSN: 1059-910X
    Keywords: Oligodendrocyte ; Myelin ; Myelin basic protein promoter ; Multiple sclerosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Why is it that oligodendrocytes do not normally express major histocompatibility complex (MHC) molecules? To examine the effect of aberrant MHC expression in oligodendrocytes, transgenic mice have been produced which expressed the class I MHC gene, H-2Kb, under direction of the MBP promoter [Turnley et al. (1991b) Nature, 353:566-569; Yoshioka et al. (1991) Mol. Cell. Biol., 11:5479-5486]. A proportion of these mice exhibited a shivering phenotype, with tonic seizures and early death. Oligodendrocyte function and viability was shown to be affected, resulting in severe dysmyelination of the CNS. Is this phenomenon of cell damage due to aberrant expression of MHC molecules restricted to oligodendrocytes, and could other, non-MHC molecules, when aberrantly expressed, result in similar cell damage? This paper discusses these questions and examines possible mechanisms for the oligodendrocyte damage and hypomyelination observed in these transgenic mice. Finally, the implications of aberrant MHC expression in oligodendrocytes for demyelinating diseases such as multiple sclerosis are discussed. © 1995 Wiley-Liss, Inc.
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  • 93
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    Microscopy Research and Technique 32 (1995), S. 363-363 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 94
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    Microscopy Research and Technique 32 (1995), S. 385-422 
    ISSN: 1059-910X
    Keywords: Spermatogenesis ; Germ cell degenerations ; Spermatogenic cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. A measure of efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. This measure is not influenced by species differences in testicular size; however, it is influenced by species differences in the numerical density of germ cells and in the life spans of these cells. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells), and germ cells (spermatogonia, spermatocytes, and spermatids). Activity of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number and yield four spermatids. Spermiogenesis is the differentiation of spherical spermatids into mature spermatids which are released at the luminal free surface as spermatozoa. The spermatogenic Cycle is superimposed on the three major divisions of spermatogenesis. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. Efficiency of spermatogenesis is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging of humans and animals. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells (but not Leydig cell number) are related to efficiency of spermatogenesis. In humans, efficiency of spermatogenesis is reflected in number of spermatogenic stages per cross-section and number of missing generations within each stage; however, the arrangement of stages along the tubular length does not reflect differences in the efficiency of spermatogenesis. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon, and daily sperm production per g parenchyma is a measure of its efficiency. © 1995 Wiley-Liss, Inc.
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  • 95
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    Microscopy Research and Technique 32 (1995), S. 457-458 
    ISSN: 1059-910X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A miniature vise built into a 5 mm diameter copper capsule is described that holds small pieces of prefrozen, hydrated specimens at low temperatures within the lens of the Hitachi S900 high-resolution scanning electron microscope.
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  • 96
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    Microscopy Research and Technique 32 (1995), S. 498-503 
    ISSN: 1059-910X
    Keywords: Colocalization ; In situ hybridization ; Immunocytochemistry ; Testis ; Digoxigenin ; Transition proiein-1 ; Sulfated glycoprotein-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that (1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and (2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized. © 1995 Wiley-Liss, Inc.
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  • 97
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    Microscopy Research and Technique 32 (1995), S. 357-361 
    ISSN: 1059-910X
    Keywords: Scanning electron microscopy ; Human neutrophils ; Tissue preparation ; Surface morphology ; Cell volume ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Using human leukocytes as test specimens, three different drying procedures for scanning electron microscopy: critical-point drying (CPD), Peldri II, and tetramethylsilane (TMS), were compared. All three procedures produced identical surface morphology preservation. An equal amount of volume shrinkage was observed regardless of the dehydrants and drying techniques employed. Considering the simplicity, convenience, and time saved, air-drying with TMS is by far the best choice for preparing animal cells for scanning electron microscopy. © 1995 Wiley-Liss, Inc.
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  • 98
    ISSN: 1059-910X
    Keywords: Golgi apparatus ; Kinesin ; Sertoli cells ; Microtubules ; Cell polarity ; Tight junction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Sertoli cells are polarized epithelial cells of the seminiferous epithelium which provide structural and physiological support for differentiating germ cells. They establish different basal and adluminal environments for the selective nurturing of pre- and post-meiotic germ cells within the seminiferous epithelium, segregated by the Sertoli-Sertoli cell tight junctional complex, the blood-testis barrier. Tight junction formation between epithelial cells in vitro is a critical polarizing event associated with changes in polarized targeting of membrane-specific proteins and reorganization of microtubules, centrioles, and the Golgi apparatus. To investigate whether tight junction formation is associated with organelle reorganization in Sertoli cells in vivo, we have characterized distribution patterns of Sertoli cell microtubules, the mechanoenzymes kinesin and cytoplasmic dynein, and the Golgi apparatus during tight junction formation in developing rat testis. Immunocytochemistry on samples taken at 5, 10, 15, 20, and 25 days of age was used to examine the distribution of these proteins during the extensive cellular reorganization that culminates in the formation of the blood-testis barrier at 19 days of age. Our data show that the distribution patterns reflect the extensive intercellular repositioning of tubule cells in developing seminiferous tubules, but that changes in intracellular organization are not temporally associated with formation of the blood-testis barrier. © 1995 Wiley-Liss, Inc.
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  • 99
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    Microscopy Research and Technique 32 (1995), S. 533-552 
    ISSN: 1059-910X
    Keywords: Fish ; Teleosts ; Elasmobranchs ; Spermatogenesis ; Testis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In this paper we present the state of knowledge on cell-cell interactions in the testis of two groups of anamniote vertebrates - teleosts and elasmobranchs - which include most fish. In these fish, the structural organization of the testis differs fundamentally from that which characterizes amniotes in which the germinal tissue is located in tubules open at both ends and consists of a permanent population of Sertoli cells associated with successive stages of germ cell development. In fish, the spermatogenic unit of testis is the spermatocyst, which corresponds to one germ cell or to a clone of isogenetic germ cells, enclosed by one or several Sertoli cells, which form the wall of the cyst. In fish testis, the Sertoli cells do not represent a permanent population of cells. Although both are of the cystic type, the teleost and elasmobranch testes are differently organized. In elasmobranchs, primary spermatogonia and Sertoli cells lie initially free within the interstitial tissue, before becoming sequestered by a basement membrane; the testis is then composed of a mass of spermatocysts which contain many Sertoli cells, each being associated with a clone of germ cells. In contrast, in teleosts, the cysts are confined to large elongated structures limited by a basement membrane. These structures are either lobules originating under the albuginea or tubules which, in contrast to those of mammals, are anastomosed. In the lobules, the spermatocysts start to develop at the blind end of the lobules and migrate towards the efferent system, whereas in the tubules, the spermatocysts are located against the basement membrane, all along the tubules and do not migrate. In elasmobranchs, unlike teleosts, Leydig cells are either absent from the interstitial tissue or rare and undifferentiated and their role in steroid production is at best marginal.While many studies have focused on topographical and functional interactions between the diverse cell types present in mammalian testis, only a few studies have brought particular attention to these aspects in fish. In fish, like in mammals, testicular cell-cell interactions are based on structural elements and chemical factors. Occasionally, various adhering junctions have been observed, essentially in teleosts, between Sertoli cells, between Sertoli cells and germ cells, between germ cells themselves, and interstitial cells. Furthermore, in some teleost species, using horseradish peroxidase or lanthanum salts, the presence of tight junctions between Sertoli cells has been correlated to the occurrence of a Sertoli barrier. In these species, the barrier develops after meiosis so that only haploid germ cells are shielded from the vascular system. In fish, recent development of techniques which enable the preparation and in vitro culture of enriched populations of testicular cells and of spermatocysts, has allowed investigations on functional aspects of cell-cell interactions. In particular, data have been obtained, in the trout, on the control of spermatogonia proliferation by Sertoli cell-conditioned media and, in the dogfish, on the steroidogenic activity of Sertoli cells, in relation to the differentiation stage of the associated germ cells. Furthermore information exists, in the trout, showing that intratubular macrophages may participate in the re-initiation of spermatogonial proliferation.In conclusion, the cytoarchitecture of fish testis, as compared to that of mammals, presents original feátures which provide unique opportunities to develop fruitful studies for a better understanding of the complex control mechanisms underlying testicular function in vertebrates. © 1995 Wiley-Liss, Inc.
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  • 100
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    Microscopy Research and Technique 31 (1995), S. 300-307 
    ISSN: 1059-910X
    Keywords: TEM ; Ion beam ; Chemical jet ; Diamond knife ; SAED ; Alumina ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Industrial materials, such as alumina, often pose difficulties in their preparation for TEM examination. Composite and particulate materials are particularly difficult to prepare using conventional thinning techniques, i.e., ion beam and chemical jet thinning. Ultramicrotomy (UM) can be used to produce TEM specimens with a uniform thickness and an unaltered composition.Some crystalline materials, i.e., alumina hydrate, were difficult to section due to conflict between the cutting direction and cleavage planes. Sectioning was successful when these two directions were mutually parallel or perpendicular. At other orientations shattering occurred. Microcrystalline particulate materials, i.e., calcined alumina, were sectioned successfully in particles 〈 70 μm in diameter.The phases found in industrial alumina particles were gamma, delta, theta, and alpha alumina. Gamma alumina consisted of fine-grained, equiaxed crystallites. The selected area electron diffraction (SAED) patterns indicated poor crystallinity with a distinct hexagonal texture. Delta and theta alumina appeared as an undifferentiated intermediary microstructure of elongated grains. The SAED patterns indicated poor crystallinity, but without a distinct texture. Alpha alumina was found to be a coarse-grained crystalline phase with high diffraction contrast. SAED patterns consisted of fine, randomly oriented spots.Considerable variation was observed in the distribution of phases. In some specimens, discrete particles of gamma and alpha predominated. In others, particles were a mixture of phases repre-sentative of the bulk composition. To characterise these samples, TEM of numerous whole particles was required. Ultramicrotomy was the only preparation technique capable of producing such samples. © 1995 Wiley-Liss, Inc.
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