bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quick‐Freeze, deep‐etch studies of renal basement membranes
Authors: Kubosawa Hitoshi , Kondo Yoichiro 
Published: February 2005

AbstractThe fine structure of the renal (i.e., glomerular, tubular, and capillary) basement membranes was re‐evaluated with the aid of a deep‐etch replica method. The structure of the laminae rarae interna and externa of the rat glomerular basement membrane (GBM) and laminae lucida of other basement membranes were basically identical in that 6 to 8 nm fibrils were interconnected to form a three‐dimensional, polygonal network. By contrast, all of the laminae densa examined were composed of closely packed granules, and a filamentous substructure was identified only in a limited area. These granular components were demonstrated to be an integral component of the lamina densa. From additional observations on the trypsinized bovine GBM, it appeared that the basic structure of renal basement membranes was almost identical, namely, that a three‐dimensional fibrillar meshwork existed throughout the individual layers to form a structural framework upon which fine particles were variably attached. In addition, we observed some of the fine structure of the pars fibroreticularis; the laminae densa of the tubular and capillary basement membranes continued to the fibrillar meshwork resembling the structural backbone of the glomerular basement membrane. The network was sometimes directly connected to the extracellular matrix, but more often changed into a rough fibrillar framework and connected to the extracellular matrix. © 1994 Wiley‐Liss, Inc.
Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis
Authors: Huang Cheng X. , Canny Martin J. , Oates Kenneth , McCully Margaret E. 
Published: February 2005

AbstractA procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully‐hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at ‐80°C in cryo‐ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (‐90°C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo‐preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al‐coated planed face is the increased accuracy of energy‐dispersive X‐ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face. © 1994 Wiley‐Liss, Inc.
Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes
Authors: van Kuppevelt Toin H. M. S. M. , Veerkamp Jacques H. 
Published: February 2005

AbstractThe application of cationic probes for the ultrastructural detection of proteoglycans in basement membranes is reviewed. Proteoglycans are highly negatively charged macromolecules due to their glycosaminoglycan side chains. The interaction of cationic probes with proteoglycans is of an electrostatic nature. Methods are discussed to increase the specificity of probes for proteoglycans. The use of phthalocyanin‐like dyes such as Cuprolinic blue, according to the critical electrolyte concentration method, results in a selective staining of proteoglycans. Enzymatic or chemical digestions, however, should be done to validate the proteoglycan nature of the dye‐positive granules/filaments, and to establish the class of proteoglycan. The value of cationic probes in basement membrane research on development and pathology is discussed. The potential for deducting molecular information from the ultrastructural appearance of stained proteoglycans is indicated. © 1994 Wiley‐Liss, Inc.
Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy
Authors: Stearns Rebecca C. , Katler Marshall , Godleski John J. 
Published: February 2005

AbstractThe detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique. © 1994 Wiley‐Liss, Inc.
Vascular basement membrane components and the lesions of Alzheimer's disease: Light and electron microscopic analyses
Authors: Perlmutter Lynn S. , Myers Martha A. , Barrón Ernesto 
Published: February 2005

AbstractAlzheimer's disease (AD) is one of several systemic and cerebral diseases that involve the abnormal deposition of fibrillar proteins called amyloids. All amyloids share conformational and staining characteristics, as well as an association with resident tissue macrophages and two extracellular matrix components [heparan sulfate proteoglycan (HSPG) and amyloid P component]. Vascular, glomerular, and Schwann cell basement membrane pathologies have been documented in many forms of amyloidosis, and often amyloid fibrils fuse to and project from the basement membrane in these diseases. The present report demonstrates the vascular basement membrane (VBM) alterations in AD autopsy samples, and details the methodologies used.Electron microscopy reveals the fusion of amyloid fibrils with the VBM and the alteration of the VBM in the absence of amyloid accumulation. Double‐labelling and pre‐embed immuno‐electron microscopy techniques demonstrate the colocalization of amyloid P component and VBM components with amyloid, and also reveal that amyloid P component is not localized to the cerebral VBM. Finally, a novel correlative light/electron microscopy technique demonstrates the association between amyloid P component and cerebral resident tissue macrophages, the microglia. Taken together, these data suggest that the physicochemical processes of amyloid formation, rather than amyloid deposition, may be responsible for VBM pathology. © 1994 Wiley‐Liss, Inc.
High voltage immunoelectron microscopy of complement receptor type 3–mediated capping and internalization of group a streptococcal cell walls by Human Neutrophils
Authors: Pryzwansky Katherine B. 
Published: February 2005

AbstractThe mechanism of human neutrophil clearance of peptidoglycan group A–specific polysaccharide polymers derived from streptococcal cell walls (PG‐APS) was investigated by high voltage immunoelectron microscopy (HVEM) in order to determine how neutrophils process this highly inflammatory bacterial debris. Neutrophil monolayers were incubated from 5–30 min with serum‐opsonized PG‐APS. Cells were lightly fixed with 0.5% glutaraldehyde, and the PG‐APS was localized on the neutrophil surface by immunogold using antibodies to N‐acetyl‐glucosamine and 15 nm colloidal gold coupled to goat anti‐rabbit IgG. Neutrophils were viewed unsectioned by stereo HVEM. Patches of PG‐APS were distributed randomly on the plasmalemma of well‐spread neutrophils within 5 min. In polarized cells, PG‐APS was densely localized on the uropod and retraction fibers. Within 15 min, PG‐APS was predominantly concentrated into a large aggregate, measuring approximately 1 μm in diameter, near the cell margin or nucleus. The aggregate of PG‐APS was engulfed in the vicinity of the indentation of the nucleus (hof). Intact microfilaments were required for aggregation and internalization of PG‐APS. Binding of PG‐APS was dependent upon complement fixation. Furthermore, PG‐APS elicited an increase in density of complement receptor type 3 (CR3, C3bi receptor) on the neutrophil surface as determined by morphometry of immunogold labeled anti‐CR3. When cells were stained for both PG‐APS and CR3, co‐localization was observed, and stereomicroscopy revealed clusters of CR3 in areas associated with phagocytosis. These data suggest that neutrophils use an efficient mechanism for removal of bacterial debris. Unlike whole streptococci which are phagocytosed at multiple sites, these bacterial cell walls are first collected into a large aggregate, or cap, which is then internalized at one site. © 1994 Wiley‐Liss, Inc.
Age‐Related changes in cartilage proteoglycans: Quantitative electron microscopic studies
Authors: Buckwalter Joseph A. , Roughley Peter J. , Rosenberg Lawrence C. 
Published: February 2005

AbstractBiochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age‐related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate–rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age‐related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age‐related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age‐related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate–rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age‐related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules. © 1994 Wiley‐Liss, Inc.
Z‐contrast imaging of supported Pt and Pd clusters
Authors: Bradley S. A. , Cohn M. J. , Pennycook S. J. 
Published: February 2005

AbstractHigh‐angle annular dark‐field or Z‐contrast microscopy was used to demonstrate that well dispersed metal supported catalysts consist of nanometer sized clusters. Depending upon the impregnated metal, different cluster sizes were observed. Grouping of Pd clusters could also be confirmed by analytical electron microscopy. © 1994 Wiley‐Liss, Inc.
Characterization of simian immunodeficiency virus (SIV) infected AA‐2 cells by SEM and immunoelectron microscopy
Authors: Norton William N. , Brown Charles R. , Lewis Mark G. , Rippy Marian K. , Martin Jennifer E. , Zack Philip M. 
Published: February 2005

AbstractThe ultrastructural features of AA‐2 cells infected with either of two strains of simian immunodeficiency virus (SIVMne‐E11S or SIVSMM‐PBj) were examined by scanning electron microscopy (SEM). Transformed CD4+ human B lymphocytes (AA‐2) were inoculated with SIV and observed at 2, 4, and 7 days post‐inoculation (dPI). Infected AA‐2 cells were distinguished by the progressive loss of microvilli, and variable numbers of free or protruding spherical particles measuring 90–120nm in diameter along the cell surface. Syncytial cell formation (complexes of fused cells) and necrotic cells were evident at each time point with the most numerous observations at 7 dPI. While the distribution and severity of the viral induced changes increased with time and affected virtually all cells by 7 dPI, the alterations were detected sooner and were more pronounced in SIVSMM‐PBj infected cells. This finding is consistent with the in vivo data from primate studies using the same strains of SIV. Syncytial cells exhibited slight to moderate indentations which appeared to coincide with the boundaries of individual cells forming the complex. The plasma membrane of syncytial cells was relatively smooth and lacked microvilli. Spherical particles and buds protruding from the plasma membrane were predominate features of syncytial cell surfaces. By the employment of antisera generated against whole SIVMne‐E11S, both transmission and scanning immunoelectron microscopy confirmed the identity of the spherical structures as free and budding SIV virions. © 1994 Wiley‐Liss, Inc.
Chondrogenic differentiation in cultures of embryonic rat mesenchyme
Authors: Langille Robert M. 
Published: February 2005

AbstractThe morphology and fine structure of day 12 rat embryonic mesenchyme from forelimb bud, mandibular arches, and frontonasal prominence is described as the cells undergo chondrogenesis in high density, micromass culture. The cultures began as a multilayered “pavement” of flattened mesenchymal cells, 3–4 deep, with moderate intercellular space but little identifiable electron‐dense extracellular matrix. Pre‐cartilage condensations, which consisted of aggregates of cells which had rounded up, displaying little or no intercellular space, formed within the first 24 h in limb mesenchyme and after an additional 24 h in mandibular and frontonasal cultures. Gap junctions occur between these cells, indicating a phase of direct cell‐cell communication. Chondrogenesis within these aggregates began within the next 24 h in limb cultures but was delayed an additional 24–48 h in the frontonasal and especially in mandibular cultures. The aggregates in both limb and mandibular mesenchyme form discrete nodules bordered by a perichondrium consisting of 2–3 layers of flattened cells. Evidence from late stage mandibular cultures suggests that chondroblasts are added to the nodules from the perichondrium, as occurs in vivo. By contrast, the frontonasal cartilage is initially unbordered and forms anastomosing trabecular arrays. Some of these arrays fuse into larger structures with time, but others become surrounded by proceeds. The sequence of cartilage matrix production, as revealed in long‐term facial cultures, appears to occur in three stages, an early phase in which the extracellular matrix consists primarily of proteoglycans, followed by a phase of homogeneous collagen‐proteoglycan matrix and a mature, territorial matrix. In all three cultures the cartilage ultimately produced resembles mature rat hyaline cartilage with chondroblasts surrounded by a territorial matrix of type II collagen and proteoglycan granules. © 1994 Wiley‐Liss, Inc.
Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization
Authors: Iwamoto Masahiro , Yagami Kimitoshi , Shapiro Irving M. , Leboy Phoebe S. , Adams Sherrill L. , Pacifici Maurizio 
Published: February 2005

AbstractDuring the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin. A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of ostenection (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17–18 sternum (US cells) and grown in monolayer in standard serum‐containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen‐positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3‐week‐old cultures with RA (10–100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA‐treated cultures. Electron microscopy confirmed the formation of large matrix‐associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells). In these cells, RA failed to induce high levels of APase activity, ON and OP gene expression, and mineralization; instead, it greatly promoted cell proliferation. Thus RA appears to have major, stage‐specific effects on the maturation program of chondrocytes. The retinoid rapidly induces expression of late maturation genes and activates mineralization of the cartilage matrix. © 1994 Wiley‐Liss, Inc.
Adenosine 5′‐triphosphate promotes mineralization in differentiating chick limb‐bud mesenchymal cell cultures
Authors: Boskey Adele L. , Doty Stephen B. , Binderman Itzhak 
Published: February 2005

AbstractWhen chick limb‐bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3–4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high‐energy phosphates adenosine 5′‐triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5–5 mM β‐glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells.In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H‐leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is present. It is suggested that ATP promotes mineral deposition by providing a high‐energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes. © 1994 Wiley‐Liss, Inc.
Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes
Authors: Hunziker Ernst B. 
Published: February 2005

AbstractGrowth plate chondrocytes play a pivotal role in promoting longitudinal bone growth. The current review represents a brief survey of the phenomena involved in this process at the cellular level; it delineates the contributions made by various activities during the course of the chondrocyte life cycle, notably proliferation and hypertrophy, and illustrates how the relative contributions may be modulated according to the particular needs of an organism at critical phases of growth. The cellular mechanisms by which a few well characterized growth‐promoting substances exert their influences are discussed in the light of recent findings pertaining to epiphyseal plate chondrocytes in vivo. © 1994 Wiley‐Liss, Inc.
Improved ultrastructural preservation of rat ciliary body after high pressure freezing and freeze substitution: A perspective view based upon comparison with tissue processed according to a conventional protocol or by osmium tetroxide/microwave fixation
Authors: Eggli Peter S. , Graber Werner 
Published: February 2005

AbstractConventional fixation of the delicate, highly folded rat ciliary body and its iridial extension, as well as of vitreal structures, is associated with the induction of a number of artifacts, thus limiting the reliability of morphological interpretations. Improved ultrastructural preservation may be achieved by microwave heating in combination with osmium tetroxide fixation. This protocol, although simple and cheap, yields results, particularly with respect to the extracellular matrix compartment between inner and outer ciliary epithelial cells, which are not greatly inferior to those obtained by implementing the sophisticated high pressure freezing and freeze substitution technique. The latter affords good to very good ultrastructural preservation of epithelium and stromal components, such as blood vessels, neural elements, smooth muscle cells, fibrocytes, and free cells, up to a depth of 50–100 μm from the tissue surface. Its superiority over osmium tetroxide/microwave fixation is revealed in the cytoplasmic, intraorganellar, and vitreal matrix compartments, which incur no obvious losses. © 1994 Wiley‐Liss, Inc.
Fabrication of Au/p‐Si Schottky barrier for EBIC study
Authors: Zhang Xiao , Joy David 
Published: February 2005

AbstractA simple method for the fabrication of a Au/p‐Si Schottky barrier suitable for electron beam induced current (EBIC) study has been developed. The mechanical and electrical properties of the fabricated Au/p‐Si Schottky barriers have been tested, and EBIC measurements of the dislocation contrast have been conducted using the fabricated Schottky barriers. © 1994 Wiley‐Liss, Inc.