bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural evidence for a binding substance to the sweet‐tasting protein thaumatin inside taste bud pores of rhesus monkey foliate papillae
Authors: Menco Bert Ph.M. , Hellekant Göran
Published: February 2005

Thaumatin is a protein that tastes intensely sweet only to Old World monkeys and to higher primates, including man. Here we used pre‐embedding ultrastructural methods to study the distribution of thaumatin in apical regions of Rhesus monkey foliate papillae, using thaumatin conjugated to 5 nm gold particles. With freeze‐substitution we saw that gold‐labeled thaumatin bound to an electron‐opaque, sponge‐like secretory substance inside the taste bud pores. Labeled thaumatin was found at the surface of the secretory substance even deep inside the pore, where other, unlabeled cellular structures surrounded the substance. With freeze‐fracture deep‐etching the secretory substance that bound the thaumatin‐gold particles appeared coarsely granular. There was no labeling of any other taste bud pore structure, including microvilli and small membranelined vesicles. Pre‐incubation with an excess of unlabeled thaumatin inhibited binding with goldlabeled thaumatin. The results suggest that the secretory substance had the greatest affinity of all taste pore structures to the sweet‐tasting compound under our experimental conditions. Therefore, gustatory reception probably involves various taste compound binding structures, microvilli, and also secretory substances like the one described here which bound thaumatin. We speculate that the secretory substance may bind taste stimuli and serve as an intermediate between stimuli and receptors. It could be involved in stimulus removal or delivery or both. © 1993 Wiley‐Liss, Inc.
HVEM ultrastructural analysis of mouse fungiform taste buds, cell types, and associated synapses
Authors: Kinnamon John C. , Henzler David M. , Royer Suzanne M.
Published: February 2005

We have used high voltage electron microscopy and computer‐generated three‐dimensional reconstructions from serial sections to elucidate the structure of taste bud cells and their associated synapses in fungiform taste buds of the mouse. Five fungiform taste buds (two of which were serially sectioned) were examined with the high‐voltage electron microscope (HVEM). We identified the synaptic connections from taste cells onto sensory nerve fibers and classified the presynaptic taste cells based on previously established ultrastructural criteria. From those data we have distinguished dark, intermediate, and light cells in murine fungiform taste buds. Synapses in murine fungiform taste buds are fewer in number, but contain many more vesicles than synapses in either foliate or circumvallate taste buds. Synapses in mouse circumvallate and foliate taste buds typically contain a few to several synaptic vesicles per section, whereas fungiform synapses may have in excess of 100 vesicles per profile. The significance of these differences in the numbers of synapses and synaptic structure between fungiform and circumvallate/foliate synapses is not known. Based on the small number of synapses observed in fungiform taste buds, we speculate that fungiform taste buds have only a few cells transducing sensory stimuli at any given time. Alternatively, communication of sensory information from the taste receptor cells to the afferent nerve fibers may be mediated by some other mechanism(s) in addition to classical chemical synapses. © Wiley‐Liss, Inc.
Sample preparation technique for cross‐sectional transmission electron microscopy of quantum wire structures
Authors: Chen Yu‐Pei , Reed Jason D. , O'Keefe Sean S. , Schaff William J. , Eastman Lester F.
Published: February 2005

A novel cross‐sectional sample preparation technique for quantum wire (QWR) structures is described. By coating a thin layer of Au with a designed pattern on the sample as a marker to indicate the position of the wire pattern, the location of the thinned area can be controlled precisely. An example of applying this technique to an InGaAs/GaAs QWR structure is demonstrated. This technique can also be applied to any other small dimensional structures or devices with specific regions of interest. © Wiley‐Liss, Inc.
Selected area polishing for precision TEM sample preparation
Authors: Liu J. B. , Tracy B. M. , Gronsky R.
Published: February 2005

A selected area mechanical polishing technique has been developed to improve the precision of cross‐sectional TEM sample preparation, based upon the early work of Benedict and colleagues [Benedict et al. (1990) MRS Symp. Proc. Vol. 199, p. 189]. TEM samples were made from a pre‐selected section through the middle of a 1 μm wide band of transistors extending laterally for more than 1 mm by precise control over the plane of polish with a corresponding reduction in sample preparation time. To illustrate the application of this technique, a uniformly thin, electron transparent TEM sample of a single, specific, failed transistor is obtained from a 4 mm by 10 mm device array. © Wiley‐Liss, Inc.
Transcellular and paracellular pathways in lingual epithelia and their influence in taste transduction
Authors: Simon Sidney A. , Holland Virgil F. , Benos Dale J. , Zampighi Guido A.
Published: February 2005

The lingual epithelium is innervated by special sensory (taste) and general sensory (trigeminal) nerves that transmit information about chemical stimuli introduced into the mouth to the higher brain centers. Understanding the cellular mechanisms involved in eliciting responses from these nerves requires a detailed understanding of the contributions of both the paracellular and transcellular pathways. In this paper we focus on the contribution of these 2 pathways to the responses of salts containing sodium and various organic anions in the presence and absence of amiloride. Electrophysiological recordings from trigeminal nerves, chorda tympani nerves, and isolated lingual epithelia were combined with morphological studies investigating the location (and permeability) of tight junctions, the localization of amiloride‐inhibitable channels, and Na‐K‐ATPase in taste and epithelial cells. Based on these measurements, we conclude that diffusion across tight junctions can modulate chorda tympani and trigeminal responses to sodiumcontaining salts and rationalize the enhancement of taste responses to saccharides by NaCl. © 1993 Wiley‐Liss, Inc.
Electron microscopic immunocytochemistry of glutamate‐containing nerve fibers in the taste bud of mudpuppy (Necturus maculosus)
Authors: Lu Kuo‐Shyan , Roper Stephen D.
Published: February 2005

The presence of glutamate immunoreactivity (glu‐IR) in the nerve fibers of the mudpuppy taste bud was investigated by electron microscopy. Pre‐embedding staining with avidinbiotin‐peroxidase complex (ABC) and post‐embedding staining with 5 mm colloid gold conjugates were used separately to identify immuno‐stained structures. We have found the following: 1) the majority of the nerve fibers innervating the mudpuppy taste bud are unmyelinated; 2) about 85% of nerve fibers located at the base of the taste bud and about 60% of the nerve fibers located between the taste cells show glu‐IR by pre‐embedding staining; 3) there is a preferential staining of the glu‐IR in the nerve fibers of the mudpuppy taste bud; and 4) the distribution of the colloidal gold particles in the nerve fibers is 1.5 to 2 times denser than that of the staining in the connective tissue background or cellular profiles of taste cells. From the distribution and pattern of the nerve fibers obtained in the thick and thin sections, we conclude that the mudpuppy taste bud is innervated by glutamate‐containing unmyelinated nerve fibers. © 1993 Wiley‐Liss, Inc.
Mechanisms of gastrulation and tail formation in ascidians
Authors: Swalla Billie J.
Published: February 2005

Ascidian embryos are useful for examining how events that occur during fertilization and cleavage affect gastrulation because they gastrulate early in development, during the seventh cleavage. In ascidians, both dorsal‐ventral and anterior‐posterior axes are determined before first cleavage. The dorsal‐ventral axis is fixed along the animal‐vegetal axis of the fertilized egg following the first phase of ooplasmic segregation, perhaps due to determinants moved to the vegetal pole in concert with the myoplasm and plasma membrane components. The first ooplasmic movements appear to be driven by the actin network in the cortical myoplasm. The anterior‐posterior axis becomes apparent after the second phase of ooplasmic segregation, when the cortical myoplasm becomes detached from the egg membrane, and moves to the posterior pole of the embryo. This movement is dependent on microtubules and has been attributed to the formatior and movement of the sperm aster. A major component of the cortical myoplasm, p58, is co‐localized along the microtubules emanating from the sperm aster. Gastrulation begins during the seventh cleavage with the invagination of the large endodermal cells at the vegetal pole of the embryo. The neural plate appears as a thickening of the epidermis on the dorsal side of the larva during the ninth cleavage; then the neural folds are formed, join, and close, elaborating the neural tube. Following neurulation, the tail is elongated as the neural tube and notochord cells intercalate at the midline of the embryo. Investigations using anural (tailless) ascidian larvae suggest that some of the processes underlying elongation can be restored by the zygotic genome. Although ascidian larvae contain fewer cells and cell types than vertebrate embryos, ascidian gastrulation and morphogenesis appear to employ similar mechanisms to those in vertebrate embryos. The extent of our current knowledge about the mechanisms involved in gastrulation and tail formation is summarized, and further experiments are suggested to explore the molecular mechanisms underlying these processes. © 1993 Wiley‐Liss, Inc.
Immunohistochemistry of a human type I pneumocyte‐associated protein in lung
Authors: Singh Jagjit , Singh Gurmukh , Katyal Sikandar L. , Wong‐Chong Mari‐Lou , McCloskey Carol A. , Gottron Shirley A.
Published: February 2005

A mouse monoclonal antibody to a human lung lavage protein was raised using proteins, with the potential ability to bind surfactant, as the immunogen. The proteins were isolated from cadaver lung lavage. The antibody was tested for its reactivity with lung and other organs. It reacted with type I pneumocytes and some of the nonciliated cells in the surface epithelium of distal bronchioles. Staining was also seen in the cells surrounding the glandular structures, superficial keratinocytes of the skin, endothelium, and nerve sheath cells. With the exception of bronchiolar cells, the stained cells have a squamous morphology, and this protein may serve as a marker or determinant of this characteristic of cells. In pathologic lungs some of the cells in air spaces with “bronchiolarization” of the epithelium exhibited staining for the protein. It could not be ascertained whether the stained cuboidal cells were reactive type II pneumocytes or distal bronchiolar cells. The intraalveolar material in pulmonary alveolar proteinosis did not show remarkable staining for the protein. Even though the protein is not unique to type I pneumocytes, it may serve as a marker for these cells in the study of their development and biology. © 1993 Wiley‐Liss, Inc.
Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells
Authors: Voorhout W. F. , Weaver T. E. , Haagsman H. P. , Geuze H. J. , Van Golde L. M. G.
Published: February 2005

Surfactant proteins A, B, and C (SP‐A, SP‐B, and SP‐C) are synthesized in alveolar type II cells. SP‐B and SP‐C are both synthesized as large precursor molecules that are proteolytically processed to their mature sizes. In a previous immunoelectron microscopic study, we showed that precursor SP‐B is processed to its mature size in multivesicular bodies. In the present study, using a specific antibody aginst precursor SP‐C, we demonstrate that precursor SP‐C is present in the same intracellular compartments of the biosynthetic pathway, i.e., endoplasmic reticulum, Golgi complex, and multivesicular bodies, as precursor SP‐B. Since mature SP‐C is known to be present in multilamellar bodies, this suggests a biosynthetic routing and site of processing of this protein similar to those of SP‐B. Double‐labeling experiments using antibodies against SP‐A, precursor SP‐B, precursor SP‐C, and an antibody against HA I, an adaptor protein involved in the budding of transport vesicles from the Golgi complex, showed that the different surfactant proteins traverse and exit the Golgi complex via the same route. The surfactant proteins do not exit the Golgi complex via HA I‐positive coated buds or vesicles. These data are in accordance with the concept that SP‐A, SP‐B, and SP‐C are transported together through the same biosynthetic pathway via multivesicular bodies to multilamellar bodies. © 1993 Wiley‐Liss, Inc.
Aerosolized fluorescent microspheres detected in the lung using confocal scanning laser microscopy
Authors: Pinkerton Kent E. , Gallen John T. , Mercer Robert R. , Wong Viviana C. , Plopper Charles G. , Tarkington Brian K.
Published: February 2005

Aerosolized fluorescent microspheres were used to study particle deposition in site‐specific regions of the lung with confocal laser scanning microscopy. A nebulizer was used to aerosolize microspheres followed by passage through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. Precoating of microspheres with albumin helped to minimize displacement during vascular fixation of the lungs. Confocal laser microscopy facilitated visualization of microspheres throughout the bronchial tree, ducts, and alveoli of the lungs. The use of fluorescent microspheres and confocal laser imaging provided distinct advantages compared with other methods to study lung particle deposition due to (1) the generation of single microspheres of uniform size by nebulization, (2) easy detection of microspheres in large slabs of microdissected lung tissues, (3) excellent resolution of tissue surfaces and microspheres for an infinite number of orientations and planes of section, and (4) the ability to visualize microspheres below fluid lining layers and on surfaces that could not easily be done by other methods of microscopy.© 1993 Wiley‐Liss, Inc.
Acute lung injury during bacterial or fungal sepsis
Authors: Lechner Andrew J. , Ryerse Jan S. , Matuschak George M.
Published: February 2005

We compared physiological and ultrastructural indices of acute lung injury (ALI) during septic shock caused by taxonomically diverse pathogens to distinguish ALI due to endogenous inflammatory mediators vs. microbial exotoxins or other factors. Conscious rats were infected i.v. with gram‐negativeEscherichia coli(EC, serotype 055:B5), exotoxin‐C producing gram‐positiveStaphylococcus aureus (SA), or yeast‐phaseCandida albicans(CA, a clinical isolate). Viable inocula of 1010 EC, 1010 SA, or 109 CA caused lethal shock in <24 h, but distinct types of ALI were noted after bacteria vs. fungi. Within 0.5 h of EC infection, leukocytes marginated in the lung vasculature; by death at 6–14 h, animals were hyperoxemic but not acidemic, and showed slight interstitial edema with increased wet/dry weight ratios (W/D = 5.22 ± 0.10, mean ± SE, vs. 4.86 ± 0.07 in controls, P <0.05). Similarly mild ALI occurred after 1010 SA. In contrast, within 0.5 h of CA infection, yeast were visible within lung intravascular leukocytes. By death at 6–12 h, CA animals showed hyperoxic acidemia and moderate ALI with capillary obstruction, interstitial hemorrhage, and elevated lung W/D (5.52 ± 0.13, P <0.05 vs. controls) associated with yeast‐mycelial transformation. Prior neutropenia accelerated mortality and worsened ALI after CA, with hypoxemic acidemia, increased lung W/D (7.23 ± 0.34, P <0.05 vs. other groups), capillary occlusion, perivascular and alveolar hemorrhage, and septal disruption by mycelia. Bacteremia induced large increases in serum tumor necrosis factor‐α (TNF) and interleukin‐1α within 1.5 h, but these cytokines remained low in CA animals, even at death. Neither survival nor ALI after EC or CA was altered by pentoxifylline, which attentuated TNF production, or by cyclooxygenase inhibition with ibuprofen. Thus, overall ALI severity correlated with physiological indices of pulmonary function, but ultrastructural changes correlated better with pathogen type than circulating cytokine or eicosanoid mediators. Whereas lethal bacteremia induced early cytokinemia and mild ALI with or without bacterial exotoxins, moderate ALI apparently was mediated by fungal exotoxins during lethal candidemia, which worsened during neutropenia due to enhanced mycelial proliferation.© 1993 Wiley‐Liss, Inc.
Artifacts caused by dehydration and epoxy embedding in transmission electron microscopy
Authors: Mollenhauer Hilton H.
Published: February 2005

Epoxy resins are the principal embedding media for the preservation of tissues to be sectioned and examined by transmission electron microscopy. Their primary advantages are good ultrastructural preservation, little or no shrinkage, ease of sectioning, and reasonable stability in the electron beam. However, epoxy resins also have disadvantages; namely, some are toxic, they may mask antigenic sites to a greater extent than do some other embedding resins, and they do not penetrate tissues as well as less viscous embedding formulations. Some unusual characteristics may also be revealed, for example, as shrinkage of organelles, as problems in poststaining sections, and as movement of tissue elements within the block and section. Some of the properties of epoxy resins are discussed in this report. © 1993 Wiley‐Liss, Inc.
The granular convoluted tubule (GCT) cell of rodent submandibular glands
Authors: Gresik Edward W.
Published: February 2005

The granular convoluted tubule (GCT) is a segment of the duct system of all rodents, situated between the striated and intercalated ducts. It has the peculiar property of synthesizing a large variety of biologically active polypeptides whose role in saliva remains unknown. The literature on the fine structure of GCT cells is critically reviewed. Some recent developments on endocrine regulation of the structure and contents of rodent GCT cells are summarized, with emphasis on EGF, NGF, renin, and kallikrein proteases. A survey of the distribution of GCT cells in several vertebrate families is presented. © 1994 Wiley‐Liss, Inc.
Dynamics of mitochondria in living cells: Shape changes, dislocations, fusion, and fission of mitochondria
Authors: Bereiter‐Hahn J. , Vöth M.
Published: February 2005

Mitochondria are semi‐autonomous organelles which are endowed with the ability to change their shape (e.g., by elongation, shortening, branching, buckling, swelling) and their location inside a living cell. In addition they may fuse or divide. These dynamics are discussed. Dislocation of mitochondria may result from their interaction with elements of the cytoskeleton, with microtubules in particular, and from processes intrinsic to the mitochondria themselves. Morphological criteria and differences in the fate of some mitochondria argue for the presence of more than one mitochondrial population in some animal cells. Whether these reflect genetic differences remains obscure. Emphasis is laid on the methods for visualizing mitochondria in cells and following their behaviour. Fluorescence methods provide unique possibilities because of their high resolving power and because some of the mitochondria‐specific fluorochromes can be used to reveal the membrane potential. Fusion and fission often occur in short time intervals within the same group of mitochondria. At sites of fusion of two mitochondria material of the inner membrane, the matrix compartment seems to accumulate. The original arrangement of the fusion partners is maintained for some minutes. Fission is a dynamic event which, like fusion, in most cases observed in vertebrate cell cultures is not a straightforward process but rather requires several “trials” until the division finally occurs. Regarding fusion and fission hitherto unpublished phase contrast micrographs, and electron micrographs have been included. © 1994 Wiley‐Liss, Inc.
Interactions between brain mitochondria and cytoskeleton: Evidence for specialized outer membrane domains involved in the association of cytoskeleton‐associated proteins to mitochondria in situ and in vitro
Authors: Leterrier J. F. , Rusakov D. A. , Nelson B. D. , Linden M.
Published: February 2005

The surface distribution of several proteins (porin, hexokinase, and two proteins associated with microtubules or actin filaments) on the outer membrane of brain mitochondria was analyzed by immunogold labelling of purified mitochondria in vitro. The results suggest the existence of specialized domains for the distribution of porin in the outer mitochondrial membrane. Similarities between the distribution of porin and the distribution of microtubule‐associated proteins bound in vitro to mitochondria suggested that mitochondria and microtubules interact by binding microtubule‐associated proteins to porin‐containing domains of the outer membrane. This hypothesis was supported by biochemical studies on outer mitochondrial proteins involved in in vitro binding of cytoskeleton elements. In vitro interactions between mitochondria and microtubules or neurofilaments were analyzed by electron microscopy. These studies revealed cross‐bridging between the outer membrane of mitochondria and the two cytoskeleton elements. Cross‐bridging was influenced by ATP hydrolysis and by several proteins associated with the surface of mitochondria or with microtubules. In addition, unidentified proteins which were recognized by antibodies to all intermediate filaments subunits were associated either with the mitochondrial surface or with microtubules. This data suggest the participation of additional cytoplasmic proteins in the interactions between cytoskeleton elements and mitochondria. © 1994 Wiley‐Liss, Inc.
Specimen preparation of free‐standing, thick‐metal, multilayered films in cross section
Authors: Wall Mark A.
Published: February 2005

A new preparation method permits the production of large‐area, electron‐transparent, transmission electron microscopy (TEM) specimens in cross section of free‐standing, thick, multilayered structures. Such production often has been difficult in the past because of large chemical differences between the component layers in the multilayer. This difference usually results in a large difference in thinning rates between the layers. A unique combination of electroplating, lapping, dimpling, and low‐angle ion milling is a successful and reproducible technique for producing high‐quality TEM specimens of these complex materials. Procedures and results presented here are for a 304 stainless‐steel/copper multilayer having a repeat period of 20 nm and a total thickness of 20 μm. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
A morphological view on mitochondrial protein targeting
Authors: van der Klei Ida J. , Veenhuis Marten , Neupert Walter
Published: February 2005

Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co‐translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established.

The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites.

In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles.

Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites.

Based on these ultrastructral data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites. © 1994 Wiley‐Liss, Inc.
Electron microscopic studies of magnetosomes in magnetotactic bacteria
Authors: Bazylinski Dennis A. , Garratt‐Reed Anthony J. , Frankel Richard B.
Published: February 2005

Electron microscopic studies on magnetosomes in magnetotactic bacteria have revealed much information on their composition, structure, and even the formation of their mineral phase. The mineral phases of the magnetosomes are of two general types: iron oxides and iron sulfides. Iron oxide‐type magnetosomes contain particles of the ferrimagnetic mineral magnetite (Fe3O4) while the iron sulfide‐type contain ferrimagnetic greigite (Fe3S4), greigite and non‐magnetic pyrite (FeS2), or possibly ferrimagnetic pyrrhotite (Fe7S8). Regardless of their composition, the crystalline particles in magnetosomes have a narrow size range: approximately 35 to 120 nm. Magnetite crystals in this size range are single‐magnetic‐domains and confer a permanent magnetic dipole moment to the cell. The single‐domain size range for greigite is not known but is probably similar to that for magnetite.

The morphology of the particles in the bacterial magnetosomes appears to be species‐specific. Morphologies of magnetite crystals in different species of magnetotactic bacteria include cubooctahedra, parallelepipedal (truncated hexahedral or octahedral prisms), and tooth‐ or bullet‐shaped (anisotropic). Morphologies of greigite particles include cubo‐octahedra and rectangular prismatic. The greigite‐pyrite particles are generally pleomorphic with no consistent crystalline morphology. A membrane has been shown to surround the particles in some organisms and may be involved in the formation of the crystalline phase while also providing physical constraints on the size and the shape of the crystal. These results clearly indicate that the biomineralization process involved in the bacterial magnetosome, a good example of a self‐assembled structure on a nanometer scale, is highly controlled by the organism. © 1994 Wiley‐Liss, Inc.
Structure of butterfly scales: Patterning in an insect cuticle
Authors: Ghiradella Helen
Published: February 2005

All butterfly and moth scales and bristles are made of non‐living insect cuticle. Each is the product of a single epithelial cell, and all share the same basic architecture. However, some are highly specialized, and their cuticle is further elaborated into stacks of thin‐films, lattices, or other minute structures, many of which first came to our attention because they interact, with light to produce structural colors. The scale cell forms the scale by extruding a projection of itself and secreting around it the outer epicuticle, a thin cuticular envelope which will form the outermost layer of the scale. The inner layers of cuticle, collectively called the procuticle, are secreted thereafter and go on to form the lattices, pillars, or other internal structures of the scale. We believe that the pattern‐forming mechanisms used by the cell to shape the cuticle into its finished form include elastic buckling of the outer epicuticle to produce external folds, and “masking” of certain areas of the original epicuticular envelope to produce thin spots which will break through to become windows. Varied though they be, all insect cuticular patterns have common basic elements, which suggests that our findings may be generalized to other highly patterned insect cuticles, particularly those formed by single cells. © 1994 Wiley‐Liss, Inc.
Cryo‐electron microscopy of deoxy‐sickle hemoglobin fibers
Authors: Lewis Michael R. , Gross Leon J. , Josephs Robert
Published: February 2005

Deoxy‐sickle hemoglobin (HbS) polymerizes in vivo into long helical fibers which fill the red cell and make it rigid. This impedes red cell passage through the capillaries and is responsible for the clinical manifestations of sickle cell disease. Images of individual and laterally associated HbS fibers were obtained by electron microscopy of frozen hydrated specimens. Each fiber possesses variable pitch, having from 6° to 12° rotation per unit cell. Laterally associated HbS fibers display systematic inter‐fiber contacts in spite of their pitch variations, and exhibit better order than isolated fibers. This suggest that inter‐fiber contacts can act to couple fibers mechanically and might therefore be a factor in rigidifying red cells in vivo. Fiber variability was attributed to local torsional variations with a standard deviation of 2.5°, but which are weakly coupled over a length of 2.25 unit cells. Variable pitch produces structural changes of as large as 5 Å azimuthally and 6 Å axially in HbS fiber unit cells. © 1994 Wiley‐Liss, Inc.
Quick‐Freeze, deep‐etch studies of renal basement membranes
Authors: Kubosawa Hitoshi , Kondo Yoichiro
Published: February 2005

The 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

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

The application of cationic probes for the ultrastructural detection of proteoglycans in basement membranes is reviewed. Proteoglycans are highly negatively charged macromolecules due to their glycosaminoglycan side chains. The interaction of cationic probes with proteoglycans is of an electrostatic nature. Methods are discussed to increase the specificity of probes for proteoglycans. The use of phthalocyanin‐like dyes such as Cuprolinic blue, according to the critical electrolyte concentration method, results in a selective staining of proteoglycans. Enzymatic or chemical digestions, however, should be done to validate the proteoglycan nature of the dye‐positive granules/filaments, and to establish the class of proteoglycan. The value of cationic probes in basement membrane research on development and pathology is discussed. The potential for deducting molecular information from the ultrastructural appearance of stained proteoglycans is indicated. © 1994 Wiley‐Liss, Inc.
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

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

Alzheimer's disease (AD) is one of several systemic and cerebral diseases that involve the abnormal deposition of fibrillar proteins called amyloids. All amyloids share conformational and staining characteristics, as well as an association with resident tissue macrophages and two extracellular matrix components [heparan sulfate proteoglycan (HSPG) and amyloid P component]. Vascular, glomerular, and Schwann cell basement membrane pathologies have been documented in many forms of amyloidosis, and often amyloid fibrils fuse to and project from the basement membrane in these diseases. The present report demonstrates the vascular basement membrane (VBM) alterations in AD autopsy samples, and details the methodologies used.

Electron microscopy reveals the fusion of amyloid fibrils with the VBM and the alteration of the VBM in the absence of amyloid accumulation. Double‐labelling and pre‐embed immuno‐electron microscopy techniques demonstrate the colocalization of amyloid P component and VBM components with amyloid, and also reveal that amyloid P component is not localized to the cerebral VBM. Finally, a novel correlative light/electron microscopy technique demonstrates the association between amyloid P component and cerebral resident tissue macrophages, the microglia. Taken together, these data suggest that the physicochemical processes of amyloid formation, rather than amyloid deposition, may be responsible for VBM pathology. © 1994 Wiley‐Liss, Inc.
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

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

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

High‐angle annular dark‐field or Z‐contrast microscopy was used to demonstrate that well dispersed metal supported catalysts consist of nanometer sized clusters. Depending upon the impregnated metal, different cluster sizes were observed. Grouping of Pd clusters could also be confirmed by analytical electron microscopy. © 1994 Wiley‐Liss, Inc.
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

The 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

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