bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of environmental scanning electron microscopy in the development of detergents and personal products
Authors: Hoyberg Karen , Kruza Kenneth G.
Published: February 2005

Environmental scanning electron microscopy enables the observation of samples in their natural state with no preparation. Unilever Research employs this technology in the research and development of detergents and personal products with much success. The results presented are from various studies which demonstrate the versatility and utility of the technique. Vacuum sensitive samples such as microcapsules were successfully characterized, the efficacy of dental products was shown by monitoring an enamel surface before and after treatment, and the performance of an adhesive was assessed by dynamic experimentation. © 1993 Wiley‐Liss, Inc.
Imaging fluid/solid interactions in hydrocarbon reservoir rocks
Authors: Uwins Philippa J. R. , Baker Julian C. , Mackinnon Ian D. R.
Published: February 2005

The environmental scanning electron microscope (ESEM) has been used to image liquid hydrocarbons in sandstones and oil shales. Additionally, the fluid sensitivity of selected clay minerals in hydrocarbon reservoirs was assessed via three case studies: HCl acid sensitivity of authigenic chlorite in sandstone reservoirs, freshwater sensitivity of authigenic illite/smectite in sandstone reservoirs, and bleach sensitivity of a volcanic reservoir containing abundant secondary chlorite/corrensite. The results showed the suitability of using ESEM for imaging liquid hydrocarbon films in hydrocarbon reservoirs and the importance of simulating in situ fluid‐rock interactions for hydrocarbon production programmes. In each case, results of the ESEM studies greatly enhanced prediction of reservoir/borehole reactions and, in some cases, contradicted conventional wisdom regarding the outcome of potential engineering solutions. © 1993 Wiley‐Liss, Inc.
Environmental scanning electron microscopy for dynamic corrosion studies of stainless steel piping used in UHP gas distribution systems
Authors: Gerristead W. R. , Link L. F. , Paciej R. C. , Damiani P. , Li H.
Published: February 2005

An ElectroScan ESEM was used for in situ corrosion studies on cold drawn electropolished 316L stainless steel tube surfaces in the as‐received and passivated conditions. Corrosion product was removed as it formed and the tube surface was viewed before, during, and after corrosive attack. The corrosion process was followed in situ, and the sample features most susceptible to corrosion (draw lines, inclusions, etc.) were identified. In addition, X‐ray photoelectron spectroscopy (XPS) was used to study the changes in surface chemistry after corrosive attack. This information provided clear evidence of relevant corrosion mechanisms and relative corrosion susceptibility. © 1993 Wiley‐Liss, Inc.
Gastrulation in the mouse embryo: Ultrastructural and molecular aspects of germ layer morphogenesis
Authors: Tam Patrick P. L. , Williams Elizabeth A. , Chan W. Y.
Published: February 2005

Ultrastructural studies and lineage analyses of gastrulating mouse embryos have revealed that differnt morphogenetic tissue movements are involved in the formation of the three definitive germ layers. Definitive ectoderm is formed by epibolic expansion of the pre‐existing progenitor population in the embryonic ectoderm. Formation of the mesoderm and the endoderm is initiated by cellular ingression at the primitive streak. The mesodermal layer is established by cell migration and cell sheet spreading, but the endoderm is formed by replacing the original primitive endodermal population. To this date, genes that are expressed during mouse gastrulation mostly encode cell surface adhesion or signalling molecules, growth factors and their receptors, and putative transcriptional factors. Their precise role during gastrulation remains to be investigated. © 1993 Wiley‐Liss, Inc.
Morphogenesis of the avian trunk neural crest: Use of morphological techniques in elucidating the process
Authors: Erickson Carol A.
Published: February 2005

Morphological data generated from light and electron microscopy form the basis of our understanding of avian morphogenesis. Because chicken embryos are readily and cheaply obtained and are easily accessible for experimental manipulation, morphogenetic processes have been studied extensively in this species. Such studies have allowed us to identify the cells involved during morphogenesis, observe the shape changes or cellular translocations that accompany a morphogenetic process, and determine the timing of these events. Elucidation of the molecular basis of morphogenesis has awaited the integration of several additional approaches. Among these are experimental embryology, which has allowed us to understand cellular behavior associated with morphogenesis; immunocytochemistry, which has identified the macromolecular cues that regulate cell movements and the environmental factors that control them; and molecular techniques, which will permit us eventually to clarify the genetic regulation of morphogenesis. Although current research in development is heavily biased towards molecular biology, morphological studies continue to frame the questions that are now being addressed using molecular techniques.

This review focuses on the cells of the neural crest as a model system where questions of avian morphogenesis have been profitably addressed. © 1993 Wiley‐Liss, Inc.
Clara cell differentiation in the mouse: Ultrastructural morphology and cytochemistry for surfactant protein A and clara cell 10 kD protein
Authors: Ten Have‐Opbroek Ank A. W. , De Vries Erwin C. P.
Published: February 2005

The morphologic and functional differentiation of the nonciliated columnar (Clara) cell, one of two secretory cell types in distalmost bronchioles in mammals, was studied in the mouse. Lungs from embryos (16–19 days of developmental age, dDA; birth on day 19), postnatal animals (5–20 days postnatally dPN), and adult animals were investigated by transmission electron microscopy, using standard staining procedures and immunogold (GAR‐Au10) labeling for SP‐A and Clara cell 10 KD antigen (CCA). At 16 dDa, all the columnar epithelial cells lining prospective distalmost bronchioles lacked distinctive features. By 17 dDa, some cells displayed a few cilia or apical dense granules. At 18 dDa, many nonciliated columnar cells had apical protrusions, as are seen in adult Clara cells. Apical concentrations of glycogen observed in nonciliated columnar cells perinatally were absent by 5 dPN, whereas apical dense granules became more abundant. Profiles of smooth and rough endoplasmic reticulum (ER), which had been randomly distributed, exhibited a selective, adult distribution at 20 dPN (apical vs. basal cytoplasmic domains). Labeling for SP‐A and CCA, which was almost absent between 17 and 19 dDa, reached adult levels at the same time. The two proteins differed in distribution. SP‐A predominated in adluminal cytoplasmic areas, where it was found over dense granules, vesicles, and multivesicular bodies; it was also present in bronchiolar lumens and intercellular spaces but not in rough ER or Golgi apparatus. In contrast, CCA showed a more uniform distribution; it was present over the same structures as SP‐A and in the synthetic organelles. Ciliated columnar cells were virtually devoid of SP‐A and CCA. We conclude that mouse Clara cells acquire a mature phenotype by 20 dPN. They are likely to be involved in recycling and/or degradation of SP‐A that is internalized from airway lumens through their apical or lateral cell borders; furthermore, they synthesize the Clara cell 10 kD protein. These two Clara cell functions (first detectable late prenatally) reach mature levels by 20 dPN. © 1993 Wiley‐Liss, Inc.
Role of alveolar macrophage chemotaxis and phagocytosis in pulmonary clearance responses to inhaled particles: Comparisons among rodent species
Authors: Warheit David B. , Hartsky Mark A.
Published: February 2005

Alveolar macrophages (AM) play an important role in clearing inhaled particles from the lung. The mechanisms through which macrophages identify particles that have been deposited in the alveolar regions is not well understood, although macrophage motility and phagocytic functions appear to be prerequisites for efficient clearance of inhaled materials. In previous studies, we assessed the mechanisms of macrophage‐mediated clearance of inhaled particles using a rat model. In this regard, it appears that one mechanism by which rat alveolar macrophages are recruited to sites of particle or fiber deposition is through complement activation and consequent generation of chemotactic factors by the inhaled particulates. Whether this mechanism is operative in other rodent species remains an unanswered question. The current studies were undertaken to compare pulmonary clearance responses in several rodent species exposed to carbonyl iron (CI) particles. In vitro and in vivo pulmonary clearance responses were evaluated using one strain each of mouse, hamster, rat, and guinea pig. In vitro studies showed that hamster AM had the greatest phagocytic activity and that rat AM migrated best to complement‐dependent chemotactic factors. Subsequently, groups of animals from each species were exposed to CI particles for 1 or 6 hr at aerosol concentrations of 100 mg/m3. Particle deposition patterns in the distal lung were nearly identical for all species, although enhanced numbers of CI particles were deposited on alveolar duct bifurcations of either rats or mice compared to hamsters, and particle deposition in guinea pigs was substantially lower. Time course studies showed that enhanced numbers of rat AM migrated to deposition sites and phagocytized particles, and this correlated with increased numbers and percentages of phagocytic macrophages recovered by lavage (P <0.01). In vivo phagocytic rates were the lowest in the mouse, and this correlated with reduced phagocytic rates in vitro. It is concluded form these studies that the rat may be the most efficient rodent species in clearing inhaled iron particles. In addition, it is conceivable that hamster AM are recruited to sites of particle deposition by a noncomplement‐mediated mechanism. © 1993 Wiley‐Liss, Inc.
Surfactant and inhaled particles in the conducting airways: Structural, stereological, and biophysical aspects
Authors: Gehr Peter , Geiser Marianne , Hof Vinzenz Im , Schürch Samuel , Waber Urs , Baumann Markus
Published: February 2005

We have investigated the displacement into the sol phase of inhaled particles deposited in the intrapulmonary conducting airways. Hamsters inhaled an aerosol of monodisperse polystyrene particles of 6 μm diameter. Their lungs were fixed by intravascular perfusion, and light and electron microscopy was used to study the epithelial coating. The surfactant film at the wall‐air interface was investigated by measuring its surface tension. The number of particles retained was determined stereologically. In addition we investigated the displacement of spherical particles in vitro on a DPPC monolayer in a Langmuir‐Wilhelmy surface balance and determined the surface tension in vivo in the horse trachea by video bronchoscopy, applying the droplet spreading method. We found that particles deposited onto a surfactant film were pulled into the aqueous subphase, and we concluded that surface forces due to the airway surfactant likely displace deposited particles into the periciliary fluid (sol phase). Comparing lungs fixed immediately after inhalation with lungs fixed 24 hr after inhalation revealed that 86% of the particles retained in the intrapulmonary conducting airways immediately after inhalation had been cleared within 24 hr. One‐third of the particles of the lungs fixed immediately after inhalation was phagocytized. The combination of structural and stereological analyses with in vitro and in vivo measurements has led to new insights into the role of airway surfactant with respect to the fate of inhaled particles, which may have important consequences regarding the effects of hazardous particles, which may have important consequences regarding the effects of hazardous particles. These studies may also help to evaluate the deposition pattern and clearance of therapeutic particles, with important implications for their therapeutic use. © 1993 Wiley‐Liss, Inc.
In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium
Authors: Harkema J. R. , Hotchkiss J. A.
Published: February 2005

Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin‐instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti‐rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S‐phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU‐labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. We did not observe a neutrophilic influx in the nasal tissues of neutrophil‐depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU‐labeling index were significantly increased compared to salineinstilled controls. In controls. In contrast, non‐neutrophil‐depleted rats instilled with endotoxin had a marked neutrophilic influx, but no significant differences in the number of nasal epithelial cells at 6 or 24 h, compared to controls. In addition, the BrdU‐labeling index in neutrophil‐sufficient rats was increased only 6 h after the last instillation, compared to controls. These results indicate that 1) endotoxin induces an increase in epithelial DNA synthesis that is neutrophil‐independent, and 2) without a neutrophilic influx, there is sustained epithelial proliferation, resulting in a hyperplastic epithelium after repeated endotoxin exposure. © 1993 Wiley‐Liss, Inc.

© 1993 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 step section method for full histopathological assessment of carcinogen‐affected target tissue during respiratory carcinogenesis
Authors: Hammond William G. , Teplitz Raymond L. , Benfield John R.
Published: February 2005

Studies of carcinogenesis that are not limited to overt neoplasms but also involve evaluations of preneoplastic stages require histopathological assessment of the entire carcinogen‐affected tissue so that the true nature and sequence of the progressive process can be determined. The customary serial sectioning approach achieves this goal, but at an inordinate logistic cost. In studies of hamster bronchial carcinogenesis, a step section method was compared to a quasi‐random approach and to the customary serial section method. The step section method achieved the same diagnostic completeness as serial sectioning, but at a two orders of magnitude reduction in costs. © 1993 Wiley‐Liss, Inc.
Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical‐point drying methods for scanning electron microscopy of biological specimens
Authors: Bray D. F. , Bagu J. , Koegler P.
Published: February 2005

Three different drying methods, critical‐point drying (CPD), Peldri II, and hexamethyldisilazane (HMDS), were compared using representative animal( rat kidney, trachea, duodenum, lung, and red blood cells) and plant( leaves from ten species of monocotyledons and dicotyledons) specimens. All three drying methods produced identical results with animal specimens. Plant specimens showed signs of shrinkage regardless of which drying method was employed. The order of preservation quality from best to worst for leaves was CPD > Peldri II > HMDS, with the CPD method providing substantially better results in all but one case. Postfixation of leaves with osmium tetroxide resulted in poorer preservation in all instances. Peldri II caused complete extraction of leaf cuticular wax, while both both CPD and HMDS showed minimal extraction compared with samples air dried directly from acetone. These results indicate that HMDS provides a time‐saving and inexpensive alternative to CPD for animal specimens. Plant specimens, particularly those containing cells with large central vacuoles, are adequately preserved only with the CPD method. In addition, postfixation with osmium should be avoided when processing plant specimens for scanning electron microscopy. © 1993 Wiley‐Liss, Inc.
A method to detect areas high in sulfhydryl groups in mouse epithelium
Authors: Kawabe Thomas T. , Buhl Allen E.
Published: February 2005

We have recently modified a non‐fluorescent, non‐radioactive histochemical method to detect sulfhydryl (S‐H) groups in tissues. This method was originally intended to detect chloramphenicol acetyltransferase (CAT) in transgenic mice. Temporal developmental differences in the keratinization of mouse digits can be seen in the staining pattern of the skin about the toes of neonatal mice. The basal cells of the epidermis exposed to the air show intense staining while the epidermis that is still attached to an adjacent toe shows no staining. The degree of S‐H presence can be determined by the tissues' resistance to blocking of the S‐H groups by iodoacetic acid. Areas that contain very high numbers of S‐H groups still show staining following blocking by iodoacetic acid. We have found that this method shows clear differences in the S‐H distribution of various epithelium, including skin, hair, nails, and tongue epithelium. © 1993 Wiley‐Liss, Inc.
A review of electron probe X‐ray microanalysis studies of salivary gland cells
Authors: Izutsu Kenneth T. , Cantino Marie E. , Johnson Dale E.
Published: February 2005

Electron probe X‐ray microanalysis (EPXMA) has now been successfully applied to several salivary gland preparations. This paper briefly reviews the principles underlying this technique and the specific sample preparation procedures which permit accurate measurement of elemental concentrations in the various intracellular spaces. Findings from salivary gland studies indicate that cytoplasmic and nuclear spaces of nonstimulated acinar cells have high concentrations of K and P, and low concentrations of Mg, Ca, and S; and that mature secretory granules have high concentrations of Ca and S, and relatively low concentrations of K and P. No consistent differences have been found between the elemental concentrations of mucous and serous secretory granules. In vivo and in vitro EPXMA studies of the elemental changes associated with secretory granule maturation indicate there are at least two stages in this process: an early stage during which granule S concentration increases in parallel with mass density as condensing vacuoles mature into secretory granules, and a late stage during which granule mass density and protein content increase with no further elemental concentration changes. Findings from other in vivo and in vitro studies indicate that secretory granule membranes are permeable to Na, K, and Cl ions because the granular concentrations of these elements are altered by electrochemical gradients. Recent EPXMA results indicate that cells stimulated with parasympathomimetic agonists have decreased K and Cl concentrations, and increased Na concentrations. Furthermore, the magnitude of these changes are quantitatively consistent with changes measured using radio‐isotope equilibration and other techniques. In contrast, cells stimulated with the β‐adrenergic agonist, isoproterenol, have increased concentrations of Na and Cl, but unchanged K concentrations. In terms of pathology, Na concentration is found to be elevated in secretory granules from labial salivary glands of patients with cystic fibrosis, but the pathophysiological consequences of this change are not known. Thus, studies utilizing EPXMA have obtained detailed and unique information about physiological and pathophysiological changes in elemental concentrations of intracellular organelles in salivary gland cells. © 1994 Wiley‐Liss. Inc.
Ontogeny of the cytoskeleton during mammalian oogenesis
Authors: Gallicano G. Ian , McGaughey Robert W. , Capco David G.
Published: February 2005

Mammalian oogenesis is a process which requires a variety of changes in the structure and function of the specialized female germ cell. Evidence suggests that the cytoskeleton may mediate several of these structural and functional changes. In this review we evaluate what is known of cytoskeletal function during oogenesis, with emphasis on specialized cytoskeletal features in mammals. Existing investigations suggest that the oocyte, as a highly specialized cell, contains unique cytoskeletal elements which exhibit functions restricted to the process of early development. © 1994 Wiley‐Liss, Inc.
Ultrastructural changes during meiotic maturation in mammalian oocytes: Unique aspects of the human oocyte
Authors: Sathananthan A. Henry
Published: February 2005

This paper reviews the process of peri‐ovulatory oocyte maturation and the ultrastructural organization of the human egg and compares it with that of the mouse. The main thrust of the paper is on the human, since there are several reviews on the mouse. Both preovulatory and postovulatory events at fertilization, as well as some of the aberrant features of maturation are covered. Some changes induced by oocyte culture and cooling in the human are also included. The report attempts to focus on unique features of the human oocyte and shows a variety of ultrastructural differences between human and murine oocytes, which may well reflect differences in their physiology and biochemistry. Based on these differences and further observations on the process of fertilization of both species, particularly with respect to the inheritance of paternal centrioles, it is concluded that the mouse may not be a suitable modle for the development and refinement of current procedures in human assisted reproductive technology. © 1994 Wiley‐Liss, Inc.
The internal compartmentation of rat‐liver mitochondria: Tomographic study using the high‐voltage transmission electron microscope
Authors: Mannella Carmen A. , Marko Michael , Penczek Pawel , Barnard David , Frank Joachim
Published: February 2005

The three‐dimensional organization of the internal compartments of conventionally fixed and embedded rat‐liver mitochondria has been determined by tomographic reconstruction from tilt‐series images collected on the Albany high‐voltage electron microscope. The results indicate that the inner membranes of these organelles are predominantly tubular in the orthodox (expanded matrix) conformation, as previously suggested by scanning electron microscopy. In the condensed (contracted matrix) conformation, the intracristal space opens up into large irregularly shaped compartments which are connected to each other and to the external (intermembrane) space by tubes with approximately the same diameter (20 nm) as those observed in the orthodox state. These results raise several questions, in particular about the nature of the structural transitions that occur in the cristae during matrix expansion and contraction, and about the influence of inner‐membrane shape on the diffusion of ions and metabolites between the intracristal and intermembrane compartments. © 1994 Wiley‐Liss, Inc.
Allografts of CNS tissue possess a blood‐brain barrier: III. Neuropathological, methodological, and immunological considerations
Authors: Broadwell Richard D. , Baker Belinda J. , Ebert Paul S. , Hickey William F.
Published: February 2005

Development of a blood‐brain barrier (BBB) within mammalian CNS grafts, placed either intracerebrally or peripherally, has been controversial. Published data from this laboratory have emphasized the presence or the absence of a BBB within solid mammalian tissue or cell suspension grafts is determined intrinsically by the graft and not by the surrounding host parenchyma (e.g., brain, kidney, testis, etc.). Nevertheless, correctly interpreting whether or not a BBB exists within brain grafts is manifested by methodologies employed to answer the question and by ensuing neuropathological and immunological consequences of intracerebral grafting. The present study addresses these issues and suggests misinterpretation for the absence of a BBB in brain grafts can be attributed to: (1) rupture of interendothelial tight junctional complexes in vessels of CNS grafts fixed by perfusion of the host; (2) damage to host vessels and BBB during the intracerebral grafting procedure; (3) graft placement in proximity to inherently permeable vessels (e.g., CNS sites lying outside the BBB) supplying the subarachnoid space/pial surface and circumventricular organs such as the median eminence, area postrema, and choroid plexus; and (4) graft rejection associated with antigen presenting cells and the host immune response. The latter is prevalent in xenogeneic grafts and exists in allogeneic grafts with donor‐host mismatch in the major and/or minor histocompatibility complex. CNS grafts between non‐immunosuppressed outbred donor and host rats of the same strain (e.g., Sprague Dawley or Wistar rats) can be rejected by the host; these grafts exhibit populations of immuonohistochemically identifiable major histopatibility complex class I+ and class II+ cells (microglia, macrophages, etc.) and CD4+ T‐helper and CD8+ T‐cytotoxic lymphocytes. PC12 cell suspension grafts placed within the CNS of non‐immunosuppressed Sprague Dawley rats are rejected similarly. Donor cells from solid CNS grafts placed intracerebrally and stained immunohistochemically for donor major histocompatibility complex (MHC) class I expression are identified within the host spleen and lymph nodes; these donor MHC expressing cells may initiate the host immune response subsequent to the cells entering the general circulation through host cerebral vessels damaged during graft placement. Rapid healing of damaged cerebral vessels is stimulated with exogenously applied basic fibroblast growth factor, which may prove helpful in reducing the potential entry of donor cells to the host circulation. These results have implication clinically for the intracerebral grafting of human fetal CNS cell suspensions. © 1994 Wiley‐Liss, Inc.
Lamina lucida of basement membrane: An artefact
Authors: Chan Franky L. , Inoue Sadayuki
Published: February 2005

In tissues prepared with chemical fixation followed by conventional dehydration, basement membranes have been observed to be laminated structures composed of a lamina lucida and lamina densa as well as a poorly limited transitional zone referred to as the pars fibroreticularis. Scattered attempts in the application of new techniques of tissue preparation such as cryofixation or freeze substitution for the study of the basement membrane structure have been made in recent years. From these studies, the possibility has arisen in which basement membranes are composed of only the lamina densa without a lamina lucida. In recent studies in this laboratory, the attempt was made to determine whether or not this lamina lucida is an artefact, and if so, which step in the conventional method of tissue preparation is responsible for its formation. Basement membranes from diverse sources in the mouse and rat including the testis, ductus epididymis, eye, thyroid, kidney, and skin, were observed after either cryofixation by slam freezing followed by freeze substitution, or aldehyde fixation followed by freeze substitution. The basement membranes after preservation with either of these two methods were composed of only the lamina densa with no lamina lucida. It indicates that an artefactual formation of the lamina lucida occurs during dehydration in conventional tissue preparation rather than during chemical fixation. In view of the well known superiority of freeze substitution over conventional dehydration, the lamina lucida of the basement membrane is likely to be an artefact. Therefore, it is concluded that the lamina lucida is an artefact formed during conventional tissue preparation, and in its original condition in the living state, the basement membrane is composed of a single layer made up of lamina densa material. © 1994 Wiley‐Liss, Inc.
Ultrastructure of developing kidney glomerular basement membranes: Temporal changes in binding of anti‐laminin lgG and cationized ferritin
Authors: Abrahamson Dale R. , John Patricia L. St.
Published: February 2005

In vivo labeling of infant rat and mouse glomerular basement membranes (GBMs) with polyclonal anti‐laminin IgGs results in binding across the full widths of GBMs at all stages of development. These stages include the pre‐fusion, double basement membranes found beneath endothelial cells and podocytes in early glomeruli, and the subepithelial matrix outpockets where newly synthesized GBM is spliced into fused basement membrane during glomerular maturation. Identical binding results are obtained either with peroxidase or post‐embedding immunogold techniques. Although injected cationized ferritin also binds abundantly to all developing GBMs, it quickly disappears and, 24 hours after injection, is generally absent from GBMs but remains within mesangial matrices. Injection of newborn mice with monoclonal anti‐laminin IgGs results in dense labeling of pre‐fusion GBMs but post‐fusion GBMs and subepithelial outpockets are weak‐negative. Although masking can not be excluded, these results indicate that laminin epitopes are removed during GBM fusion and splicing, either by isoform substitution or proteolytic processing. The loss of bound cationized ferritin is believed to occur mainly through rapid turnover of GBM proteoglycans. © 1994 Wiley‐Liss, Inc.
Morphology of the basement membrane
Authors: Merker Hans‐Joachim
Published: February 2005

The aim of this contribution is to summarize our knowledge of the morphology of the basement membrane (BM). The first step in this direction is the attempt to define this term. The BM is composed of the Lamina lucida, densa, and fibroreticularis. Subsequently, the historical development of this term is discussed. Our main interest is, of course, focused on the description of the BM‐structure up to the macromolecular level and the special forms of this structure. This is supplemented by discussing its chemical composition and establishing a relationship between morphology and biochemistry. The obtained findings yielded some indications as to the molecular composition of the BM which may serve for the construction of “models.” The composition of the Lamina lucida (L.l.) and the Lamina or Pars fibroreticularis (L.f.) must be discussed separately, since, if present, they show a different and strongly varying structure (L.f.). An important aspect is the function of this extracellular layer which comprises mechanical tasks up to inductive effects. Finally, the concepts of the formation of the BM, especially of the Lamina densa (L.d.), are summarized. It obviously consists of a sequence of individual steps which starts with expression and secretion of the L.d.‐components and is followed by an induction of integrin expression. © 1994 Wiley‐Liss, Inc.
Quantitative X‐ray microanalysis of P, Ca, and S in the mucus secretory granules of the cryofixed frog palate epithelium
Authors: Wagner Denis , Puchelle Edith , Hinnrasky Jocelyne , Girard Pascal , Balossier GéRard
Published: February 2005

In respiratory epithelium, the mucus is densely packed inside the secretory granules (SG) of secretory cells (SC) before being released by exocytosis in the airway lumen. We have previously shown that the frog palate is a representative model of respiratory epithelium and that rapid cryofixation is a very effective technique in preserving the integrity of the mucus SG. The concentration of phosphorus (P), sulphur (S), and calcium (Ca) were analysed inside the SG of the SC of frog palate after quick freezing, cryosubstitution, and embedding in Lowicryl resin at low temperature. The experiments were carried out using X‐ray microanalysis conducted with energy dispersive spectrometry (EDS) at 100 kV. The quantitation was carried out using the continuum method with reference to Agar standards. The cryofixation permitted us to distinguish two types of SG depending on whether they were electron dense (serous cells) or electron‐lucent (mucous cells). A significant (P < 0.001) difference in the S concentration was observed between the individual serous (239 ± 79 mmol.kg−1) and the mucous SG (161 ± 48 mmol.kg−1). No significant difference could be identified in the Ca concentration between the two SG phenotypes. In the serous SG, the P content was high (41 ± 17 mmol.kg−1) compared with the mucous SG where it was not measurable. The comparison of the three element concentrations in each type of secretory cells showed that significant differences in concentration of S and Ca concentration could be observed from one SC cell to another. A significant correlation (r = 0.76, P < 0.01) was observed between the S concentration and the topographical position of the SG inside the SC, the more proximal to the lumen, the higher the S concentration, suggesting that the maturation of the SG involves an increase in the protein content possibly due to a maturation process before the mucus exocytosis. Therefore, these results suggest that the elemental composition of granules varies according to the phenotype of the secretory cells and that changes in the S content from one SG to another or even inside the same cell may reflect a differential state in the functional activity of the secretory cells. © 1994 Wiley‐Liss, Inc.
Role of the basement membrane in neurogenesis and repair of injury in the central nervous system
Authors: Choi Ben H.
Published: February 2005

Although remarkable progress has been made during the last two decades concerning the biosynthesis, expression and assembly of extracellular matrix (ECM) macromolecules in nonneural cells, we are still far from a complete understanding of the role and function of the ECM and the basement membrane (BM) in the central nervous system (CNS). With the aid of correlative light and electron microscopic, Golgi and immunohistochemical studies of the developing neural tube of both early human fetus and mouse, we have shown that the establishment of the pial‐glial barrier (PGB) is one of the earliest histogenetic events in neurogenesis. This is accomplished by coordinated interaction among the processes of radial glia, various ECM components, and mesenchymal cells at the pial surface, with the formation of a BM that tightly abuts the glia limitans. The PGB and the BM appear to be critical to the migration and final positioning of neurons and to the differentiation of the laminar cortical pattern within the developing neopallium. This hypothesis is further supported by our study of the brain of a human newborn infant in whom multiple sites of disruption of the BM and PGB resulted in abnormal neuronal migration and massive ectopia of neurons within the subarachnoid space, with abnormal cortical lamination. Finally, studies of the experimental cryogenic injury to the neonatal rat cerebrum have shown that the final positioning of neurons within the developing cortical plate appears to depend largely on the reconstitution of the BM and PGB, which presumably provide crucial positional signals for migrating neurons. Also, one of the essential reparative features seen following cryogenic injury to the adult rat cerebrum is an orderly and dynamic interaction between various ECM components and neural cells, resulting in the formation of the BM. © 1994 Wiley‐Liss, Inc.
Surface expression and distribution of Fc receptor III (CD 16 molecule) on human natural killer cells and polymorphonuclear neutrophils
Authors: Fernández‐Segura E. , García J. M. , López‐escámez J. A. , Campos A.
Published: February 2005

Human natural killer cells and polymorphonuclear neutrophils constitutively express the low‐affinity IgG Fc receptor (FcγRIII, CD16 molecule). To investigate cell surface morphology, antigenic receptor density, and topographical distribution of FcγRIII on the plasma membrane of natural killer cells and polymorphonuclear neutrophils, conventional scanning electron microscopy (SEM), flow cytometry, and immunoscanning electron microscopy were used. FcγRIII was detected with an indirect immunogold labeling procedure, and receptors were visualized in the backscattered and secondary electron imaging mode of SEM. Natural killer cells showed a cell surface morphology compatible with lymphocytic differentiation characterized by microvilli and microridges. Polymorphonuclear neutrophils showed surface features characterized by ridges with folds and scattered short microvilli. Natural killer cells displayed a lower cell labeling density, whereas polymorphonuclear neutrophils showed a high level of expression of FcγRIII on the plasma membrane by quantitative analysis with SEM in the backscattered electron imaging mode. Flow cytometry analysis confirmed these findings. Analysis of the topographical distribution of FcγRIII antigenic receptor sites by SEM in the backscattered and secondary electron imaging modes showed that FcγRIII on natural killer cells are randomly distributed, whereas FcγRIII are located on ridges and folds of the plasma membrane of polymorphonuclear neutrophils. These observations suggest that natural killer cells and polymorphonuclear neutrophils differ in their levels of expression and topographic distribution of FcγRIII on the plasma membrane. This different spatial distribution of FcγRIII would provide morphological evidence of certain cellular functions mediated by natural killer cells and polymorphonuclear neutrophils. © 1994 Wiley‐Liss, Inc.
Endocytosis of three serum proteins of a multigene family and of arachidonic acid in human lectin‐stimulated T lymphocytes
Authors: Geuskens M. , Torres J. M. , Esteban C. , Uriel J.
Published: February 2005

Alpha‐fetoprotein (AFP), serum albumin (SA), and vitamin D binding protein (DBP) are members of a multigene family of proteins showing high structural homology. AFP and SA exhibit a reciprocal relation during development and carry mostly fatty acids, while DBP carries vitamin D and its metabolites in the plasma. Covalent conjugates of these proteins with horseradish peroxidase (HRP) were used to follow by cytochemistry, at the electron microscope level, the protein uptake and intracellular pathways in peripheral blood human lymphocytes stimulated to blast formation by phytohemagglutinin (PHA). Transferrin (Tf), an iron‐binding plasma protein, was used as a control.

Combined with the results of competition and saturability experiments reported elsewhere, the ultrastructural observations are in favour of a specific endocytosis of the four proteins through cell surface receptors. Tf and AFP enter the cells via small vesicles and endosomes and move to multivesicular bodies (MVBs) and tubular vesicular elements located in the Golgi‐centrosphere region to be finally recycled back into the medium. A noncovalent conjugate of AFP‐HRP with 3H arachidonic acid [3H‐(20:4)] is strongly internalized at 37°C in PHA‐stimulated lymphocytes; the autoradiographic labelling, localized in cellular membranes and mostly in lipid droplets, was only occasionally associated with organelles where the presence of AFP‐HRP was cytochemically detected.

SA, which competes with AFP for a common binding site on the surface of activated T cells, is endocytosed through small vesicles, endosomes, and MVBs before being released in a degraded form from the cells, in agreement with the localization of SA‐HRP in lysosome‐like organelles. DBP‐HRP is poorly internalized through noncoated vesicles, endosomes, and MVBs and is finally routed to lysosomes.

The physiological role of AFP and SA would be to mediate the transfer of fatty acids into cells, while that of DBP would be to facilitate the intracellular delivery of vitamin D. © 1994 Wiley‐Liss, Inc.
Scanning and transmission electron microscopy of clonal peripheral blood lymphocytes in low grade non‐Hodgkin's B‐cell lymphomas with predominant splenomegaly
Authors: Soligo Davide , Quirici Nadia , Caneva Lorenza , Baldini Luca , Cro Lilla , Deliliers Giorgio Lambertenghi
Published: February 2005

Peripheral blood mononuclear cells (PBLs) from 14 patients with low grade non‐Hodgkin's B‐cell lymphomas with predominant splenomegaly were studied by means of scanning (SEM) and transmission electron microscopy (TEM). All patients had peripheral blood and bone marrow involvement, the absence of lymphoadenopathy, and, except in one case, immunophenotypic features of a malignant proliferation of mature spleen B‐cells arising from outside the germinal center, but not consistent with CLL or HCL. Several distinctive cytological features were observed in PBLs of the different subgroups. The SEM surface features of PBLs in patients with intermediate differentiation lymphocytic lymphoma (IDL) (five cases), lymphoplasmacytoid immunocytoma (LP‐IC) (two cases), and mixed small and large cells malignant lymphoma (one case) were characterized by the presence of numerous well‐developed microvilli. Some distinctive TEM ultrastructural features were also seen in the different cases. In the two cases of splenic lymphoma with villous lymphocytes (SLVL), SEM revealed large and elongated surface microvilli generally arising from two or three poles of the cells. This surface morphology, confirmed by TEM analysis, may be pathognomonic of this disease. Four additional cases, tentatively classified as small lymphocytic lymphoma on the basis of immunophenotypic data, were extremely heterogeneous at both SEM and TEM analysis. The ultrastructural features revealed by SEM and TEM may be useful for the more precise characterization of this heterogenous group of diseases, which is generally difficult to define even when immunophenotypic and molecular approaches are used. © 1994 Wiley‐Liss, Inc.
Calculation of overall tilt angles for a double tilt holder in a TEM
Authors: Kelly Patrick M. , Wauchope Corinna J. , Zhang Xuezhi
Published: February 2005

A simple formula has been derived for the tilt angle of a specimen in terms of the two tilt angles of a side entry, double tilt holder in a transmission electron microscope. An expression for calculating the direction of the apparent tilt axis in relation to the observed diffraction pattern has also been derived. The accuracy and reproducibility of specimen tilting has been assessed experimentally. © 1994 Wiley‐Liss, Inc.
Proteoglycan synthesis by cultured human chondrocytes
Authors: Aulthouse Amy Lynn , Becker Konrad , Beck Michael
Published: February 2005

Iliac crest biopsies are important in the detection of human skeletal dysplasias. Therefore, culture of these cells may serve as a valuable method for studying proteoglycan metabolism in chondrocytes of individuals with skeletal abnormalities. Morphological and biochemical studies were performed on human iliac crest chondrocytes grown in monolayer and in agarose gels. Two proteoglycan populations of different hydrodynamic size and glycosaminoglycan composition were synthesized by cells grown in monolayer. Chondrocytes cultured in an agarose gel for 2 weeks synthesized proteoglycans identical to those of the native tissue with respect to hydrodynamic size and glycosaminoglycan chain length. However, the ratio of chondroitin‐6‐sulfate to chondroitin‐4‐sulfate was higher than in the native tissue. This ratio was not influenced by different sulfate concentrations in the medium. Moreover, treatment with ascorbic acid did not influence proteoglycan synthesis; however, there was a pericellular accumulation of proteoglycans. © 1994 Wiley‐Liss, Inc.
Preservation and contrast without osmication or section staining
Authors: Locke Michael
Published: February 2005

Conventional treatment of tissues for sectioning and transmission electron microscopy uses aldehyde fixation and osmium tetroxide postfixation. Although the result is aesthetically pleasing, osmication destroys some cell components and reduces the chemical activity of others, such as reactions with antibodies and lectins. We have found that aldehyde fixation followed by uranyl acetate preserves and contrasts most structures and visualizes some that are not easily seen after osmication. Aldehyde/UA treated tissues have enough contrast to be observed without section staining while retaining some of the chemical activity that is lost through osmication. Sections of tissues with good preservation and contrast can be used for immunogold and lectin‐gold labelling of at least some molecules. © 1994 Wiley‐Liss, Inc.
A negative contrast stain for ultra‐thin frozen sections
Authors: Himmelhoch Stanley
Published: February 2005

Ultra‐thin frozen sections are ideal substrates for immunolabeling in high resolution electron microscopy. However, visualization of subcellular structures is inferior to that obtained with corresponding plastic sections. Although negative staining is generally effective and even superior to positive staining, the accumulated stain is often too heavy, obscuring morphology and markers used for immunocytochemical localization of antigens. This paper describes the development of a modified negative contrast staining technique in which a high concentration of uranyl acetate is mixed with methyl cellulose at a low pH. Application of this stain to cryosections of cells and tissue resulted in improved visualization of morphological structures characterized by negative images of membranes and cell organelles. Use of this stain is advantageous for morphological and immunocytochemical studies involving ultra‐thin frozen sections. © 1994 Wiley‐Liss, Inc.
Correlated alkaline phosphatase histochemistry and quantitative backscattered electron imaging in the study of rat incisor ameloblasts and enamel mineralization
Authors: Gomez Santiago , Boyde Alan
Published: February 2005

The different functional conditions of pre‐ameloblasts, secretory ameloblasts, and maturation ameloblasts in 9 day rat incisors were recognized using high resolution light microscopic alkaline phosphatase histochemistry: Digital backscattered electron imaging was performed using the block surfaces from which thin sections were taken for histochemical study. It was possible to correlate exact locations in histochemical sections with positions in the block face at all stages of enamel mineralization from early secretion through late maturation. The first steep increase in the rate of mineralization of completed enamel matrix occurs after the first transition from smooth ended ameloblasts to ruffle ended ameloblasts. In the 9 day rat incisors used for this purpose, there are only two smooth to ruffle cyclical transitions, and the width of successional smooth ended bands of ameloblasts in the maturation cycling process is always narrow. Nevertheless, there seems to be a good correlation between mineralization increase and the acquisition of the high alkaline phosphatase activity in the deeply enfolded distal cytoplasm of the ruffle‐ended maturation stage ameloblasts. © 1994 Wiley‐Liss, Inc.