bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel cell culture technique for electron microscopy
Authors: Wang Fen , Ledford Lindsay B. , Head Jonathan F. , Elliott Robert L.
Published: February 2005

A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed. The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ. Electron micrographs showed cells in their natural state of contact with one another. The plasma membrane and intracellular organelles were well preserved. This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them. © 1993 Wiley‐Liss, Inc.
Morphological effects of diabetes on the granular ducts and acini of the rat submandibular gland
Authors: Anderson Leigh C. , Suleiman Ahmed H. , Garrett John R.
Published: February 2005

Effects of experimental diabetes on rat submandibular glands have been documented, but earlier reports suggested that diabetes caused an extensive cellular degeneration and a replacement of the parenchymal cells by fibrous connective tissue. Such observations, however, are difficult to reconcile with the relatively normal physiological responsiveness of the gland (Anderson and Suleiman, 1989). This study, therefore, reexamined the histological, histochemical and ultrastructural effects of streptozotocin‐induced diabetes on rat submandibular glands. The tissues were examined at 3 weeks, and 3 and 6 months after the induction of diabetes, and compared with glands from age‐matched controls by both light and electron microscopy. Light microscopically, the proportional volumes of the acini and granular ducts remained constant in control rats at about 48% and 38% respectively. In diabetic animals the volume density of the acini increased progressively to 62%, whereas that of the granular ducts decreased to 20%. The diameter and number of granular ducts were reduced in diabetic animals, but acinar cell profile area was only affected 6 months after the induction of diabetes. Ultrastructurally, there was an accumulation of lipid in the acinar cells and, with increasing duration of diabetes, the number of autophagic structures in both the acini and the granular ducts increased. Although there was evidence of some cellular degeneration it was never excessive. Morphometry showed that the volume density of secretory granules within the acinar cells was unaffected, but there was a significant reduction in the volume density of secretory granules within the granular ducts. Thus, in the rat submandibular gland the greatest effect of streptozotocin‐induced diabetes was to cause hypotrophic changes in the cells of the granular ducts. The relative contributions of a direct effect of insulin insufficiency and the hypogonadal effects of diabetes, however, are not known. © 1994 Wiley‐Liss, Inc.
Regulation of ovarian follicular development: A review of microscopic studies
Authors: Roy Shyamal K.
Published: February 2005

Follicular development in the mammalian ovary is a complex process regulated by an orchestrated action of the pituitary gonadotropins, e.g., follicle stimulating hormone (FSH) and luteinizing hormone (LH), and local ovarian factors, such as peptide growth factors and steroids. The mechanism of endocrine/paracrine/autocrine regulation of folliculogenesis (i.e., cell proliferation and functions) has been addressed largely by biochemical means. However, the availability of immunological and molecular tools now enables us to undertake critical microscopic studies revealing the ovarian cell‐type specific synthesis and/or accumulation of many of these local peptide modulators, their roles in the proliferation and differentiation of follicular cells, and their regulation by gonadotropins and local factors. The objective of this review is to provide a comprehensive yet complete picture of the endocrine/autocrine regulation of mammalian folliculogenesis as revealed by microscopic studies. Efforts have been made to include adequate research information relevant to update our understanding of the process of follicular development; however, to maintain the brevity, many equally important studies could not be included. This review confirms that FSH and LH are still the primary stimuli for follicular development. However, it is clear that the actions of these hormones at the cell level involve a host of peptide factors which are produced locally by different follicular cell types and which are powerful modulators of gonadotropin actions. The temporal and spatial expression of the genes of these modulators, the synthesis of active factors, their interactions, and the dynamics of their receptors on the follicular cell surface may be the ultimate determinants of cellular events which are crucial to coordinated growth and differentiation of follicular cells leading to folliculogenesis and ovulation. © 1994 Wiley‐Liss, Inc.
Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes
Authors: Van Blerkom Jonathan , Davis Patrick W.
Published: February 2005

This study examined the effects of cryopreservation on cellular organization, chromosomal complement, and developmental potential of immature and mature mouse and human oocytes. Chromosomal analyses were performed by DNA fluorescence microscopy and karyotyping on the same metaphase II‐stage oocytes before and after freezing. Cellular analyses involved electron microscopy, time‐lapse video recording, and fluorescent‐probe microscopy of cortical granules. The findings demonstrate that while profound cytoplasmic, nuclear, and nucleolar alterations occur in the immature oocyte during cryopreservation, an apparently normal nucleus and cytoplasm is re‐established progressively after thawing and culture. The resulting oocytes mature at high frequency and for the mouse, are fertilizable and capable of normal preimplantation of embryogenesis. Cryopreservation of mature mouse and human oocytes is not accompanied by a significant increase in the frequency of aneuploidy. However, cryopreserved human oocytes, while fertilizable, arrest development during the early cleavage stages and display aberrant patterns of cytokinesis. The possible etiologies of developmental failure in the human embryo that may be related to oocyte cryopreservation, as well as the potential benefits of cryopreservation of the immature oocyte, are discussed with respect to clinical and commercial applications. © 1994 Wiley‐Liss, Inc.
Mitochondrial matrix granules: Their behavior during changing metabolic situations and their relationship to contact sites between inner and outer mitochondrial membranes
Authors: Jacob Wim A. , Bakker Annette , Hertsens Robert C. , Biermans Walter
Published: February 2005

Since their discovery in the early fifties mitochondrial granules have been the subject of many researches. Some twenty years ago two hypotheses on their function were introduced. Peachey thought that the granules were a sink of cations and that they would eventually regulate the concentrations of these ions. Alternatively, Barnard thought that the granules were precursors of the mitochondrial inner membrane. There are only a few data on organic constituents of the granules. Phospholipids (e.g., cardiolipin) glycoprotein or lipids, calcium precipitable lipoprotein, cytochrome c oxidase seem to be present in the granules. There has been much debate on whether calcium is present or not. Reports are mostly based on X‐ray microanalysis, the result of which depends on preparation techniques.

In heart muscle in stimulating situations the NMG (native matrix granules) move towards the inner membrane and are incorporated in it. They appear to create contact sites between inner and outer mitochondrial membranes in which enzymes can function efficiently.

It is hypothetized that the system, NMG‐contact sites, forms the structural basis of a regulatory mechanism, by which cells can cope with a high and sudden energy demand. © 1994 Wiley‐Liss, Inc.
Normal and abnormal development of the blood‐brain barrier
Authors: Farrell Catherine L. , Risau Werner
Published: February 2005

The blood‐brain barrier is responsible for the maintenance of the neuronal microenvironment. This is accomplished by isolation of the brain from the blood by the tight junctions that join endothelial cells in cerebral microvessels, and by selective transport and metabolism of substances from blood or brain by the endothelial cells. This review describes the growth and maturation of the brain vasculature, and the development of the special properties of the endothelia at the blood‐brain interface. Evidence suggests that the development of the unique properties of the brain microvasculature is a consequence of tissue‐specific interactions between endothelial cells of extraneural origin and developing brain cells. The cellular and molecular mechanisms that control these processes are as yet unknown but this review will include experimental studies which have used in vivo and in vitro systems to investigate what factors may be involved, and some pathological conditions in which abnormal barrier development is thought to be an important aspect of the disease process. © 1994 Wiley‐Liss, Inc.
Use of fluorescent europium chelates as labels in microscopy allows glutaraldehyde fixation and permanent mounting and leads to reduced autofluorescence and good long‐term stability
Authors: Sevéus Lahja , Väisälä Mikko , Hemmilä Ilkka , Kojola Hannu , Roomans Godfried M. , Soini Erkki
Published: February 2005

In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time‐resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross‐linking fixation with glutaraldehyde was used and the signal‐to‐background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long‐term stability for the samples with glutaraldehyde fixation and permanent mounting. © 1994 Wiley‐Liss, Inc.
Comparative study of freeze‐substitution techniques for x‐ray microanalysis of biological tissue
Authors: Påsgård Eva , Lindh Ulf , Roomans Godfried M.
Published: February 2005

A comparative study of some aspects of freeze‐substitution as a preparative method for X‐ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X‐ray microanalysis of thin sections of mouse pancreas were used. Freeze‐substitution for 3 weeks in diethy ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze‐substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze‐substitution in acetone resulted in noticeable loss and redistribution of ions and freeze‐substitution in methanol gave very poor results. © 1994 Wiley‐Liss, Inc.
Cytoskeleton of cartilage cells
Authors: Benjamin M. , Archer C. W. , Ralphs J. R.
Published: February 2005

The cytoskeleton of chondrocytes consists of microfilaments made of actin, microtubules made of tubulin, and intermediate filaments made of a variety of subunits. Actin filaments are not prominent in vivo but may form in vitro. In culture, changes in filament polymerisation are important in determining cell shape, initiating chondrogenesis, and maintaining the chondrogenic phenotype. Microtubules, besides their role in cell division, organise the distribution of organelles and are involved in secretory transport mechanisms in collagen and proteoglycan synthesis. A variety of intermediate filaments may be present, frequently forming large whorled aggregates. The filaments include vimentin, cytokeratins, and glial fibrillary acidic protein. These may occur at different depths in articular cartilage. Vimentin accumulates during development of some fibrocartilages with increased mechanical loading. Together with other elements of the cytoskeleton, intermediate filaments could form part of a mechanotransduction system by which cells respond to external forces and sense changes in their external environment. © 1994 Wiley‐Liss, Inc.
Automated image analyzing system for the quantitative study of living cells in culture
Authors: van Opstal W. Y. Xu‐ , Ranger C. , Lejeune O. , Forgez P. , Boudin H. , Bisconte J. C. , Rostene W.
Published: February 2005

A fully automated image analyzing system was developed for the quantitative study of cells in culture. It was able to count cells, to classify cells according to their morphological characteristics and to follow cell culture development. A specific procedure was designed to process Hoffman modulation contrast images. It detects local gray level differences while using conditional dilation techniques. We were able to successfully detect aggregated unstained cells, presently a technical limit in image segmentation. Living cells can be studied in a noninvasive and nondestructive way with this system. An improved automatic focusing algorithm was developed which ensured an accurate prediction of the optimal focus position. A strictly defined sampling procedure was applied to estimate unbiasedly cell density and obtain precisely cell contours. The evaluation of the system was carried out on Chinese hamster ovary (CHO‐NTR) cell cultures treated with a newly developed neurotensin agonist JMV449. Chinese hamster ovary cell division was found to be retarded 20 hours after the JMV449 treatment, while the morphology of CHO‐NTR cells has already undergone significant changes 12 hours after the treatment. This image analyzing system provides the possibility to follow cell culture development (e.g., cell density evolution, cell morphological changes) under various experimental conditions. © 1994 Wiley‐Liss, Inc.
Analysis of the mechanism for acetylcholine release at the synapse formed between rat sympathetic neurons in culture
Authors: Mochida Sumiko , Nonomura Yoshiaki , Kobayashi Haruo
Published: February 2005

Superior cervical ganglion neurons (SCGNs) were isolated from 7‐day‐old rat SCG and cultured in MEM containing horse serum, fetal calf serum, and nerve growth factor. In this culture condition, it is well known that the SCGNs form cholinergic synapse. In 3–4 weeks cultured neurons, immunofluorescent staining for synaptophysin, a small synaptic vesicle associated protein, showed the presence of synaptophysin as small dots on the surface of the soma. Postsynaptic potentials could be recorded in 50–80% of the neurons responding to evoked action potentials elicited in neighboring neurons. Because of its relatively large cell size and the short distance to the terminal, this synapse is a useful model for studying the mechanisms of acetylcholine (ACh) release by introducing substances such as antibodies or selective inhibitors into the presynaptic neuron by means of the whole‐cell clamp technique.

In this model synapse we tested the possible role of myosin in ACh release. The distribution of myosin was studied by the immunofluorescent staining technique. Myosin was recognized by the anti‐myosin II IgG at the same synaptic terminals that showed the presence of synaptophysin with its antibody. The functional blockade of myosin by the antibody itself, and that of myosin light chain kinase (MLCK) by a pseudosubstrate inhibitor of MLCK, SM‐1, or by a selective inhibitor of MLCK, wortmannin, induced depression of synaptic transmission in a dose‐dependent manner. These indicate that phosphorylation of myosin by MLCK may be necessary for ACh release mechanisms. © 1994 Wiley‐Liss, Inc.
Classification of the enteric nerve cells of the porcine small intestine into two subpopulations using enzyme histochemical techniques
Authors: De Ridder E. , Weyns A.
Published: February 2005

Using acetylcholinesterase (AChE), nicotinamide adenine dinucleotide diaphorase (NADHd), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) enzyme histochemical techniques, the ganglionated plexuses of the porcine enteric nervous system were investigated in small intestine whole‐mount preparations. Both AchE and NADHd techniques revealed a majority of the neurons in the ganglia of all three major plexuses. The AchE technique also demonstrated clearly the axodendritic networks of the plexus myentericus. Intraganglionic blank areas revealed the localization of negative cell groups. A very high correlation was found between the activity of both enzymes in one neuron, although this correlation was certainly not linear. Many neurons exhibited a stronger signal for one enzyme. A very small part of the positive nerve cells showed intense staining for both AchE and NADHd. The NADPHd technique demonstrated that the NADPHd‐positive neurons fill the negative intraganglionic spaces in the ganglia. Double staining with the two other enzymes showed virtually no colocalization of NADPHd with either NADHd or AchE in the porcine jejunal enteric ganglia. Little negative intraganglionic spaces were seldom found, leaving room for perhaps still more negative enteric neurons. Based upon these results we suggest that the enteric neurons of the porcine small intestine can be subdivided into AchE‐NADHd and NADPHd subpopulations. Since the latter colocalizes with the neuronal NO synthase enzyme, we further suggest a subdivision of the enteric nerve cells into AchE‐NADHd and NOS‐NADHd neurons. © 1994 Wiley‐Liss, Inc.
Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy
Authors: Klimaschewski Lars , Tran Thang D. , Nobiling Rainer , Heym Christine
Published: February 2005

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double‐labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL‐ or VIP‐positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP‐positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL‐ and VIP‐positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 μm, GAL‐immunoreactive neurons are predominantly of small and intermediate size (22.2 μm), whereas VIP occurs mainly in larger neurons (26.1 μm). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre‐embedding immuno‐electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate‐limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL‐ or VIP‐positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target‐derived factors are discussed. © 1994 Wiley‐Liss, Inc.
Synaptic morphology of substance P terminals on catecholamine neurons in the commissural subnucleus of the nucleus tractus solitarii in the rat
Authors: Chen I‐Li , Cusick Catherine G. , Weber Joseph T. , Yates Robert D.
Published: February 2005

The ultrastructure of substance P‐containing nerve terminals synapsing on catecholamine neurons in the rat commissural subnucleus of the nucleus tractus solitarii (NTScom) was studied using a double immunocytochemical labeling technique. Although there were numerous tyrosine hydroxylase‐immunoreactive (TH‐I) somata present, substance P immunoreactive (SP‐I) cell bodies were only occasionally found in the NTScom. At the light microscopic level, many SP‐I terminals were seen closely associated with TH‐I dendrites and somata. At the electron microscopic level, SP‐I terminals synapsing on TH‐I structures were also readily encountered. SP‐I terminals contained small, clear, and predominantly spherical vesicles (32 ± 4 nm diameter), as well as large dense‐cored vesicles approximately 100 nm in diameter. Postsynaptic TH‐I dendritic profiles of various calibers and somata were encountered. These postsynaptic TH‐I structures often showed postsynaptic densities. The morphological features of the SP‐TH synapses in the present study, that is, the size of synaptic vesicles and the presence of postsynaptic densities, are quite different from those of central carotid sinus afferent synapses reported in our previous study [Chen et al. (1992), J. Neurocytol., 21:137–147]. Therefore, most of the SP terminals of the SP‐TH synapses in the NTScom appear not to originate from the carotid sinus afferents. SP‐I second‐order neurons of the carotid sinus afferent pathway [Chen et al. (1991), J. Auton. Nerv. Syst., 33:97–98] may be one of the possible sources of such terminals. © 1994 Wiley‐Liss, Inc.
Intersection of first order laue zone lines along the [100] direction in an fcc material
Authors: Curzon A. E.
Published: February 2005

An analytical result is given for the wavelength at which two or more first order Laue zone (FOLZ) lines intersect at a single point on the positive x axis when a face centred cubic crystal is observed in the convergent beam mode with the beam axis parallel to the [001] direction. The position of the intersection along the x axis is also given. The method uses the kinematic approximation of electronic diffraction and the exact hyperbolic form of the FOLZ lines, so that the accuracy is good even for lines that intersect at small angles. © 1994 Wiley‐Liss, Inc.
Effects of vasectomy on the epididymis
Authors: Flickinger Charles J. , Howards Stuart S. , Herr John C.
Published: February 2005

Common principles can be discerned in the response of the epididymis to vasectomy, despite species differences. Increases in the size and number of lysosomes are the most frequent changes in the epididymal epithelium. The presence or absence of additional alterations such as changes in the height of the epithelium may be related to variations in distensibility of the vas deferens and epididymis. Direct measurements by micropuncture of epididymal and seminiferous tubule hydrostatic pressure indicate that, contrary to dogma, increased pressure in the distal epididymis after vasectomy is not generally transmitted to the seminiferous tubules. The epididymal interstitium shows microscopic changes indicative of chronic inflammation, with infiltration of macrophages, lymphocytes, and plasma cells, and rats with these lesions have higher antisperm antibody levels than animals lacking epididymal changes. Macrophages and neutrophils may enter the duct through the epididymal epithelium, at sites of rupture of the duct, and in the efferent ductules. Cyst‐like spermatic granulomas occur in virtually all species where the epididymis or vas deferens ruptures with escape of spermatozoa. The sites and timing of granuloma formation may depend on the mechanical properties of the tract in different species, and they are probably important in the immune response to vasectomy. Postvasectomy sera in Lewis rats recognize a consensus repertoire of dominant autoantigens that closely resembles the antigens bound by sera from rats immunized with isologous spermatozoa. There are multiple routes for disposal of the sperm that continue to be produced after vasectomy. © 1995 Wiley‐Liss, Inc.
Structural investigations of recently discovered high Tc superconductors
Authors: Van Tendeloo G. , Krekels T. , Amelinckx S. , Babu T. G. N. , Greaves C. , Hervieu M. , Michel C. , Raveau B.
Published: February 2005

A short overview is given of the possibilities of electron microscopy in the determination of the local, atomic scale structure of high Tc superconducting materials. Examples include the detection of weak oxygen ordering, description and characterization of deformation modulations in layered superconductors, and analysis of very long period superstructures. The ordering principles for tetrahedral chains in Ga‐, Co‐, or Al‐substituted YBCO are discussed and their complex defect structures are described. The incommensurate modulation in YBCO‐based materials containing SO4‐tetrahedra, centered on the Cu(1) sites of the CuO‐chain plane is attributed to the ordering of b‐oriented SO4‐rich chains in the Cu(1)‐S‐O layer; the structure is described in terms of an SO4‐concentration wave. As examples of the new mercury‐based superconducting family we discuss Y0.6Ca0.4Ba2Hg1−xMxCu2O6+y, which crystallizes in the space group P4/mmm with a = 0.3870(1) nm, c = 1.2537(1) nm. This cuprate belongs to the 1212 series; susceptibility measurements show a Tc (onset) of 90K, with a diamagnetic volume fraction of 27% at 4.2K to be reached. A second example is related to the compound Tl2HgBa4Cu2O10+y, in which ordering between single Hg layers and double Tl layers is observed. © 1995 Wiley‐Liss, Inc.
HREM study of the crystal chemistry of the alkaline‐earth cupric oxides stabilized at high pressure
Authors: Hiroi Z.
Published: February 2005

The crystal chemistry of the alkaline‐earth cupric oxides stabilized at high pressure was studied by high‐resolution electron microscopy. Two families of superconductors, (Ca1−ySry)1−xCuO2 and Srn+1Cun O2n+1+δ (n = 1, 2), and one series of semiconductors, Srn−1Cun+1 O2n, are reported. Their structural characteristics are systematically interpreted on the basis of the simple infinite‐layer structure seen in SrCuO2. Microstructures and their relevance to high‐Tc superconductivity are discussed, particularly in the alkaline‐earth‐deficient infinite‐layer phase. © 1995 Wiley‐Liss, Inc.
Shadow images for in‐line holography in a STEM instrument
Authors: Wang Shi‐Yao , Cowley J. M.
Published: February 2005

In a dedicated STEM instrument equipped with a field emission gun, shadow images are easily obtained and have many uses. They are very sensitive to misalignment of the instrument and astigmatism, and therefore can be used for rapid and accurate alignment of the microscope. For crystalline materials, the shadow image contains both the bright‐field and dark‐field images. It is a summation of the transmitted and diffracted beams, and is basically a kind of Gabor's in‐line hologram. Under small or medium defocus, shadow images of a thin, well‐orientated crystalline specimen take the characteristic form of Ronchigrams, which offer a unique means to calibrate the microscope operation parameters, such as the spherical aberration coefficient Cs and defocus settings of the objective lens, with high accuracy. With the calibrated values of Cs and δ, a transfer function of the objective lens may be generated. In the stage of numerical reconstruction, by adapting this transfer function to the experimentally recorded hologram the lens aberration introduced in forming the hologram may be corrected and an improved resolution may be achieved for electron microscope images. © 1995 Wiley‐Liss, Inc.
Freeze‐fracture study of Bodo sp. (Kinetoplastida: Bodonidae)
Authors: Vommaro Rossiane Claudia , Attias Márcia , De Souza Wanderley
Published: February 2005

Freeze‐fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free‐living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows. © 1995 Wiley‐Liss, Inc.
Development and postnatal regulation of adult myoblasts
Authors: Yablonka‐Reuveni Zipora
Published: February 2005

The myogenic precursor cells of postnatal and adult skeletal muscle are situated underneath the basement membrane of the myofibers. It is because of their unique positions that these precursor cells are often referred to as satellite cells. Such defined satellite cells can first be detected following the formation of a distinct basement membrane around the fiber, which takes place in late stages of embryogenesis. Like myoblasts found during development, satellite cells can proliferate, differentiate, and fuse into myofibers. However, in the normal, uninjured adult muscle, satellite cells are mitotically quiescent. In recent years several important questions concerning the biology of satellite cells have been asked. One aspect has been the relationship between satellite cells and myoblasts found in the developing muscle: are these myogenic populations identiacal or different? Another aspect has been the physiological cues that control the quiescent, proliferative, and differentiative states of these myogenic precursors: what are the growth regulators and how do they function? These issues are discussed, referring to previous work by others and further emphasizing our own studies on avian and rodent satellite cells. Collectively, the studies presented indicate that satellite cells represent a distinct myogenic population that becomes dominant in late stages of embryogenesis. Moreover, although satellite cells are already destined to be myogenic precursors, they do not express any of the four known myogenic regulatory genes unless their activation is induced in the animal or in culture. Furthermore, multiple growth factors are important regulators of satellite cell proliferation and differentiation. Our work on the role of one of these growth factors [platelet‐derived growth factor (PDGF)] during proliferation of adult myoblasts is further discussed with greater detail and the possibility that PDGF is involved in the transition from fetal to adult myoblasts in late embryogenesis is brought forward. © 1995 Wiley‐Liss, Inc.
Confocal microscopy of thick sections from acrylamide gel embedded embryos
Authors: Germroth Patricia G. , Gourdie Robert G. , Thompson Robert P.
Published: February 2005

The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three‐dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10–15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 μm. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water‐soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2–3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water‐miscible media. © 1995 Wiley‐Liss, Inc.
Immunogold studies on peroxisomes: Review of the localization of specific proteins in vertebrate peroxisomes
Authors: Usuda Nobuteru , Hanai Toru , Nagata Tetsuji
Published: February 2005

Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A‐gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research. © 1995 Wiley‐Liss, Inc.
Quantitative immunocytochemical study of secretory protein expression in parotid glands of rats chronically treated with isoproterenol
Authors: Vugman Ithamar , Hand Arthur R.
Published: February 2005

Chronic treatment of mice and rats with isoproterenol (IPR) causes marked hypertrophy and hyperplasia of the salivary glands, and alters the expression of several secretory proteins. We used quantitative postembedding immunogold labeling to study the cellular responses in the rat parotid gland during daily (up to 10 days) injections of IPR and during recovery (up to 14 days) after cessation of IPR treatment. Labeling densities of acinar cell secretory granules with antibodies to amylase and protein SMG‐B1 (cross‐reactive with the rat homologue of Parotid Secretory Protein, PSP) fell to 10% of control levels after 8–10 IPR injections, then increased during recovery, paralleling previous biochemical determinations of changes in protein and mRNA levels. With antibodies to proline‐rich proteins (PRP), labeling densities initially fell, then subsequently showed considerable variability, but never exceeded control levels. These results contrast with biochemical determinations showing a marked induction of PRP synthesis, and may have both immunological and structural explanations.

Occasional intercalated duct cells located close to the acini underwent differentiation toward an acinar‐like phenotype as a result of IPR treatment. After 1–2 IPR injections, the secretory granules of these cells labeled with antibodies to amylase and PRP. Subsequently, the granules appeared electron‐lucent and were increased in size and number. These observations support earlier work, suggesting that intercalated duct cells may differentiate into other gland cell types.
Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells
Authors: Gómez‐Pascual Amparo , Londoño Irene , Ghitescu Lucian , Desjardins Michel , Bendayan Moöse
Published: February 2005

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho‐cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A‐gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde‐fixed, osmiumpostfixed, and Epon‐embedded kidneys. Cationic BSA‐DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium. © 1995 Wiley‐Liss, Inc.
Cellular functions during activation and damage by pathogens: Immunogold studies of the interaction of bacterial endotoxins with target cells
Authors: Risco Cristina , da Silva Pedro Pinto
Published: February 2005

Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gramnegative bacteria that act on numerous cellular functions through the processes of cell activation and damage. The molecular mechanisms involved in the “endotoxic phenomenon” are not defined yet, although extensive studies have been carried out. Immunogold and electron microscopy (EM) have contributed to identify the primary target cells of endotoxins and the subcellular systems that receive the direct action of these bacterial agents. Here, we review our studies on immunogold detection of endotoxins in cellular and subcellular systems. The analysis of the interaction between endotoxins and cells was focussed on the following aspects: (1) morphological characteristics of the LPS aqueous suspensions used in experimental work; (2) binding of endotoxins to the plasma membrane of type II pneumocytes and alveolar macrophages (two of their cellular targets), and influence of the state of aggregation of the LPS; (3) movement and distribution of endotoxins inside the cell, from the plasma membrane to the nucleoplasm; and (4) interaction of LPS with microtubules and its effects on the integrity of the microtubular network. These approaches provide information at the molecular level as well as data for the establishment of physiological models of endotoxicity. © 1995 Wiley‐Liss, Inc.
Immunogold labeling of oncogenic and tumor related proteins
Authors: Rulong Shen , Zhou Renping , Tsarfaty Ilan , Hughes Steven , Woude George Vande , da Silva Pedro Pinto
Published: February 2005

Immunogold labeling electron microscopy technique has been used to study the ultrastructural localization of oncogenic proteins: Mos, Met, Ski, and the tumor‐associated protein, Muc1, as well as their relationship with other tumor‐related proteins. By pre‐ and postembedding immunogold labeling electron microscopy techniques, we showed that the Mos protein pp39mos colocalized with microtubule bundles, suggesting that microtubulin or microtubule‐associated protein(s) may be the substrate of Mos. Met protein was labeled at the microvilli of the lumen that are formed in cultured T47D cells, implying its potential involvement in lumen formation. Ski localization experiments revealed a unique globular structure “Ski body” that is present inside the nucleus of interphase chicken embryo fibroblast infected with Ski cDNA FB29 and FB2–29. Ski bodies were also found scattered in the cytoplasm of metaphase FB29 and FB2–29 Ski expressing chicken embryo fibroblasts. In T47D cells, tumor‐associated protein Muc1 was associated with both the plasma membrane and the membranes of secretory vesicles in the cytoplasm. In MUC1 infected NIH3T3 cells, however, labeling showed that in addition to the plasma membrane and the membranes of secretory vesicles, some Muc1 gold spheres were seen inside the secretory vesicles, suggesting that the subcellular localization of the protein may vary in different cell types. © 1995 Wiley‐Liss, Inc.
Origin and migratory pathways of the eleven epithelial cell types present in the body of the mouse stomach
Authors: Karam Sherif , Leblond Charles Philippe
Published: February 2005

The secretions of the mammalian stomach are produced by cells present in invaginations of the epithelium, which in the mouse are straight tubules referred to as “zymogenic units.” These units comprise four regions, namely pit, isthmus, neck, and base, in which there are several cell lineages with different phenotypes and migratory pathways. In the isthmus, stem cells designated “undifferentiated granule‐free cells” undergo division so as to maintain their own number and produce several differently oriented progenitors: (1) “Pre‐pit cell precursors” are characterized by prosecretory Golgi vesicles with a uniform, fine particulate content. They give rise to “pre‐pit cells” defined by the presence of few dense mucous granules. These cells migrate outward from the isthmus to the pit, where they become the dense granule‐rich “pit cells” which populate the pit region and migrate to the gastric surface where they are lost. (2) “Pre‐neck cell precursors” are identified by prosecretory Golgi vesicles containing an irregular dense center and a light rim. They give rise to “pre‐neck cells” defined by a few mucous secretory granules with a clear‐cut core. These cells migrate inward from the isthmus to the neck where they become “neck cells,” which contain many such granules. Even though neck cells are mature mucus‐producers, they are not end cells. As they enter the base region, they become “prezymogenic cells” whose phenotype gradually changes from mucous to serous. These cells eventually lose the ability to produce mucus and thus become the typical zymogenic cells that populate the base region. (3) “Pre‐parietal cells” are classified into three variants, which probably come from three different sources, that is, pre‐pit cell precursors, pre‐neck precursors, and the undifferentiated granule‐free cells themselves. The preparietal cells mature into parietal cells which migrate either outward to the pit or inward to the neck and base. As a result, parietal cells are scattered in the four regions of the unit. (4) Precursors of “entero‐endocrine” and “caveolated” cells give rise in the isthmus to these cells, which may also migrate outward or inward. © 1995 Wiley‐Liss, Inc.
Development and application of a dry ultramicrotomy technique for the preparation of galvanneal sheet coatings
Authors: Barreto Marie Paule , Veillette René , L'espérance Gilles
Published: February 2005

The formability of galvanneal steel sheets used in the automotive industry is influenced by the presence and distribution of brittle and difficult to distinguish Zn‐Fe intermetallics in the coating. Characterization of these intermetallics requires a high spatial resolution technique such as analytical transmission electron microscopy (ATEM). Sample preparation by ion milling is impossible due to iron redeposition, and traditional ultramicrotomy using water affects the coating chemistry. A technique based on dry ultramicrotomy has therefore been developed.

To optimize the technique, different parameters (knife angle, cutting medium, thickness setting on the ultramicrotome, cutting speed) have been investigated for the preparation of galvanneal coatings and pure Al sections. Results show that dry cutting does not affect the coating chemistry but shortens the life of the knife. Knife quality (cleanliness, sharpness and absence of defects) is a major factor to obtain good dry sections. The best results for the more ductile pure Al are obtained with a 35° knife whilst for the harder galvanneal coating it is recommended to use a 55° knife. These results suggest that the sectioning mechanism for the harder material involves more a cleavage‐fracture mechanism whilst a greater amount of shear is involved when sectioning relatively ductile Al. The optimum parameters for sectioning galvanneal coatings are established and results obtained by parallel electron energy loss spectrum imaging and energy dispersive X‐ray spectrometry in the TEM are given. This study shows that with a good control of all the sectioning parameters it is possible to obtain good sections repeatedly and rapidly. © 1995 Wiley‐Liss, Inc.
Ultramicrotomy of industrial alumina
Authors: Antonovsky A.
Published: February 2005

Industrial materials, such as alumina, often pose difficulties in their preparation for TEM examination. Composite and particulate materials are particularly difficult to prepare using conventional thinning techniques, i.e., ion beam and chemical jet thinning. Ultramicrotomy (UM) can be used to produce TEM specimens with a uniform thickness and an unaltered composition.

Some crystalline materials, i.e., alumina hydrate, were difficult to section due to conflict between the cutting direction and cleavage planes. Sectioning was successful when these two directions were mutually parallel or perpendicular. At other orientations shattering occurred. Microcrystalline particulate materials, i.e., calcined alumina, were sectioned successfully in particles < 70 μm in diameter.

The phases found in industrial alumina particles were gamma, delta, theta, and alpha alumina. Gamma alumina consisted of fine‐grained, equiaxed crystallites. The selected area electron diffraction (SAED) patterns indicated poor crystallinity with a distinct hexagonal texture. Delta and theta alumina appeared as an undifferentiated intermediary microstructure of elongated grains. The SAED patterns indicated poor crystallinity, but without a distinct texture. Alpha alumina was found to be a coarse‐grained crystalline phase with high diffraction contrast. SAED patterns consisted of fine, randomly oriented spots.

Considerable variation was observed in the distribution of phases. In some specimens, discrete particles of gamma and alpha predominated. In others, particles were a mixture of phases repre‐sentative of the bulk composition. To characterise these samples, TEM of numerous whole particles was required. Ultramicrotomy was the only preparation technique capable of producing such samples. © 1995 Wiley‐Liss, Inc.
Ultramicrotomy of diamond films for tem cross‐section analysis
Authors: Swab P.
Published: February 2005

Ultramicrotomy has been used to prepare TEM cross‐sections of typical hard dielectric, semiconductor, and metal coatings, providing a critical capability in the study of structure‐property relationships of thin films. Ultramicrotomy of thin film coatings requires meticulous attention to technique and handling. The sample to be microtomed must be very small, well bonded to the epoxy embedding medium, and precisely oriented. In this article we report the ability to microtome TEM cross‐sections of diamond and cubic boron‐nitride (cBN) coatings. © 1995 Wiley‐Liss, Inc.
Microscopy of the gap junction: A historical perspective
Authors: Severs Nicholas J.
Published: February 2005

Gap junctions were discovered more than three decades ago, and since this time, enormous strides have been made in understanding their structure and function. This article summarises the part played by microscopy, within the context of multidisciplinary research, in the historical development of our knowledge of the gap junction. © 1995 Wiley‐Liss, Inc.