bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme‐gold affinity labelling of cellulose
Authors: Berg R. Howard , Erdos Gregory W. , Gritzali Mikelina , Brown Ross D.
Published: February 2005

The enzyme‐linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15–30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme‐gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4‐β‐mannans or 1,3‐β‐xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4‐β‐glucans in chitin, by much lower labelling density when done at 4°C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I‐, CBH II‐, and EG‐linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's. The cellulase‐gold probe remained active for at least 4 weeks at 4°C and much longer when frozen at −80°C in 20% glycerol. This technique should prove useful in studies of cellulose degradation and cellulose deposition and of the interaction of cellulose with other wall components.
Relationship between structure and function in distal tubule and collecting duct
Authors: Madsen Kirsten M. , Verlander Jill W. , Tisher C. Craig
Published: February 2005

The relationship between structure and function in the distal tubule and collecting duct has been studied with morphologic and physiologic techniques, including morphometric analysis, to identify functionally distinct cell populations. The distal tubule, including the thick ascending limb (TAL) and the distal convoluted tubule (DCT), is involved in active reabsorption of sodium chloride. It is characterized by extensive invaginations of the basolateral plasma membrane, numerous mitochondria, and high Na‐K‐ATPase activity, features characteristic for an epithelium involved in active transport. Between the distal tubule and the collecting duct is a transition region, the connecting segment or the connecting tubule (CNT), which exhibits species differences with respect to both structure and function. The collecting duct includes the cortical (CCD), the outer medullary (OMCD), and the inner medullary (IMCD) collecting ducts. Principal cells are present throughout the collecting duct, whereas intercalated cells are located mainly in the CCD and OMCD. Morphometric analysis combined with micropuncture and microperfusion studies has provided evidence that the CNT and principal cells are responsible for potassium secretion in the connecting segment and the CCD. The OMCD is a main site of hydrogen ion secretion, and morphometric studies have provided evidence that the intercalated cells in this segment secrete hydrogen ion at least in the rat. Two configurations of intercalated cells exist in the CCD—a type A and a type B. The A cells are similar in ultrastructure to the intercalated cells in the OMCD and are believed to be involved in hydrogen ion secretion. The function of the B cells remains to be established. The inner two‐thirds of the IMCD corresponds to the papillary collecting duct, which has a high permeability to urea. The relationship between structure and function in the IMCD has not been studied in detail. This review emphasizes the role of morphometric analysis in establishing the relationship between structure and function in the distal nephron.
Three‐dimensional reconstruction of cells from serial sections and whole‐cell mounts using multilevel contouring of stereo micrographs
Authors: Marko Michael , Leith Ardean , Parsons Donald
Published: February 2005

A comprehensive computer‐graphics‐based system (STERECON) is described for tracing and digitizing contours from individual or stereopair electron micrographs. The contours are drawn in parallel planes within the micrographs. Provision is also made for tracing and digitizing in full three‐dimensional (3‐D) coordinates in any direction along linear structures such as cytoskeletal elements. The stereopair micrographs are viewed in combination with the contours being traced on a graphics terminal monitor. This is done either by projecting original electron micrograph (EM) negatives onto a screen and optically combining these images with contour lines being drawn on the monitor, or by first digitizing the images and displaying them directly on the monitor along with the contour lines. Prior image digitization allows computer enhancement of the structures to be contoured. Correction and alignment routines are included to deal with variable section thickness, section distortion and mass loss, variations in photography in the electron microscope, and terminal screen curvature when combining projected images with contour lines on the monitor. The STERECON system organizes and displays the digitized data from successive sections as a 3‐D reconstruction. Reconstructions can be viewed in any orientation as contour stacks with hidden lines removed; as wire‐frame models; or as shaded, solid models with variable lighting, transparency, and reflectivity. Volumes and surface areas of the reconstructed objects can be determined. Particular attention was paid to making the system convenient for the biological user. Users are given a choice of three different stereo‐viewing methods.
Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth
Authors: Baba Norio , Baba Misuzu , Imamura Masao , Koga Masatada , Ohsumi Yoshinori , Osumi Masako , Kanaya Koichi
Published: February 2005

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).
Light, scanning, and transmission electron microscopy of a single population of detergent‐extracted cardiac myocytes in vitro
Authors: Delcarpio Joseph B. , Underwood Elizabeth G. , Moses R. L.
Published: February 2005

A technique for performing light, scanning, and transverse transmission electron microscopy on cultured cells grown within a single tissue culture flask is described. Permanent light microscopy slides are obtained by removing selected portions of the plastic tissue culture vessel and mounting them on glass slides with an aqueous mounting solution. The images obtained from these slides are superior to viewing through the bottom of the flask with an inverted stage microscope. For scanning electron microscopy, selected areas are also cut from the remainder of the vessel and prepared for viewing. The final portion of the culture container is transferred and attached to a new tissue culture vessel and prepared for transmission electron microscopy using alcohol instead of acetone and propylene oxide during dehydration, infiltration, and embedding.
Detection of small displacement of atoms in crystals by atom resolution electron microscopy
Authors: Hashimoto Hatsujiro , Kuwabara Masago , Takai Yoshizo , Tsubokawa Sumiyuki , Yokota Yasuhiro
Published: February 2005

Four kinds of works on the detection of displacement of atoms in crystals are shown. The irregular small displacement of atoms has been detected, with an accuracy of about 0.1 Å around dislocations and stacking faults in Au crystals as shown by their electron microscope images. The displacement of the atomic images is recorded by aberration‐free focus (AFF). Even when the periodic displacement of atoms in SiC and TaS2 crystals is around 0.1% of the lattice constant, this displacement has been revealed as the weak‐contrast anomaly in the images. Using in situ observations by a TV system attached to an electron microscope, the rapid movements of atoms that have taken place within 1/30 sec have been recorded. Using the technique of successive subtraction of the images by TV system and image sigma, only the images of moving atoms in Au crystal have been recorded each 1/10 sec.
Quick‐freeze, deep‐etch, rotary‐shadow views of the extracellular matrix and cortical cytoskeleton of Xenopus laevis eggs
Authors: Larabell Carolyn A. , Chandler Douglas E.
Published: February 2005

The quick‐freeze, deep‐etch, rotary‐shadow technique provides a powerful tool to study the structural dynamics of extracellular matrices. Using this technique, we show that the extracellular investments of the Xenopus laevis egg are multilayered and securely anchored to the egg surface. The cortical cytoskeleton within the egg contains embedded cortical granules with surrounding endoplasmic reticulum and is capped by a thin reticular sheet that contacts the inner surface of the plasma membrane. The extracellular matrix undergoes three distinct changes at fertilization: (a) formation of a “smooth” layer below the vitelline envelope (VE), (b) transformation of the VE itself to an altered VE composed of concentric fibrous sheets, and (c) formation of a dense, “briar‐patch”‐like fertilization layer at the upper surface of the VE.
Advantages of the use of intact plant tissues in freeze‐fracture electron microscopy
Authors: Platt‐Aloia K. A. , Thomson W. W.
Published: February 2005

The use of whole, intact plant tissue for freeze‐fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artifical (phospholipid) membrane preparations. This is not to imply that these exminations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three‐dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution.

It is our intention that this survey of freeze‐fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.
Ultrathin (1 nm) vertically shadowed platinum‐carbon replicas for imaging individual molecules in freeze‐etched biological DNA and material science metal and plastic specimens
Authors: Ruben George C.
Published: February 2005

Single molecule resolution in beam‐sensitive, uncoated, noncrystalline materials has heretofore not been possible except in thin (≤150 Å) platinum‐carbon (Pt‐C) replicas, which are resistant to electron beam destruction. Previously, the granularity of metal film replicas limited their resolution to ≥20 Å. This paper demonstrates that Pt‐C film granularity and resolution are a function of the method of replication and other controllable factors. Low‐angle 20° rotary, 45° unidirectional, and vertical 9.7 ± 1 Å Pt‐C films deposited on mica under the same conditions were compared. Vertical replication had a 5 Å granularity, the highest resolution, and evenly coated the whole surface. A 45° replication had a 9.5 Å granularity, a slightly poorer resolution, and a discontinuous surface coating. The use of 20° rotary replication proved to be unsuitable for high‐resolution imaging, with 20–25 Å granularity and resolution two to three times poorer. Vertical and 45° Pt‐C replicas can visualize the deep‐etched DNA helix and the 13.3 Å 32 helix of pectin in a gel. The DNA double helix, the complex structures of sol‐gel glasses, Immobilon filters (polyvinylidene fluoride), a polymethacrylate plastic, the metal oxide surfaces of 440c stainless steel, and aluminum are illustrated. This high‐resolution vertical Pt‐C replica technique can image in the context of solutions, gels, or solids, single molecular chains 3–7 Å wide, their associations, and their conformation. Included in the present article are first time descriptions for removing replicas from metals and plastics and for making high‐magnification photographic prints of normal contrast using a reversal rephotographic process.
Hepatocyte fine structure during maturation and senescence
Authors: Schmucker Douglas L.
Published: February 2005

Aging is accompanied by a myriad of changes in cell structure, function, and composition. The fact that much of the information concerning age‐related alterations in cellular morphology is qualitative precludes meaningful correlations with biochemical changes in order to enhance data interpretation. The mammalian liver has been subjected to both qualitative and quantitative evaluations of hepatocyte structure as a function of aging, i.e., development, maturation, and senescence. Although these data are characterized by considerable variability and, in some instances, blatant contradictions, there exists sufficient agreement in several parameters to permit a consensus in the inbred rat model. Certainly the volume of individual hepatocytes increases with age, and many of the organelle compartments reflect this change. While old rats exhibit a high incidence of polyploidy, there is no definitive evidence to demonstrate a concomitant increase in the binuclear hepatocyte index. Several specific hepatocellular organelles undergo changes in their relative volume or surface area that appear to correlate with functional alterations. The volume density of the lysosomal compartment enlarges significantly during senescence and is accompanied by increased activities of several constituent hydrolases. The hepatic concentration of smooth‐surfaced endoplasmic reticulum declines markedly with aging, as does the yield of liver microsomes and the activities of several microsomal enzymes, e.g., mono‐oxygenases and glucose‐6‐phosphatase. However, the responses of the majority of hepatocyte organelles to aging is varied and inconsistent based on the limited data currently available.
Immunocytochemistry of neurotransmitter receptors
Authors: Wenthold R. J. , Altschuler R. A. , Hampson D. R.
Published: February 2005

Over the last several years our knowledge of neurotransmitter receptors has increased dramatically as receptor types and subtypes have been identified through the development of selective antagonists, neuropharmacological studies, and radioactive ligand binding studies. At the same time major advances were made in the immunocytochemical localization of neurotransmitters and their related enzymes. However, only recently has immunocytochemistry been used to localize neurotransmitter receptors, and these studies have been limited. Four receptors have been localized in the CNS with immunocytochemistry: the nicotinic acetylcholine receptor, the beta‐adrenergic receptor, the GABA/benzodiazepine receptor, and the glycine receptor. Of these the glycine receptor has been the most thoroughly characterized. Glycine receptor immunoreactivity is highly concentrated at postsynaptic sites, and the distribution of immunoreactivity appears to correlate closely with glycinergic neurons. However, immunocytochemical studies done on other receptors suggest such a distribution may not always be the case. Some receptors may not be concentrated at postsynaptic sites, and receptor distribution may not always closely fit the distribution of the respective neurotransmitter. Work is rapidly progressing on the purification of other receptors and on the production of selective antibodies which will allow immunocytochemical studies which address these and other questions.
Ultrastructure of proteoglycans in the tectorial membrane
Authors: Santi Peter A. , Lease M. Kathryn , Harrison Robert G. , Wicker Eileen M.
Published: February 2005

The ultrastructure of proteoglycans (PGs) in the tectorial membrane (TM) of the mature chinchilla cochlea was investigated using the cationic dye Cuprolinic blue. When used at a high critical electrolyte concentration, Cuprolinic blue has been shown specifically to bind to the glycosaminoglycan residues of sulfated PGs. After Cuprolinic blue treatment, PGs were observed in the TM which were represented as rod‐shaped, electron‐dense structures. A perifibrillar, primarily orthogonal, array of PGs was associated with the type A protofibrils. These PGs were distributed in 50 nm intervals along the length of the type A protofibrils. A less common orientation was parallel to the axis of the type A protofibrils. PGs did not appear to be associated with the type B protofibrils. Based upon previous results by other investigators, the TM contains types II and IX collagen, and it appears likely that the type A protofibrils are composed of collagen type II. PGs visualized in the TM in this study thus may represent the glycosaminoglycan residue of type IX collagen which is associated with the type II collagen fibrils. Alternatively, the TM PGs may be small dermatan or chondroitin sulfate PGs.
A replica technique for extracting precipitates from zirconium alloys for transmission electron microscopy analysis
Authors: Ng‐Yelim J. , Woo O. T. , Carpenter G. J. C.
Published: February 2005

A reliable two‐stage carbon replica technique has been developed to extract precipitates from zirconium alloys. Using this technique, all precipitating phases can be extracted from Zircaloy‐2, Zr‐Cr‐Fe, and Zr‐Nb‐Fe alloys. Precipitate identification using EDS X‐ray analysis and convergent beam electron diffraction was greatly facilitated in comparison to thin foils. In addition, the sensitivity for the detection of trace elements in particles was increased using extraction replicas. The chemical compositions of the precipitates as determined from both replica and thin foils were in excellent agreement.
Lowicryl embedding of gastrointestinal tissues
Authors: Hagen Susan J.
Published: February 2005

In the present study, we modified the technique described by Altman et al. (J. Histochem. Cytochem., 32:1217–1223, 1984) for rapid embedding of tissues in Lowicryl K4M. To attain good sections of the small intestine that contained villi, crypts, submucosa, and external muscle layers, we cut 100 μm slices of the full thickness of the wall with a vibratome before embedment and then deoxygenated the resin and tissue before polymerization. The sections we obtained compared favorably with the quality of sections from conventional resins.
A simple pneumatic device for plunge‐freezing cells grown on electron microscopy grids
Authors: Cole Richard , Matuszek George , See Charles , Rieder Conly L.
Published: February 2005

A detailed design for a simple and inexpensive variable‐speed (1.0–5.8 m s−1) pneumatic plunge‐freezing device is presented. Cultured cells, grown on Formvar‐coated 75‐mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN2). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN2 creates an atmosphere below −120°C in which the transfer can be accomplished.
Traffic through the golgi apparatus as studied by radioautography
Authors: Bennett G. , Wild G.
Published: February 2005

The ability to radiolabel biological molecules, in conjunction with radioautographic or cell fractionation techniques, has brought about a revolution in our knowledge of dynamic cellular processes. This has been particularly true since the 1940's, when isotopes such as 35S and 14C became available, since these isotopes could be incorporated into a great variety of biologically important compounds. The first dynamic evidence for Golgi apparatus involvement in biosynthesis came from light microscope radioautographic studies by Jennings and Florey in the 1950's, in which label was localized to the supranuclear Golgi region of goblet cells soon after injection of 35S‐sulfate. When the low energy isotope tritium became available, and when radioautography could be extended to the electron microscope level, a great improvement in spatial resolution was achieved. Studies using 3H‐amino acids revealed that proteins were synthesized in the rough endoplasmic reticulum, migrated to the Golgi apparatus, and thence to secretion granules, lysosomes, or the plasma membrane. The work of Neutra and Leblond in the 1960's using 3H‐glucose provided dramatic evidence that the Golgi apparatus was involved in glycosylation. Work with 3H‐mannose (a core sugar in N‐linked side chains), showed that this sugar was incorporated into glycoproteins in the rough endoplasmic reticulum, providing the first radioautographic evidence that glycosylation of proteins did not occur solely in the Golgi apparatus. Studies with the tritiated precursors of fucose, galactose, and sialic acid, on the other hand, showed that these terminal sugars are mainly added in the Golgi apparatus. With its limited spatial resolution, radioautography cannot discriminate between label in adjacent Golgi saccules. Nonetheless, in some cell types, radioautographic evidence (along with cytochemical and cell fractionation data) has indicated that the Golgi is subcompartmentalized in terms of glycosylation, with galactose and sialic acid being added to glycoproteins only within the trans‐Golgi compartment. In the last ten years, radioautographic tracing of radioiodinated plasma membrane molecules has indicated a substantial recycling of such molecules to the Golgi apparatus.
Scale formation in algae
Authors: Melkonian Michael , Becker Burkhard , Becker Dieter
Published: February 2005

Scale biogenesis in algae represents a unique model system to study the transport of secretory macromolecules through the Golgi apparatus (GA) and their exocytosis. The larger scales can be visualized in the light microscope, and thus the kinetics of scale assembly, transport, and secretion can be studied in vivo. In addition, scales are osmiophilic and readily visible in conventional transmission electron microscopy; thus, details of scale assembly and sorting can be studied without invoking immunolabeling techniques. The following are distinctive features of scale biogenesis in algae: (1) transport of scales through the GA‐stack occurs by cisternal progression; (2) scale secretion may be very rapid (in some cases a single GA‐cisterna leaves the stack every 15–20 s); (3) sorting of different scale types does not occur in the GA, but in a post‐GA‐compartment. Recent progress in the analysis of scale formation in the green flagellates Tetraselmis and Scherffelia is reviewed.
Role of the golgi apparatus in cellular pathology
Authors: Morré Dorothy M.
Published: February 2005

The Golgi apparatus response to pathological disorders is predominantly as an intermediary component of membrane biogenesis where it is involved in processing, sorting and secretion of materials via secretory granules, and in the formation of lysosomes. A common initial response of the Golgi apparatus to any stress is an alteration or cessation of secretory activity. In the transformed cell, the Golgi apparatus is altered both morphologically and biochemically, suggesting a shift from a secretory to a membrane‐generating mode of functioning. However, since fewer or less well‐developed Golgi apparatus are frequently found in transformed cells, analytical methods of membrane isolation developed for normal tissues may not always yield equivalent results when applied to tumors. Cell surface alterations characteristic of malignant cells may result from modifications occurring at the level of the Golgi apparatus. Some lysosomal dysfunctions may result from underglycosylation of acid hydrolases by the Golgi apparatus. The use of cell‐free systems between endoplasmic reticulum and Golgi apparatus or within Golgi apparatus cisterane is providing a new approach to the elucidation of the role of the Golgi apparatus in normal as well as pathological states.
Polarity of the surface and cortex of the amphibian egg from fertilization to first cleavage
Authors: Stewart‐Savage J. , Grey Robert D. , Elinson Richard P.
Published: February 2005

The anuran egg is polarized along its animal–vegetal axis and becomes bilaterally symmetrical before first cleavage. Functional sperm entry is regionally restricted to the animal hemisphere of the egg, and functional sperm entry does not occur after egg activation. This regional and functional restriction in sperm entry correlates with the presence of long, slender microvilli and with the presence of the filamentous component of the glycocalyx. After sperm fusion, the egg undergoes activation, including a depolarization of the membrane potential and exocytosis of granules in the cortex. Both of these activation responses are the result of a propagated increase in intracellular calcium. The egg's ability to undergo a propagated activation response develops after germinal vesicle breakdown and depends on the development of the cortical endoplasmic reticulum. Once activated, the radial symmetric egg acquires bilateral symmetry due to a rotation of the egg cortex relative to the inner cytoplasm. A transient array of parallel microtubules forms near the vegetal cortex and may be part of the motor driving the cortical rotation.
Ultrastructural immunocytochemistry of the adenohypophysis in the rat: A review
Authors: Kurosumi Kazumasa
Published: February 2005

Immunocytochemistry has made great strides in the morphology of endocrine glands, especially the adenohypophysis, because the localization of hormones can be clearly demonstrated by this method in the microscopic preparations both for light and electron microscopy. In the adenohypophysis, electron microscopic immunocytochemistry is useful for identifying the producer cell of each hormone. The second contribution is its application to the cell biology of secretion mechanisms. The pituitary hormones, their precursors, derivatives, and fragments were artificially synthesized and their antibodies were produced. Using these antibodies the intracellular sites of synthesis, condensation, processing, and sorting were studied under the electron microscope. The ultrastructure of each cell organelle and its alteration due to the changing function was studied. It was proved that the intracisternal granules in the thyroidectomy cells contain thyroid‐stimulating hormone (TSH). The trans‐Golgi network or GERL contains a peculiar supporting structure, intracisternal skeleton. Transport of secretory granules may be performed in relation to the microtubules, actin, and some related substances. The most frequently observed mode of hormone release in the adenohypophysis is exocytosis. Sometimes multigranular exocytosis occurs. Vesiculation of membrane around the secretory granules often occur inward or outward. The inward vesiculation forms pinocytotic vesicles, through which the membrane material may be retrieved. The outward vesiculation forms vesicle‐like fragments of cytoplasm being discarded to the extracellular space. By these mechanisms the surface area of the cell is maintained constantly.
Ultrastructure of spontaneous and transplanted pituitary tumors in laboratory animals
Authors: Lloyd Ricardo V.
Published: February 2005

The present review examines ultrastructural studies of pituitary tumors in laboratory animals. Such studies have been facilitated by the development of newer techniques such as immunocytochemistry and in situ hybridization. Using these methods it has been possible to correlate morphological features with function by characterizing specific pituitary hormones in secretory granules and analyzing the messenger ribonucleic acids produced by specific cell types. Analyses of spontaneous and experimentally induced tumors have provided a great deal of insight into the function and ultrastructure of these neoplasms. The description of tumor cells producing more than one hormone has been facilitated by immunocytochemical analyses. Studies of transgenic models of pituitary hyperplasia and tumor development are expanding our knowledge about the ultrastructure and about other aspects of pituitary tumors in a more controlled experimental setting. The production of various hormones by the same pituitary tumors and even the same tumor cells indicates that complex regulatory mechanisms must be analyzed by a combination of ultrastructural and other techniques to learn more about growth and hormone secretion in pituitary neoplasms.
Design and operation of a simple environmental chamber for rapid freezing fixation
Authors: Bailey S. M. , Chiruvolu S. , Longo M. L. , Zasadzinski J. A. N.
Published: February 2005

Rapid freezing is the most important step in sample preparation for freeze‐fracture and other cryotechniques for electron microscopy. We present the design and operation of a simple environmental chamber coupled to a plunger‐driven freezing device that has provided simple and reliable freezing from temperatures and humidities other than ambient. The chamber can be constructed and operated with equipment and techniques common to most electron microscopy labs. Temperature control of ±0.1°C and relative humidities of >90% were provided over the range −5–60°C. Typical electron micrographs showing well preserved structures comparable to jet‐freezing are presented.
Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane
Authors: Dekker Nusi P. , Lammel Claudia J. , Brooks Geo. F.
Published: February 2005

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.
Effects of vitamin A deficiency on the inter—Sertoli cell tight junctions and on the germ cell population
Authors: Ismail Nermine , Morales Carlos R.
Published: February 2005

When 20‐day‐old rats are placed on a vitamin A deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells, a few residual A0, A1 spermatogonia, and preleptotene spermatocytes (PL). The type A1 spermatogonia and PL spermatocytes are arrested in their G2 phase. In VAD rats type A2‐A4, intermediate (In) and B spermatogonia and all types of spermatocytes (except PL spermatocytes) and spermatids are eliminated from the seminiferous tubules. Two questions were raised in this investigation: (1) Is there, in VAD rats, any correlation between a breakdown of the blood‐testis barrier (e.g., Sertoli cell tight junctions) and germ cell loss? (2) Is the disappearance of most germinal cells due to their degeneration during spermatogenesis or to a maturation depletion process resulting from an arrest of spermatogenesis at the spermatogonial stage? To investigate these questions four groups of male Sprague‐Dawley rats (20‐days old) were fed a VAD diet for 7 to 12 weeks. The testes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate containing 2% lanthanum nitrate, an electron opaque tracer used to test the patency of Sertoli cell tight junctions. The lanthanum permeated the intercellular space of the basal compartment but was arrested by normal inter—Sertoli cell tight junctions. The seminiferous epithelium showed numerous degenerating germ cells, some being internalized by Sertoli cells as membrane‐bound phagosomes. Thus, these results indicate firstly that inter—Sertoli cell tight junctions remain intact during vitamin A deficiency, and secondly that in a first phase nonviable germinal cells degenerate during spermatogenesis and their residues are actively phagocytosed by Sertoli cells followed by a second phase where the regressed state of the seminiferous epithelium is maintained by a maturation depletion condition resulting from an arrest of spermatogonial proliferation and differentation.
Intermediate filaments in sertoli cells
Authors: Aumüller G. , Schulze C. , Viebahn C.
Published: February 2005

Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre‐ and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone‐dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone‐dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycledependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cell‐supporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad.
Ultrastructure of the neurohypophysis
Authors: Scheithauer B. W. , Horvath E. , Kovacs K.
Published: February 2005

This review summarizes our current knowledge of the ultrastructure of the human neurohypophysis and includes comments on its anatomy, physiology, and embryology. The neurohypophysis represents a unique tissue having neural and endocrine characteristics and possessing ultrastructural features distinct from those of conventional endocrine organs such as the anterior pituitary, thyroid, pancreatic islets, etc. In contrast to these glands, the neurohypophysis is composed of the processes of mature neurons. As such, it is not capable of synthesizing hormones but only of their storage and release. Neurosecretion is one of the most exciting areas of neuroendocrinology and, although spectacular progress has been achieved in elucidating the process, a number of aspects are incompletely understood. Recent evidence indicates that the magnocellular nuclei of the hypothalemus, the anatomic origin and functional basis of the neurohypophysis, produce not only vasopressin and oxytocin, the so‐called “neurohypophyseal hormones,” but a number of other biologically active peptides as well. The physiologic function of these substances is largely unknown but they may be of profound importance in endocrine homeostasis. Based on these novel findings, the role of the neurohypophysis in endocrine regulation has to be re‐evaluated.
Species‐specific localization of actin in mammalian spermatozoa: Factor artifact?
Authors: Fouquet Jean‐Pierre , Kann Marie‐Louise
Published: February 2005

Actin has been characterized and localized in sperm cells of many mammals. Nevertheless, the reported localizations obtained by different methods and/or antibodies varied from species to species and even for the same species. To clarify the question, sperm actin distribution was reinvestigated under uniform technical conditions. Immunogold post‐embedding procedures were performed using a polyclonal and two monoclonal antibodies of known specificity to localize actin in spermatids and spermatozoa of rabbit, mouse, rat, monkey, and human. In these species, actin (F‐actin) was detected with the three antibodies between the nucleus and the acrosome of round and elongating spermatids. Species‐specific changes occurred in maturing spermatids. In the rabbit, actin labeling decreased and disappeared from the tip to the base of the subacrosomal layer. In testicular and epididymal spermatozoa actin was detected only with a monoclonal antibody (Amersham) successively in the neck, postacrosomal area, and subacrosomal bulges. In mouse late spermatids a transitory labeling of the neck was detected only with the polyclonal antiactin. In testicular and epididymal spermatozoa an actin labeling was observed in the principal piece of the tail. In rat, monkey, and human sperm cells actin remained undetected. These results suggest that there is a redistribution of actin in late spermatids and spermatozoa which is a species‐specific process but not an artifact of methodological origin. Thus, a function for actin in sperm, if any, remains to be demonstrated.
Degradation, amorphization, and recrystallization of ion bombarded si(111) surfaces studied by in situ reflection electron microscopy and reflection high energy electron diffraction techniques
Authors: Claverie A. , Beauvillain J. , Fauré J. , Vieu C. , Jouffrey B.
Published: February 2005

In this paper we report the effect of noble gas ions bombardment on the degradation of atomically flat Si(111) surfaces at room and high (400°C—600°C) temperatures. Reflection high energy electron diffraction (RHEED) and reflection electron microscopy (REM) have been used to characterize the topography and structure of the as‐implanted and post annealed surface layers. It is shown that the fading of the specularly reflected beam is not directly related to the amorphization of the surface. This experimental study has also evidenced the difficulties one meets to regrow a defect‐free material after amorphization by noble gas bombardment. For high temperature for which the amorphization is not possible, the surface loses its stepped structure and turns into a monocrystalline but atomically rough surface. This roughness is a function of substrate temperature.
Ab‐initio primitive cell parameters from single convergent‐beam electron diffraction patterns: A converse route to the identification of microcrystals with electrons
Authors: Page Y. Le
Published: February 2005

A new method for the ab initio derivation of Buerger‐reduced primitive cell parameters from coordinate measurements of spots on single convergent‐beam electron diffraction (CBED) patterns is described, which does not involve trial‐and‐error. The pattern can be taken along any zone axis, and misorientations of the crystallite by as much as a few degrees are taken into account without loss of accuracy. This derivation of cell parameters by least‐squares analysis of the measurements has been automated in a program called NRCBED. Present accuracy is about 1% on lengths and 2° on angles, but could be significantly improved by modelling projector lens aberrations, or by using a microscope without a projector lens. With present technology, it is possible to obtain a CBED pattern and a semi‐quantitative energy‐dispersive X‐ray (EDX) analysis simultaneously from a single microcrystal a few hundred Ångströms across. It becomes therefore possible to identify the material of the crystal on a single CBED pattern: a cell parameter database for known compounds is searched with the primitive cell parameters obtained in the above way, and with a mask describing the EDX results qualitatively. Feasibility is demonstrated on a crystallite of CeO2 500 Ångströms across. With this new approach, trial‐and‐error should disappear from the solution of other long‐standing problems: interpretation of X‐ray powder patterns for new compounds in the presence of impurity lines, or in the case of multiple phases should become straightforward.
Background correction of intensifier camera images in the T.E.M.
Authors: Beauregard Paul
Published: February 2005

Several disadvantages of using intensified television cameras to acquire TEM images can be overcome by using background subtraction with a frame store.
Fine structure of the pinealopetal innervation of the mammalian pineal gland
Authors: Møller Morten
Published: February 2005

The mammalian pineal gland is innervated by peripheral sympathetic and parasympathetic nerve fibers as well as by nerve fibers originating in the central nervous system (central innervation). The perikarya of the sympathetic fibers are located in the superior cervical ganglia, while the fibers terminate in boutons containing small granular vesicles and a few large granular vesicles. Both noradrenaline and neuropeptide Y are contained in these neurons. The parasympathetic fibers originate from perikarya in the pterygopalatine ganglia. The neuropeptides, vasoactive intestinal peptide and peptide histidine isoleucine, are present in these fibers, the boutons of which contain small clear transmitter vesicles and larger granular vesicles. The fibers of the central innervation originate predominantly from perikarya located in hypothalamic and limbic forebrain structures as well as from perikarya in the optic system. These fibers terminate in boutons containing small clear and, in certain fibers, an abundant number of large granular vesicles. In rodents, the majority of the central fibers terminate in the deep pineal gland and the pineal stalk. From these areas impulses might be transmitted further caudally to the superficial pineal gland via neuronal structures or processes from pinealocytes. Several hypothalamic neuropeptides and monoamines might be contained in the central fibers.

The intrapineal nerve fibers are located both in the perivascular spaces and intraparenchymally. The majority of the intraparenchymally located fibers terminate freely between the pinealocytes. However, some nerve terminals make synaptic contacts with the pinealocytes and in some species with intrapineal neurons.

In fetal mammals, sympathetic, parasympathetic, and central fibers are also present. In addition, an unpaired nerve, connecting the caudal part of the pineal gland with the extreme rostral part of the mesencephalon, is present. This nerve is a homologue to the pineal nerve (nervus pinealis) observed in lower vertebrates. © 1992 Wiley‐Liss, Inc.
Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous‐liquid, and vapor fixation techniques
Authors: Grote Monika
Published: February 2005

In order to find a good compromise between preservation of ultrastructural morphology and retention of antigenicity, birch pollen grains were chemically fixed in aqueous p‐formaldehyde or glutaraldehyde, in p‐formaldehyde or glutaraldehyde dissolved in anhydrous glycerol, and in p‐formaldehyde or glutaraldehyde vapor. Representative cytoplasmic areas were inspected for the preservation of ultrastructural morphology and for their capacity to bind a monoclonal antibody against Bet v I, the major birch pollen allergen. The experiments showed that cytoplasmic morphology was best preserved after vapor fixation in p‐formaldehyde. This fixation also led to the highest degree of specific antibody binding. © 1992 Wiley‐Liss, Inc.
Comparison of nuclear hydration in boar spermatozoa before and after freeze‐thawing: Contrast analysis of cells embedded in bromide‐labeled medium
Authors: Courtens J. L. , Ekwall H. , Plöen L.
Published: February 2005

Epon labeled with bromide was used to embed ejaculated and freeze‐thawed spermatozoa, with the hypothesis that it replaces most of cell water. Image analysis of relative contrasts between sperm nuclei and the surrounding medium revealed that when used in low concentrations, bromide is mostly absorbed to the nuclear structures. For higher concentrations, the chromatin is saturated, and the increase in contrast can be used to calculate relative differences in the hydration of nuclei. Boar sperm nuclei are more hydrated after freeze‐thawing than before. © 1992 Wiley‐Liss, Inc.
An image processing/stereological analysis system for transmission electron microscopy
Authors: Poole Max C.
Published: February 2005

This study examines the feasibility of combining computer image digitization, image enhancement, and point counting stereological techniques to quantify video images from transmission electron microscopes (TEM). The essential hardware consists of an IBM PC/AT, a Matrox imaging board, a digitizing tablet, a high resolution black and white monitor, and a portable mass storage device. In addition a video camera must be mounted to the TEM. The software is written in three modules which have numerous routines for image acquisition, enhancement, and quantification. Quantification is achieved by selecting an electronic lattice and superimposing it on the cell image. A cursor is moved on the lattice (via the digitizing tablet) and the points are entered into a spreadsheet. One of the major limitations of the system was the reduced resolution inherent in the current hardware. However, sampling experiments showed that one could compensate for the reduced resolution by increasing the magnification of the digitized images, and the stereological values from digitized images compared favorably to those from electron micrographs. Furthermore, the system proved advantageous by eliminating the usual darkroom work, and in enhancing low contrast tissue. In spite of several hardware limitations, the concept of quantifying computer digitized TEM images appears promising. © 1992 Wiley‐Liss, Inc.