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PubMed1969

The "9+1" pattern of microtubules in spermatozoa of Mesostoma (Platyhelminthes, Turbellaria).

The living spermatozoa of the flatworm Mesostoma georgianum have a sperm body about 100 mu long and 0.5 mu wide, and two motile free flagella, ca. 200 mu long. In sections examined with the electron microscope, these flagella have the usual nine pairs of peripheral doublet microtubules and have a single central core unit which is connected to the A members of the doublets by spokelike structures. There are also short connections between the doublets and the flagellar membrane. In material negatively stained with phosphotungstic acid, the doublet microtubules seem to have very different elastic properties than the core; they tend to fall on the copper grids in coils of rather uniform diameter (2-4 mu), while the core is much more rigid and is often found extending alone, along a relatively straight course, for very long distances (up to 73 mu). After negative staining, the core has a striking appearance with a dense center around which are wound two hollow structures in a double helix of 45 degrees pitch. The center-to-center distance of each gyre is approximately 650 A, and the hollow structures are ca. 180 A in diameter.

Animals↗
PubMed1973

Structure of fibers of sickle cell hemoglobin.

Electron microscope studies have been conducted on individual fibers of human deoxyhemoglobin S (sickle cell hemoglobin). The fibers are obtained by injection of gelled samples into a large excess of glutaraldehyde, which quickly stabilizes the fibers by cross-linking. The fibers are negatively stained with phosphotungstic acid or shadowed with platinum-carbon. The fibers are approximately 200 A in diameter, and display long and short helical striations with an opposite handedness. The long striations occur at an angle of about 15 degrees from the fiber axis, and complete one turn around the helix at a distance of about 2 x 10(3) A along the fiber axis. The short striations occur at an angle of about 80 degrees from the fiber axis, with a spacing of about 65 A, and complete one turn around the helix at a distance along the fiber axis of about 130 A. The structure of the fiber appears to be a sextuple helix in terms of the long striations, and a double helix in terms of the short striations. The shadowed samples are consistent with a left-handed screw sense for the short striations, thus implying a right-handed sense for the long striations. A structural model incorporating these features is compatible with the atomic structure of hemoglobin, with individual molecules oriented with their dyad axis of symmetry perpendicular to the fiber axis and their alpha(1)-beta(1) pseudo-dyad axis roughly parallel to the fiber axis. This orientation places the two beta-6 regions of each molecule (sites of the sickle cell mutation) in contact with the beta-6 regions of the molecules above and below along the long striations. Both the long and short striations are accounted for by individual hemoglobin molecules arranged in double helical arrays with 6.4 molecules per turn in each array.

Hemoglobin, Sickle↗
PubMed1973

Immunoferritin localization of intracellular antigens: the use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining.

A general method for the ultrastructural localization of intracellular proteins and antigens by immunoferritin techniques has been developed. The method involves direct staining of ultrathin sections of mildly glutaraldehyde-fixed and frozen tissues cut by means of a cryo-ultramicrotome. Bovine pancreatic sections were cut, mounted on grids, and stained with ferritin-rabbit antibovine RNase conjugates. After negative staining with 0.2% phosphotungstic acid, electron micrographs revealed specific labeling of all of the zymogen granules and the cisternae of the rough endoplasmic reticulum. No significant labeling was seen in the nucleus, mitochondria, or cell sap regions. The observation that no significant labeling was found in any region of rat pancreatic sections was consistent with the fact that rat RNase is immunologically non-crossreactive with bovine RNase. In addition, the labeling seen in bovine pancreas was completely absent if the sections were first incubated with free antibody. The method used here avoids prolonged fixation, dehydration, and other harsh chemical or physical treatments, and should extend the usefulness of immunoferritin techniques to the intracellular localization of many protein antigens beyond previously available methods.

Animals↗
PubMed1975

Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

The kinetochores of isolated HeLa cell chromosomes attached to an electron microscope specimen grid, fixed in formaldehyde, and stained with alcoholic phosphotungstic acid are visible as dark, preferentially stained structures distinct from the chromatin with which they are associated. When unfixed chromosomes are immobilized by attachment to grids and incubated with chick brain tubulin, microtubules are observed to assemble onto the kinetochores. This demonstrates the competence of kinetochores in isolated chromosomes to act in vitro as microtubule assembly sites and suggests that they also possess this capacity in vivo. In addition, the results provide a possible means for isolating and characterizing kinetochores.

Chromosomes↗
PubMed2005

Retrobulbar rhabdomyosarcoma in a budgerigar (Melopsittacus undulatus).

This report describes a retrobulbar rhabdomyosarcoma in a 7-year-old male budgerigar (Melopsittacus undulatus). The bird was presented with orbital distension and exophthalmia of the left eye and died during surgery. Necropsy revealed a tan to grey retrobulbar mass compressing all components of the left eye with obscured vision. In histopathological examination, the mass was composed of sheets of spindle-shaped cells with pleomorphic nuclei, numerous bizarre mitotic figures and mononucleated or multinucleated giant cells and also typically strap cells. Neoplastic cells had no cross-striations in sections stained by phosphotungstic acid haematoxylin. The tumour cells did not invade surrounding tissues, including the retina, and there was no metastasis to other organs. Immunohistochemically, tumour cells were positive for desmin, vimentin and alpha-smooth muscle actin, but were negative for S-100 protein and pancytokeratin.

Animals↗
PubMedDate not supplied

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics.

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.

Aluminum↗
PubMedDate not supplied

The effect of prenatal and postnatal lead exposure on neonatal synaptogenesis in rat cerebral cortex.

Pregnant rats were exposed to drinking water with lead (Pb) concentrations of 0, 30, or 200 mg/l. The resultant pups were sacrificed at 11, 15, and 21 d of postnatal age for the determination of synapses/mm3 in parietal cortex. Synaptic counts from electron micrographs of ethanol phosphotungstic acid stained cortical slices were counted by four observers who were blinded as to treatment (control or 200 mg Pb/l drinking water). A greater than fourfold increase in synaptic counts was observed in layers I, II, and III of rat pups parietal cortex between 11 and 21 d of age. Pb treatment depressed synaptic counts maximally at 15 d of age. However, Pb-exposed pups displayed essentially the same synaptic counts as controls by 21 d of age. In a cross-fostering design, it was shown that prenatal exposure to Pb completely accounted for the delays in synaptogenesis. No significant depression of synaptic counts was observed in pups exposed only during the postnatal period. Blood lead concentrations (Pb X B) were determined during gestation and suckling in both mother and offspring. A dramatic peripartum (partum plus and minus 4 d) peak in Pb X B was seen in mother and pup. Pup Pb X B peaked at 80 micrograms/dl at exposures of 200 mg Pb/l drinking water. In addition to being dose-dependent, blood Pb levels resulting from the same concentration of Pb in drinking water displayed a significant dependence on litter at time-points between birth and 1 yr of age. These data indicate that the substantially elevated blood Pb concentrations that are evident at partum in pups prenatally exposed to Pb might be responsible for the postnatally observed delay in synaptogenesis.

Age Factors↗
PubMed1955

An electron microscope study of the epidermis of mammalian skin in thin sections. I. Dermo-epidermal junction and basal cell layer.

1. Basal epidermal cells and their junction with the dermis, as revealed in thin sections of osmium-fixed human and rodent skin, were studied with the electron microscope. Phosphotungstic acid staining was occasionally used to increase the electron density of membranous and filamentous structures. 2. Along the dermo-epidermal junction in all skin specimens there is a sub-microscopic ( approximately 350 A thick) membrane following the basal contours of the epidermal cells, but separated from them by an approximately 300 A space. No epidermal or dermal filaments can be seen to cross it and except in embryonic skin it has no associated band of amorphous material. It is called the "dermal membrane" to distinguish it from the thicker membrane and associated material commonly called the "basement membrane." 3. In adult human skin the basal cell membrane facing the dermal membrane is continuous with, or adjacent to regularly spaced groups of small dense rodlets at which tonofilaments are attached and appear to terminate. Less dense spherical granules are also found in the cellular ectoplasm and are unattached to filaments. 4. The connective tissue fibers in the upper dermis were narrower (>==300 A) than, but displayed the same period (>==350 A) as the collagen fibers in the deeper dermis. The related fibers in embryonic human skin were even narrower (150 to 250 A). In accordance with the views of others that these are "young" collagen with great affinity for polysaccharide, they are called collagen fibers. 5. The same cytoplasmic components are found in all basal epidermal cells: mitochondria, many filaments, many submicroscopic particulates, and only very occasional vesicles of the endoplasmic reticulum. Adult human cells possess pigment granules and intercellular bridges, in addition. Before keratinization is evident, no intercellular bridges and little or no cytoplasmic filaments are visible. The scarcity of vesicles of the endoplasmic reticulum and the prevalence of submicroscopic particulates (80 to 150 A) distributed at random through the cytoplasm, support the view (19) that basophilia and cytoplasmic nucleoprotein are associated with the particulates. 6. Cytoplasmic filaments are <100 A wide and are directed toward the dermo-epidermal junction and to intercellular bridges (when they occur). Because of their obvious identity as the submicroscopic constituents of tonofibrils, they were called "tonofilaments" and believed to represent the keratin-like fibrous protein "epidermin" (29). Bundles of them appear identical with Herxheimer fibers when, particularly in thick skin, they extend from the outer nuclear membrane to the granules spaced along the dermo-epidermal junction. 7. In human skin, adjacent basal cells are separated by simple cell membranes and connected by intercellular bridges. The filaments of one cell are attached to dense elongate granules in these bridges which are separated by a narrow less electron-dense space from a matching granule to which the filaments of the neighboring cell are attached. No filaments have been observed to cross the space in these bridges. In between the bridges adjacent cell membranes may separate from each other leaving an intercellular space. Although occasional basal cells possess a large perinuclear area devoid of cytoplasmic filaments, none following the classical histological description of "clear" cells were noted. 8. Pigment granules are extremely electron-dense with irregular angular outline. Their smallest diameter was significantly greater in Negro than in white skin while the lengths also appeared greater in the former skin type.

Electrons↗
PubMed1985

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.

Animals↗
PubMed1961

The fine structure of inhibitory synapses in the crayfish.

Physiological investigations have shown that the synaptic input to the sensory neuron of the stretch receptor in the abdominal muscles of the crayfish is purely inhibitory. This neuron was chosen, therefore, as a site in which to study the fine structure of inhibitory synaptic endings. It was hoped that this fine structure might (a) provide a morphological prototype for the study of more complex synaptic systems and (b) reflect the inhibitory mechanisms. Stretch receptors were fixed in situ in buffered OsO(4), dehydrated, and embedded in Araldite. Both cross and longitudinal sections were examined after staining with phosphotungstic acid. The inhibitory endings were easily identified by their great similarity to previously described excitatory endings. Small circular profiles (synaptic vesicles) about 460 A in diameter and an accumulation of mitochondria were consistently observed within the presynaptic endings. An increased osmiophilia of pre- and postsynaptic membranes, where they were in apposition, was also seen. The only observed difference between these inhibitory endings and excitatory endings, described by other authors, was the variable presence of a latticework of 230 A tubules in the connective tissue immediately adjacent to the inhibitory endings. Inhibitory endings were observed on all parts of the sensory neuron except the axon.

Animals↗
PubMed1962

Optical and electron microscopic changes in ultraviolet irradiated chromosome segments.

Chromosome segments of urodele cells lose some substance after irradiation with about 10(-1) ergs/micro(2) of heterochromatic ultraviolet light. These segments stain faintly or negatively with the Feulgen and pyronine-methyl-green methods and weakly with the Alfert-Gesch-wind stain for basic protein. In the living cells, Perry found in these chromosome segments a decrease of 50 to 60 per cent in absorption at 2400, 2600, and 2800 A, i.e., in the region of intense chromosomal absorption that is maximal at 2600 A. Apparently the material lost contains DNA (?DNP) and we call the process DNA-steresis. In such cells, fixed in neutral formalin in Tyrode's solution and stained with phosphotungstic acid, electron microscopy shows that the unirradiated parts of the chromosomes consist of (a) a homogeneous or finely fibrillar material (component-A) filling the meshes of (b) an irregular network with bars 40 to 300 A in diameter, some of which continue into a similar interchromosomal network. DNA-steretic portions of the chromosomes consist mainly of this network and only small amounts of component-A, which presumably contains the DNA. We have not been able to demonstrate DNA-steresis with the electron microscope after primary fixation with OsO(4) or KMnO(4). Structural changes due to DNA-steresis are compared with certain nuclear changes in the mitotic cycle.

Chromosomes↗
PubMed1963

Effect of trypsin on liver microsomes.

The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated at 37 degrees C and centrifuged thereafter under the conditions of their initial isolation. The trypsin-treated microsomes and the untreated controls were fixed in unbuffered osmium tetroxide and embedded first in gelose and then in methacrylate. In the trypsin-treated microsomes, there was a removal of the ribosomes from the rough vesicles. Parallel chemical determinations showed that the total nitrogen and total phosphorus of the pellet were lowered. Particles, densely stained with phosphotungstic acid (PTA) and homogeneous in appearance, were found within microsome smooth vesicles in a fluffy layer which collects on the top of the microsome pellet. The morphology of these PTA-stained particles remained unchanged after incubation with trypsin.

Animals↗
PubMed1963

The size of the cellulose microfibril.

Recently the lateral width of the cellulose microfibril has been estimated as 30 A rather than about 150 to 200 A, by extrapolation of data from model shadowing experiments. The difference was attributed to a layer of metal deposited during shadowing. However, direct photographs of the same microfibrils parallel and perpendicular to the direction of shadowing, of unshadowed portions of microfibrils compared with shadowed portions of the same microfibrils, of silver-stained unshadowed microfibrils, and of unshadowed, unstained segments of microfibrils give no evidence of a layer of metal of this thickness in material shadowed under normal conditions. Furthermore, the evidence for microfibril strands of about 35 A in width from negative-staining experiments is subject to a bias from the form of the filaments and from variable positive adsorption of phosphotungstic acid by cellulose. Consequently, the conclusion that the true lateral width of native cellulose microfibrils is about one-fifth of the presently accepted value is not yet justified by unequivocal direct experimental evidence.

Acetobacter↗
PubMed1966

Structure of isolated plant Golgi apparatus revealed by negative staining.

Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO(4)-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.

Golgi Apparatus↗
PubMed1967

Ultrathin frozen sections. I. Methods and ultrastructural preservation.

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25-4% glutaraldehyde for 1-4 hr, are washed overnight in buffer at 3 degrees C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70 degrees C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35 degrees C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

Animals↗
PubMed1968

Structural features of mesosomes (chondrioids) of Bacillu subtilis after freeze-etching.

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.

Bacillus subtilis↗
PubMed1969

Isolation and properties of secretory granules from rat islets of Langerhans. II. Ultrastructure of the beta granule.

Beta granules isolated from rat islets of Langerhans and subjected only to phosphotungstic acid had, in negatively stained images, a 50-A periodicity. This periodicity was also observed in thin-section profiles of beta granules in intact cells. In shadowed preparations, the granules were spherical in shape and had irregular edges and surface structure. The presence of such a periodicity in the beta granule indicates that its matrix may be composed of a crystalline material.

Animals↗
PubMed1969

Ultrastructure of dyads in muscle fibers of Ascaris lumbricoides.

The dyads of Ascaris body muscle cells consist of flattened intracellular cisternae applied to the sarcolemma at the cell surface and along the length of T-tubules. In specimens prepared by conventional methods (glutaraldehyde fixation, osmium tetroxide postfixation, double staining of sections with uranyl acetate and lead hydroxide), both the sarcolemma and the limiting membrane of the cisterna exhibit unit membrane structure and the space between them is occupied by a layer of peg-shaped densities which is referred to as the subsarcolemmal lamina. The lumen of the cisterna contains a serrated layer of dense material referred to as the intracisternal lamina. In specimens fixed in glutaraldehyde, dehydrated, and then postfixed in phosphotungstic acid, with no exposure to osmium tetroxide or heavy metal stains, the membranous components of the dyads appear only as negative images, but the subsarcolemmal and intracisternal laminae still appear dense. Except for the lack of density in membranes and in glycogen deposits, the picture produced by the latter method is very much like that of tissue prepared by conventional methods.

Animals↗