Structure of human chromosomes visualized at the electron microscopic level

A newly developed technique allows cytological (light microscope level) chromosome preparations to be examined at the electron microscopic level. Ultrathin (50 nm) sections of highly condensed Hela cell metaphase chromosomes show the characteristic mitotic chromosome morphology. In addition a fibrous network (presumably chromosome fibers) can be seen within them. Fibers appear to be gathered at foci along each chromatid. Treatment of chromosomes with trypsin in a trypsin/G-banding procedure reduces the amount of staining material at the electron microscopic level and results in more prominent foci. Thicker (100 nm) sections of less condensed chromosomes prepared from human lymphocytes display a banding pattern similar to G-banding, even without pretreatment with proteases.     

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Summary Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.     

Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography

We investigated the enzyme cytochemical localization of sarcosine oxidase (SOX) in the liver and kidney of several mammals using a cerium technique. First we measured the enzyme activities in the liver and kidney of several mammals and in several organs of mice. The highest activity was found in the Chinese hamster, followed by the mouse. Therefore, we used hamster and mouse tissues for enzyme cytochemistry. The liver and kidneys were fixed by perfusion with various concentrations of glutaraldehyde for 10 min. Tissue slices were incubated in reaction medium consisting of 50 mM TRIS-maleate buffer (pH 7.8), 9 mM sodium azide, 9.8 mM sarcosine, 25 microM FAD, 2 mM cerium chloride, 0.002% saponin, and 0.003% Triton X-100 for 0.5-8 h at 37 degrees C. Optimum staining reaction was obtained in tissues fixed with 0.2% glutaraldehyde, followed by incubation for 2-4 h. Electron-dense reaction products were present exclusively in peroxisomes. Within the peroxisomes strong reactions were observed in the matrix subjacent to the limiting membrane decreasing toward the center. The staining reaction was completely inhibited by 2 mM N-bromosuccinimide. These results indicated that SOX is a peroxisomal enzyme and that the enzyme might be associated with the peroxisomal membrane.     

High-resolution scanning electron microscopic study of the mouse submandibular salivary gland.

The submandibular gland of the mouse was studied by high-resolution scanning electron microscopy, using the osmium-dimethylsulfoxide-osmium method. The three-dimensional structures of the intracellular membranous organelles of acinar cells were clearly revealed. The luminal surface of cisterns of the granular endoplasmic reticulum and Golgi apparatus exhibited particles of 8-15 nm in diameter. The secretory canaliculi presented short microvilli which were irregularly arranged. The striated duct cells were characterized by rich mitochondria arranged vertically in the basal portion. The lamellar mitochondrial cristae were noted in three-dimensional images. The luminal surface extended short microvilli, while that of the excretory duct cell presented complicated microplicae. The capillary endotheliocytes showed a few short microvilli, and their fenestrated areas were bordered by cytoplasmic crests. Fenestrae were 50-80 nm in diameter and showed a plug in their center. The basement membranes of the acini and capillaries showed a spongy structure with various strands and meshes. Collagenous fibrils crisscrossed on their surface.     

Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.     

Synthesis and electron microscopic investigation of model polyacryloynucleosides

Using the interaction of polyacrylic anhydride with uridine or N-acetyl derivatives of adenosine, cytidine, and guanosine, the water-soluble copolymers, polyacryloylnucleosides, were obtained. The acryloylnucleoside units to acrylic acid units ratio in the copolymer was usually about 1:20. The attaching of nucleosides to the polymer occurs mostly through the 5′-hydroxyl group of the sugar. The prominent feature of all polyacryloylnucleosides obtained is their fibrous structure at an ionic strength 0.2 and neutral pH. At concentrations <200 μg/ml the separate strands with a length of 0.2–1.0 μ and diameter of 30–40 Å are distinguishable. Evidently they are formed by side association of two molecules of copolymer or by folding of one molecule on itself. At higher concentrations branched multistranded structures are formed. In the same conditions polyacrylic acid alone does not form the fibrous structures. Heating of polyacryloylnucleoside solutions at 100°C and fast cooling in ice water, or raising of the pH to 12 turned the stranded structures to coils. After annealing or neutralization the stranded structures reformed. These transformations are similar to those which occur with nucleic acids. The results show that the fibrous structure of the copolymers depends on the hydrogen bonds formed by purine and/or pyrimidine bases.     

Heterogeneity of distribution pattern at the electron microscopic level of heparan sulfate in various basement membranes.

Using the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique and enzymatic digestion, we investigated the ultrastructural distribution pattern of heparan sulfate side chains of heparan sulfate proteoglycan (HSPG) in various basement membranes (nerve, capillary, oral epithelial, muscle, and dental basement membranes). Four different distribution patterns of stain deposits were identified as heparan sulfate on the basis of enzymatic degradation by heparitinase. In some basement membranes associated with tooth germs and oral epithelium, HID-TCH-SP stain deposits were regularly located at both sides of the lamina densa, but few were observed in the lamina densa itself. In nerve, muscle, and capillary basement membranes, the stain deposits were localized at the external side of the lamina densa adjacent to the underlying connective tissue, but were not found in the laminae lucida and densa. In the internal basal lamina of junctional epithelium of gingiva, the stain deposits were detected mainly in the lamina lucida region. Finally, in some dental and oral epithelial basement membranes, the stain deposits were randomly distributed throughout both laminae lucida and densa. Thus, the present study demonstrated distinct differences in heparan sulfate distribution pattern among various basement membranes, suggesting their architectural heterogeneity.     

An electron microscopic study of the human infundibulum

Summary For a limited period during the oogenesis of Protopterus, blebs of the perinuclear cistern contain, in addition to other inclusions, a special kind of microtubular elements. Most of these blebs face parts of multiple nucleolar bodies that extend toward and make contact with the inner nuclear membrane. The microtubular lumen contains a finely dispersed material of moderate electron density which seems to be in contact with this nucleolar material.Aside from these intracisternal structures there are, within both the perinuclear cytoplasm and the nucleoplasm, similar microtubular arrays without apparent connection with the nuclear envelope. These are either enclosed by membranes derived from those of the envelope or unconfined, having escaped through breaks in their respective bounding membranes. Extracisternal tubules are presumed to have passed their period of putative functional activity and to be undergoing a process of regression and subsequent disintegration.Among possible roles attributable to the intracisternal microtubular apparatus are the following: (1) It may serve for the transport of special nucleolar components to the cytoplasm, possibly to be incorporated in the matrix of developing perinuclear mitochondria; (2) it may provide openings in the nuclear membranes for the direct passage of particulate elements between nucleus and cytoplasm; (3) it may be instrumental in the breakdown of parts of the nuclear envelope prior to its restitution during the subsequent phase of oogenesis.Supported by grants NB-00840 and NB-05219 from the U.S.P.H.S.     

Scanning electron microscopic investigation of mouse cerebellar macrophages in vitro

Dispersed monolayer cultures of post-natal mouse cerebella were studied using scanning electron microscopy. Special attention was given to the morphology of the brain macrophages and their interaction with neuronal cells. The brain macrophages resemble other macrophages and mononuclear phagocytes in vitro, reported in the literature. Their ability to induce neuronal growth cones to avoid contact, results in areas devoid of neuronal processes (halos) in this culture system; this seems of considerable interest in connection with their possible developmental role in post-natal reshaping of the brain. Scanning electron microscope observations indicate that these halos are not the result of phagolysis of already established neuronal networks, but can only be induced during neuronal growth and process formation. Established, intact neurons are apparently not altered or harmed by contact with brain macrophages.     

The structure of fibrin. An electron microscopic investigation

An electron microscopic investigation on the structure of fibrin is reported. Fibrin morphology was investigated in a wide variety of experimental conditions, and by carefully controlled staining procedures. Two main band patterns A (230-Å-spaced main dark bands) and B (230-Å-spaced main light bands) are observed for stained fibrin; it is shown that the former results from the superimposition of both positive and negative staining, and the latter is given by positive staining. By suitable denaturation experiments, it was found that the fiber is composed of a regular alternation of lose and dense regions along the axis. We have assumed that the monomer of fibrin is described by the three-nodular model of Hall and Slayter, as supported by recent investigations. The monomers are arrayed according to a head-to-tail sequence along the fiber, and to a staggered lateral association. This model accounts for all the experimental observations, and predicts well the high-resolution band pattern of fibrin. It further agrees with the results of a recent work on the early stages of the fibrinogen-to-fibrin conversion.     

Scanning electron microscopic investigation of thermal damage of the teeth.

Sound teeth were heated to 200 degrees C and 1300 degrees C and the gradually developing morphological changes have been studied. The cementum structure was destroyed at about 500 degrees C, the enamel structure between 700 and 900 degrees C, whereas dentine preserved its canalicular structure even after the inorganic salts had melted at 900 degrees to 1000 degrees C. At 1300 degrees C the mineral substances of the tooth were melting into atypical, globular formations. Scanning electron microscopic examination of dental residues damaged by high temperature seems valuable from forensic, criminological as well as anthropological aspects, since the origin of the finding can be determined, from a small fragment, the material can be identified with a tooth and conclusions can be drawn concerning the temperature inducing the damage.     

Chemical components of cells at the microscopic level

Thin slices of potato tuber can be used to demonstrate, at the cellular level, the presence of protein, carbohydrate, and nucleic acids. The presence of fat can readily be demonstrated with avocado     

High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium

A mixture of dihexanoyl phosphatidylcholine and dimyristoylphosphatidylcholine in water forms disc-shaped particles, often referred toas bicelles [Sanders and Schwonek (1992) Biochemistry, 31, 8898–8905].These adopt an ordered, liquid crystalline phase, which can be maintained atvery low concentrations of the bicelles (down to 3% w/v). At thisconcentration the spacing between individual bicelles, on average, exceeds300 Å. The bicelles are shown to have a negligible effect on therotational diffusion of ubiquitin as judged by the ¹⁵NT₁ values of the backbone amides relative to those inisotropic aqueous solution. The protein exhibits a residual degree ofalignment which is proportional to the bicelle concentration, andapproximately collinear with ubiquitin's rotational diffusion tensor. Thedegree of alignment obtained offers unique opportunities for studying theprotein's structure and dynamics.     

Preliminary electron microscopic and x-ray study of the crystalline protein of diphtheria toxin]

The authors present the results of the electron microscopic and roentgenological study of the crystalline protein of diphtheria toxin. On the basis of direct measurements of the crystal image and diffraction pictures it was possible to calculate the interplane distances of the placement of molecules in the crystal. Examination in polarization microscope showed that the crystals were optically uniaxial with a direct extinction and a positive character of elongation. Molecular aggregates revealed in the protein molecules resembled the caudal part of the bacteriophage by structure.     

An electron microscopic study of the human epidermal keratinocyte

Summary 1. The epidermis of the flexor surface of the upper arm of human subjects was studied with the electron microscope. 2. The cytoplasm of the keratinocytes in the basal layer contained many tonofilaments, ribosomes and other cell organelles. The tonofilaments were arranged singly or in loose bundles and many were attached to the inner membrane of the desmosomes. Along the basal border of the cells pinocytotic vesicles could be seen at different stages of development. 3. The keratinocytes in the stratum spinosum differed from those in the basal layer in two main ways: (a) The tonofilaments were grouped together into large compact bundles known as tonofibrils and it was possible to determine a definite beading or cross banding along the length of some of the filaments. (b) The cells were assuming a flattened shape. 4. The keratinocytes in the stratum granulosum possessed large numbers of irregularly shaped keratohyaline granules. The granules were strongly osmiophilic and were always situated on a meshwork of tonofibrils. The keratohyaline granules had no internal structure. The nuclei and mitochondria showed evidence of degeneration. 5. The keratinocytes in the stratum corneum were long and flattened. The cell walls showed increased electron density and were considerably thickened. The cytoplasm was filled with closely packed fibres separated by a small amount of lucent matrix. The fibres were grouped together in bundles running in different directions within the flattened squames. The fibres had along their entire length alternating areas of high and low electron density. The keratohyalin granules had disappeared and nothing remained of the nuclei or the organelles. In the deepest cells of this region the fibres were sometimes loosely packed leaving large irregular open spaces. This area corresponded to the stratum lucidum. In the most superficial layers of the stratum corneum the fibres appeared to be breaking down so that little remained within the keratinocyte except large lucent spaces. The desmosomes showed distinct structural changes. 6. An attempt was made to correlate the structural changes in the different epidermal layers with the process of keratinization. The possible part that keratohyalin may play in the process of thickening of the cell walls was discussed. The relationship between the desmosome and its dynamic environment was considered.I wish to express my sincere thanks to Dr. David Hilding of the Department of Otolaryngology for the use of an R.C.A. electron microscope and other facilities in his laboratory. This research was supported by the United States Public Health Service and American Cancer Society grants. USPHS CA 04679-07, NB 03995.