The light and electron microscopic distribution of acid phosphatase activity in human normal oesophageal epithelium

Acid phosphatase activity in human normal oesophageal epithelium was studied with light and electron microscopic techniques. The maximum activity was found to be in the prickle and lower functional layers. Electron microscopic examination revealed activity to be localized in GERL, lysosomes and membrane coating granules. These last structures probably secreted their content into the intercellular space in the central part of the functional layer. Thick sections (0.5 micron) with tilting showed GERL to consist of anastomosing tubules.     

Mechanism of secretory granule exocytosis: can granule enlargement precede pore formation?

Secretory granules have been observed to swell during the process of exocytosis. Swelling is an indication of osmotic stress. The probable role of osmotic pressure in facilitating membrane fusion makes it necessary to determine whether granule membrane 'swelling' can occur prior to its fusion with the plasma membrane (pore formation) in the process of exocytosis. By subjecting adjacent thin and semi-thin sections of an activated granule to ultrastructural examination for membrane enlargement, and to metachromatic staining for verification of pore formation it is concluded that the perigranular membrane can indeed enlarge prior to pore formation. However, the degree of membrane enlargement can far exceed the limit of 2-3% stretching allowed under normal osmotic stress for a membrane bilayer. Such an extensive membrane enlargement, which takes place in the mechanism of exocytosis, cannot be achieved without being accompanied by the insertion of additional membrane.     

Ultrastructural Details in Germinating Sporangiospores of Rhizopus stolonifer and Rhizopus arrhizus

Electron microscope examination of sporangiospore sections from Rhizopus stolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracellular organization not previously reported. Aldehyde fixation followed by chromeosmium postfixation permitted clear depiction of ribosomes hitherto unrevealed in these cells. Mitochondria were diversiform. Spore wall structures in the two species were generally similar, but outer contours differed sufficiently to permit easy species identification in examination of sections. The spores of both species abounded in cytosomes, corresponding in size, shape, and heavy-metal "stain" affinities to spherosomes in cells of higher plants. The osmiophilic response of these spherosome-like inclusions was intensified by treatment of sections with thiocarbohydrazide solution and subsequent application of aqueous osmium tetroxide, which strengthens an assumption that they are lipid-rich. The margins of the spherosome-like inclusions in lead citrate-stained sections included dense particles, about 60 A across, whose crystalline-like arrangements suggested that protein as well as lipid was present. Frequent and close associations between the spherosome-like inclusions and various cell membranes suggested that such bodies participate in membrane elaboration during germination.     

Ultrastructural Properties of the Extra Membranes of Escherichia coli O111a as Revealed by Freeze-Fracturing and Negative-Staining Techniques

Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.     

Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens

The expression of complement receptors by human follicular dendritic cells (FDC) was investigated by immunohistochemical techniques by using polyclonal and monoclonal antibodies to antigenic determinants of CR1, CR2, and CR3. Upon optical immunohistochemical examination of frozen sections from human reactive lymph nodes and tonsils by a three-step immunoperoxidase technique, a strong staining of cell bodies and cytoplasmic extensions of FDC was observed in germinal centers with anti-CR1 and anti-CR2 antibodies. Staining for these antigens was also found on cytoplasmic extensions of FDC in the mantle zone and on the plasma membrane of B cells in the entire follicles. Staining of FDC with anti-CR2 antibody was more intense than that of B lymphocytes. Monoclonal antibodies directed against epitopes of the alpha-chain of CR3 weakly stained FDC in follicles in a similar pattern to that which was observed on adjacent sections with mouse monoclonal antibody KIM4 that only recognizes FDC in human lymph nodes. Immunoelectron-microscopy was performed on frozen sections of a lymph node involved with a centroblastic centrocytic B malignant lymphoma and a reactive tonsil with the use of rabbit F(ab')2 anti-CR1 antibodies and mouse monoclonal anti-CR2 antibody. All the plasma membrane of the cell body and cytoplasmic extensions of FDC in germinal centers and in the mantle zones homogeneously stained for CR1 and CR2 antigens. Fibroblastic reticulum cells were negative. The plasma membrane of tumoral B lymphocytes strongly stained with anti-CR1 and weakly stained with anti-CR2 antibodies. The presence of CR1, CR2, and CR3 on FDC is a unique surface characteristic of these cells that should optimally allow the cells to bind antigen/antibody complexes bearing any type of C3 fragment.     

Heparan sulfate proteoglycan is present in basement membrane as a double-tracked structure

Basement membranes contain 4.5-nm wide sets of two parallel lines, along which short prongs called "spikes" occur at regular intervals. The nature of this structure, referred to as "double tracks," was investigated in Lowicryl sections of mouse kidney and rat Reichert's membrane immunolabeled for basement membrane components using secondary antibodies conjugated to 5-nm gold particles. When the mouse glomerular basement membrane and rat Reichert's membrane were exposed to antibodies directed to the core protein of heparan sulfate proteoglycan, 95% or more of the gold particles were over double tracks, whereas after exposure of Reichert's membrane to antisera against laminin, collagen IV, or entactin, labeling of the double tracks remained at the random level. When heparan sulfate proteoglycan was incubated in Tris buffer, pH 7.4, at 35 degrees C for 1 hr, a precipitate resulted which, on electron microscopic examination, was found to consist of 5- to 6-nm wide sets of two parallel lines along which densities were observed. Immunolabeling confirmed the presence of the proteoglycan's core protein in the sets. Since double tracks were closely similar to this structure and were labeled with the same antibodies, they were likely to be also composed of heparan sulfate proteoglycan.     

The activity of Golgi-membrane galactosyltransferase in the liver of streptozotocin-diabetic rats.

The Golgi-rich membrane fraction isolated from streptozotocin-diabetic rat liver had a lower protein content than the corresponding fraction from normal liver. Its UDPgalactose-N-acetylglucosamine galactosyltransferase activity calculated per 1 g of liver or whole liver was decreased. The electron-microscopic examination of the negatively stained fraction revealed morphological changes. The morphology of the Golgi complex in thin sections of diabetic liver was also changed.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

The Use of Evan's Blue Stain to Test the Survival of Plant Cells after Exposure to High Salt and High Osmotic Pressure

The survival of plant cells may be tested rapidly and convenientlyby staining tissue sections in a solution of Evan's blue (0·5%w/v) after exposure to solutions of high salt concentrationor low osmotic potential. Living cells retain the ability toexclude Evan's blue at the plasma membrane and remain theirnatural colour. Cells damaged by salt or osmotic stress areunable to exclude Evan's blue, are stained deep blue, and arereadily distinguished upon microscopic examination.     

Biochemical activity and morphology of the Golgi apparatus in alloxan diabetic rats.

In Golgi-enriched membrane fraction isolated from alloxan diabetic rat liver, the protein content is lower but the protein composition is similar to normal. The specific activity of galactosyl transferase in this membrane fraction is higher than normal, but the total activity of the enzyme in the whole liver is normal. Great dispersion is found among the individual values. Differences in the specific activities of some other marker enzymes were also found in the Golgi-enriched membrane fraction. The morphology of the Golgi complex examined in either thin sections of Golgi-enriched fraction or ultra-thin sections of livers appears normal. A slight reduction of the rough endoplasmic reticulum is observed. The results obtained are discussed in comparison with those obtained for streptozotocin diabetic rat liver.     

The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia

The connecting cilium of rat retinal rods was studied by freeze-fracture and thin-sectioning techniques. Transverse strands of intramembranous particles could be observed on fracture face B on the ciliary plasma membrane. The strands were essentially similar to those found at the transitional zone of motile cilia ("ciliary necklace"). The larger number of intramembranous particles obscured the pattern on fracture face A of the membrane. On longitudinal sections of the cilia, beads showing a periodicity similar to the necklace strands were observed. Each bead consisted of two structures apposed to both sides of the plasma membrane. Transverse sections of the cilia revealed radial Y-shaped structures that connected each ciliary doublet with the plasma membrane. Axial tubules, central sheath, radial spokes and dynein arms were missing in the connecting cilium. Comparing the fine structure of the retinal cilia with that of motile cilia it becomes evident that the connecting cilium is analogous in structure with the transitional zone of motile cilia. The present observations suggest that periodic membrane beads along the plasma membrane on thin sections correspond to strands of necklace particles as observed on freeze-fractured membranes. The arrangement of the particles in transverse strands is probably ensured by the radial connecting structures.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)     

New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.     

SELECTIVE DEPOSITION OF LANTHANUM IN MAMMALIAN CARDIAC CELL MEMBRANES : Ultrastructural and Electrophysiological Evidence

Perfusion of beating false tendons of the dog heart with ionic lanthanum produced drastic but reversible modifications of the excitability and the transmembrane action potential of Purkinje cells. Ultrastructural examination of these cells revealed the appearance of a fine extracellular precipitate detectable on unstained sections. In addition, specimens perfused with La⁺⁺⁺ showed a striking increase in the contrast of the sarcolemma, particularly in gap junctions and in pinocytic vesicles. La⁺⁺⁺ deposits were restricted to the cytoplasmic leaflets of the sarcolemma; no precipitates were found at the plasma membrane of fibroblasts, endothelial and smooth muscle cells, or unmyelinated nerve fibers present in the same specimens. A selective deposition of La⁺⁺⁺ was also observed in the sarcolemma of atrial and ventricular cells of dog, rabbit, and cat hearts, as well as in the membrane of the transverse tubular system of ventricular cells. Both the electrophysiological effects and the ultrastructural membrane deposits produced by La⁺⁺⁺ disappeared when the specimens were subsequently perfused with phosphate-containing tyrode solution. These results tend to demonstrate that a distinctive feature of the sarcolemma of mammalian cardiac cells is the presence of regions with a high surface density of binding sites for polyvalent cations.     

Production of the Milk Agent in Cultures of Mouse Mammary Carcinoma

Thin sections of tissue cultures grown from tumors of the RIII high-breast-cancer strain mice were studied in the electron microscope. These tissues contain an abundance of particles whose morphology is consistent with biophysical measurement of the milk agent. These particles, found only extracellularly in our cultures, are formed at the cell membrane. The process of formation, as reconstructed from sections, appears to include a thickening and protrusion of the cell membrane which then evolves gradually into a dense sphere and separates from the cell in much the same manner as does influenza virus. The contents of the newly formed body are later rearranged to form a nucleoid within a membranous sac.     

Fine needle aspiration cytology of invasive micropapillary carcinoma of the breast: review of cases in a three-year period.

OBJECTIVE: To describe the fine needle aspiration cytology findings of invasive micropapillary carcinoma and correlate them with the histologic appearance. STUDY DESIGN: We reviewed the cytologic features of three cases of pure invasive micropapillary carcinoma in the files of Pamela Youde Nethersole Eastern Hospital from 1998 through 2000. Immunohistochemical study for epithelial membrane antigen was performed retrospectively on the cell block sections. Ultrastructural examination was also carried out on one of the cases. RESULTS: Two of the tumors were at pathologic stage II, and the remaining case was at stage III. Ipsilateral axillary lymph node metastases with similar morphology were seen in two of them. Cytologically, the smears were of moderate cellularity and composed of three-dimensional tumor cell balls, abortive and sometimes branching papillae, angulated tumor cell clusters, morules and occasional acini. Some of the tumor cell balls possessed scalloped borders. Focally, the tumor morules clustered together and were separated from each other by small, slitlike spaces. A small number of isolated malignant cells was also present in the background. The cell block sections showed mainly dispersed acini of tumor cells. The "reverse polarity" highlighted in histologic sections by immunohistochemical study for epithelial membrane antigen was not consistently demonstrated in the cell block material. Ultrastructural examination confirmed the focal presence of surface microvilli on the periphery of the tumor cell morules. CONCLUSION: Invasive micropapillary carcinoma of the breast possesses some subtle but distinctive cytologic features. With the help of cell block morphology and ancillary techniques, the preoperative suspicion of this rare subtype of ductal carcinoma, which carries a high propensity for lymphatic permeation, is possible.     

Determination of the pheromone-producing region that has epoxidation activity in the abdominal tip of the Japanese giant looper, Ascotis selenaria cretacea (Lepidoptera: Geometridae)

The epoxydienyl sex pheromone of Ascotis selenaria cretacea can be detected only within a rod-like abdominal tip (RAT) of the female. To clarify which part of the RAT is the sex pheromone-producing region, the RAT was morphologically divided into three sections, defined positionally from the abdomen as sections A, B, and C. GC-MS measurements clearly showed that the sex pheromone compound levels in section B were four times greater than those of the other sections. Microscopic dissection analysis revealed that section B consists of four tissues: rectum, oviduct, musculature, and intersegmental membrane. GC-MS analysis of the individual tissues revealed that approximately 90% of the sex pheromone in section B is localized in the intersegmental membrane. A cell layer was found in the intersegmental membrane after staining with propidium iodide. Furthermore, incubation of tissues dissected from section B with a deuterated trienyl pheromone precursor revealed that the labeled epoxy pheromonal component was detected exclusively in the intersegmental membrane. We have determined that the sex pheromone-producing region of A. s. cretacea is on the terminal side of the intersegmental membrane located between the 8th and 9th abdominal segments.     

A staining method using acridine orange and auramine O for fungi and mycobacteria in bovine tissue.

A rapid staining method using auramine O and acridine orange (AOAO) is described for staining mycobacteria and fungi in paraffin sections of bovine tissues. One hundred seventy-seven tissue sections from specimens divided into two general groups on the basis of previous histopathology results were examined with a fluorscent microscope. Group I, a total of 77 sections, were from 47 mycobacterial and 30 fungal granulomas. Mycobacteria were found by the AOAO procedure in 44 of the 47 tissues previously diagnosed as positive for mycobacteria. All 30 fungal granulomas previously diagnosed using convential fungal stains were positive with the AOAO procedure. Group II consisted of sections prepared from 100 granulomas in which typical mycobacterial lesions were observed by histopathologic examination but in which no mycobacteria had been detected. Using the AOAO procedure, two of these 100 granulomas were found to contain mycobacteria and two were found to contain bacterial colonies. In the remaining 96 no etiologic agent could be demonstrated.     

A Staining Method Using Acridine Orange and Auramine O for Fungi and Mycobacteru in Bovine Tissue

A rapid staining method using auramine O and acridine orange (AOAO) is described for staining mycobacteria and fungi in paraffin sections of bovine tissues. One hundred seventy-seven tissue sections from specimens divided into two general group on the basis of previous histopathology results were examined with a fluorscent microscope. Group I, a total of 77 sections, were from 47 myco bacterial and 30 fungal granulomas. Mycobacteria were found by the AOAO procedure in 44 of the 47 tissues previously diagnosed as positive for mycobacteria. All 30 fungal granulomas previously diagnosed wing conventional fungal stains were positive with the AOAO procedure. Group II consisted of sections prepared from 100 granulomas in which typical mycobacterial lesiona were observed by histopathologic examination but in which no mycobacteria had been detected. Using the AOAO procedure, two of these 100 granulomas were found to contain mycobacteria and two were found to contain bacterial colonies. In the remaining 96 no etiologic agent could be demonstrated.     

Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen

The fine structure of plasmalemmal tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen has been demonstrated by scanning and transmission electron microscopy. In addition, the three-dimensional structure of the tubular invagination has been revealed by computer-aided reconstruction. The tubular invaginations of the plasma membrane plunged into the cytoplasm everywhere from the apical, lateral, and basal surfaces of the plasma membrane. The invaginations were tubular and branched away, and their plasma membranes were reinvaginated to form numerous caveolae and occasional coated pits. Numerous caveolae were found in clusters that looked similar to a bunch of grapes and the coated pits were present at the base of the clusters. The caveolae and coated pits derived from the tubular invaginations were almost ultrastructurally identical to those derived from the surface plasma membrane. From examination of the fractured surfaces of the endothelial cells treated with the aldehyde prefix osmium-dimethyl sulfoxide-osmium method and of ultrathin sections of those infiltrated by lanthanum nitrate, the tubular invaginations were found to not penetrate any endothelial cells. A computer-aided reconstruction revealed that the caveolae derived from the tubular invaginations were in close apposition to the surface-connected canaliculi. The reaction product of Concanavalin A conjugated to horseradish peroxidase was present on the outer leaflet of the membranes of the coated pits and coated vesicles and also in the contents of the endosomes, but it was absent from any caveolae. Based on our observations, the functional significance of the tubular invaginations in sinus endothelial cells is discussed. Accepted: 13 September 1999