bluuebunny/crossref_metadata_2025 huggingface dataset:
Scanning Electron Microscopic structure and function of the fore-gut of Indian fresh water prawn,Macrobrachium lamarrei H.M. Edwards(Decapoda-Palaemonidae)
Authors: Shukla Sanjive , Sharma U. D.
Published: June 2020

The fore-gut structure of certain crustaceans is well documented. However, data on palaemonidae are scanty. The present work has been carried out on the ultrastructure of the fore-gut in relation to feeding and food movements inM. lamarrei, an omnivorous animal. The fore-gut was dissected out and slit open in various individuals for SEM studies and processed. Samples were observed under an SEM (JEOL JSM 35C) and photographed.

The number, size, shape, and arrangement of bristles vary in different zones, which is devoid of gastric mill. Bristles are either simple or compound, and are present in groups at the entrance of the esophagus from the buccal cavity, on esophageal folds, and on the wall and floor of the cardiac stomach (Figs. 1-10). The simple smallest bristles (35-53 μm) are on the esophageal folds; simple as well as compound bristles (275 μm) are present on the cardiac luminal surface.
Freeze-fracture Electron Microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes
Authors: Benga Gheorghe , Brain Anthony , Pop Victor I. , Wrigglesworth John
Published: June 2020

The intra-membrane particles (IMPs) observed on the fracture face of frozen erythrocyte membranes are thought to correspond primarily to “band 3” tetramers or dimers. Some recent studies correlating the inhibition of water diffusion in erythrocytes by p-chloromercuribenzene sulfonate (PCMBS) with the binding of 203Hgto erythrocyte membrane proteins has enabled band 3 and the polypeptides in band 4.5 to be identified as the proteins associated with the channels for water permeation in human erythrocytes. A further characterization of the effects of the incubation of human erythrocyte membranes with PCMBS and N-ethylmaleimide (NEM) has been performed as previously described. Experimental conditions have been previously described.

A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes with the sulphydryl reagents. It was found that on many of the control and NEM-treated cells, small (50-100 nm) elevated patches could be seen (Fig. 1, 2 and 3). These are present on both fracture and etch faces and are devoid of any intramembrane particles. The patch elevations were never observed in the membranes of PCMBS-treated cells (Fig. 4).
Purification of the helical filaments appearing in mitochondria
Authors: Sasaki Hiroyuki , Kurioka Susumu , Misawa Takeyuki , Suzuki Teruo
Published: June 2020

Helical filaments with characteristic morphology have been found exclusively in the outer compartment of mitochondria in the epithelial cells of renal proximal tubules of patients, and in the brain astrocytes, the ameloblasts and the epithelial cells of renal medullary tubules of the normal rats, and in the liver parenchymal cells of the rats with and without ethanol ingestion. Recently, the filaments with same helical structure found in the mitochondria of the certain parasite. Thus, the intramitochondrial helical filaments appeared widely in biological fields. However, the nature, origin and physiological significance of the helical filament are at present unknown. In the present study, to clarify the molecular arrangement of the helical filament, the filaments were purified from the mitochondriaof the liver of ethanol ingested rats in which have been confirmed to appear large amount of the helical filaments in mitochondria.

Male S-D strain rats (6-week old) were used in the present study. Rat were given 30% ethanol for 90 days with oral administration. With conventional TEM observations, the helical filaments were frequently found in mitochondria of liver cells of the rat with ethanol ingestion (Fig. 1). For purification of the helical filaments, the mitochondria were isolated from ethanol ingested rats liver cells by slightly modified Bustamante et al. method. The isolated mitochondria were ruptured by osmotic breakage.
The application of freeze-fracture in the analysis of intercellular junctions in pleuro-pulmonary tumors
Authors: Mukherjee T. M. , Swift J. G.
Published: June 2020

Thin section and freeze-fracture techniques have been used to examine the morphology of cell junctions in a variety of pleuro-pulmonary tumours with the aim of identifying features that may be of diagnostic importance or of significance in the development of the tumour. Freeze-fracture preparations are particularly useful for the analysis of cell junctions, since extensive face views of the interior of the cell membrane are exposed. This enables precise characterisation of the type of junctions present, their extent and their inter-relationships.

Freeze-fracture replicas can reveal the presence of junctions that would be difficult or impossible to detect in thin sections. For example, desmosomes are a well-known feature in thin sections of squamous cell carcinomas, but these tumours may also have focal tight junctions and gap junctions (Figs. 1,2). The tight and gap junctions can occur separately (Fig.l), or in combination (Fig. 2). Similarly, in a recent study of a case of “Ewing’s sarcoma”, replicas showed the presence of unusual, elaborate focal tight junctions, a feature never suspected from the routine thin section studies of this tumour.
Morphometric analysis and autoradiography of the smooth endoplasmic reticulum during glycogen deposition in the fetal mouse hepatocyte
Authors: Shockey Breslin Joanette , Cardell Robert R.
Published: June 2020

Analyses of adult hepatic glycogen deposition by numerous investigators have determined that the smooth endoplasmic reticulum (SER) proliferates immediately prior to glycogen deposition and during the early stages of glycogen accumulation, then decreases as glycogen levels reach their maximum, suggesting that SER participates in adult hepatic glycogen metabolism. Less is known regarding fetal hepatic glycogen synthesis and the participation of the fetal SER. The studies described here test the hypothesis that the SER functions in the synthesis of fetal hepatic glycogen. Quantitative analysis of SER and glycogen levels during hepatic glycogen synthesis tests the existence of a correlation between glycogen and SER. Newly deposited labeled glycogen is localized via autoradiography and the extent of association between labeled glycogen and SER quantified, establishing whether glycogen is necessarily deposited near membranes of SER.

Fetal mouse livers were harvested at daily intervals between days 14 and 19 of gestation, immersion fixed in 2% glutaraldehyde, 2% paraformaldehyde, post-fixed in 1 % OsO4 dehydrated in EtOH and embedded in Epon 812. Semi-thin (0.5μm) and ultra-thin sections (60 nm) were prepared for morphometric analysis.2
Electron microscopy analysis of degenerating pancreatic ß cells in transgenic mice expressing the MHC Class I antigen Dd
Authors: Stewart T. A. , Liggitt D. , Pitts S. , Martin L. , Siegel M. , Keller G-A.
Published: June 2020

Insulin-dependant (Type I) diabetes mellitus (IDDM) is a metabolic disorder resulting from the lack of endogenous insulin secretion. The disease is thought to result from the autoimmune mediated destruction of the insulin producing ß cells within the islets of Langerhans. The disease process is probably triggered by environmental agents, e.g. virus or chemical toxins on a background of genetic susceptibility associated with particular alleles within the major histocompatiblity complex (MHC). The relation between IDDM and the MHC locus has been reinforced by the demonstration of both class I and class II MHC proteins on the surface of ß cells from newly diagnosed patients as well as mounting evidence that IDDM has an autoimmune pathogenesis. In 1984, a series of observations were used to advance a hypothesis, in which it was suggested that aberrant expression of class II MHC molecules, perhaps induced by gamma-interferon (IFN γ) could present self antigens and initiate an autoimmune disease. We have tested some aspects of this model and demonstrated that expression of IFN γ by pancreatic ß cells can initiate an inflammatory destruction of both the islets and pancreas and does lead to IDDM.
On the cross-sectional shape of the cellulose crystallites in the tunicate Halocynthia papillosa
Authors: Revol J.-F. , Van Daele Y. , Gaill F.
Published: June 2020

The only form of cellulose which could unequivocally be ascribed to the animal kingdom is the tunicin that occurs in the tests of the tunicates. Recently, high-resolution solid-state l3C NMR revealed that tunicin belongs to the Iβ form of cellulose as opposed to the Iα form found in Valonia and bacterial celluloses. The high perfection of the tunicin crystallites led us to study its crosssectional shape and to compare it with the shape of those in Valonia ventricosa (V.v.), the goal being to relate the cross-section of cellulose crystallites with the two allomorphs Iα and Iβ.

In the present work the source of tunicin was the test of the ascidian Halocvnthia papillosa (H.p.). Diffraction contrast imaging in the bright field mode was applied on ultrathin sections of the V.v. cell wall and H.p. test with cellulose crystallites perpendicular to the plane of the sections. The electron microscope, a Philips 400T, was operated at 120 kV in a low intensity beam condition.
Effect of calcium on endospore ultrastructure
Authors: Stears Robin L.
Published: June 2020

Clostridium sporogenes has been designated as a test species for the closely related but pathogenic Cl. perfringens. Cl. sporogenes possess a unique morphology. High resolution EM has not been applied and little is known about its calcium distribution, thus this study presents a morphological evaluation as well as data on calcium localization, which is considered important for endospore resistance.

Cl. Sporogenes was grown on cooked meat/media. Sporulation was monitered using the malachite green method. After maximum sporulation the spores were harvested in deionized distilled water, heated for 20 minutes at 80°C to kill any remaining vegetative cells, washed 3 times with sterile deionized water and the stock suspension was stored at 2-8°C until needed. Aliquots of the spores were placed momentarily in warm agar prior to fixation then fixed in 3% gluteraldehyde (GCHO) in 0.1 M cacodylate buffer pH 7.4 to which Ca was deleted or with 1% CaCl.
Selective contrast enhancement with ruthenium tetroxide for observation of reverting yeast protoplasts
Authors: Naito Nobuko , Yamada Naoko , Sato Mamiko , Kobori Hiromi , Osumi Masako
Published: June 2020

Rutenium tetroxide (RuO4) is a strong oxidizing agent like osmium tetroxide (OsO4). Therefore its possible use as a fixative and staining agent for electron microscopy is promissing. It also has the advantage of reacting with polar lipids which do not react with OsO4. Despite these advantages, however, RuO4was not put into practical use because it is easily decomposed. Recently we used RuO4fixation to observe the glucan fibrils on reverting protoplasts by low-voltage SEM, and obtained very high resolution images of glucan fibrils (Fig. 1). The purpose of this study was to evaluate the effectiveness of RuO4as a stain for TEM. The results showed that RuO4treatment is very effective for observing the u1trastructure of reverting yeast protoplasts. The treatment provided clear images of regenerating glucan fibrils, Golgi apparatus, secretory vesicles and filasomes in the cytoplasm.
Comparative aspects of the plasma membrane structure in yeast by a new high-resolution freezereplica method and ordinary freeze-replica method
Authors: Hirano Tadashi , Tanaka Akira , Yamaguchi Masashi
Published: June 2020

The development of the ordinary freeze-replica method providing a clear three-dimensional image. And many researchers have made technical improvements in these method. However,the metal shadowing film replica method has problems.For instance, the fine structure of a surface of biological specimen can suffer thermal damage due to radiant heat derived from the high temperature necessary to bring about evaporation of the heavy metal. Also, the evaporated materials is in the form of particles (Figure 1). Accordingly, it is questionable whether these particles are a natural feature or artificially introduced by ordinary freeze-replica method. Walzthong et al. (1982) showed that the particles were an artifact or form of contamination produced under the conditions used in the ordinary freeze-replica method.
Ultrastructural localization of cell-wall antigens in Paracoccidioides brasiliensis
Authors: Edwards M. R. , Samsonoff W. A. , Ostrander G. A.
Published: June 2020

Paracoccidioides Brasiliensis is a pathogenic fungus responsible for a widespread systemic disease in Latin America. It grows at 25°C as a mold, but at 37°C produces veast cells. Previous ultrastructural studies revealed the eukaryotic structures of these cells. Here we present results from an immunoelectron-microscopic study using human antibodies from patients with paracoccidioidomycosis and rabbit anti-human IgG conjugated with gold particles (10 nm in diameter).

Yeast cells were fixed with 0.2-0.5% glutaraldehyde with 2% formaldehyde in cacodylate buffer (pH 7.2) for 5 h at room temperature and at 4°C overnight; after buffer washings, with or without 0.5% saponin, the samples were osmicated, dehydrated and embedded in epoxy resins, according to standard methods. Thin sections were incubated with normal human serum (negative control) or with human antiserum (kindly provided by A. Restrepo-Moreno, Medellin, Colombia) and treated with immunogold probes according to published protocols.
Effect of RI-331, a new antifungal antibiotic, on the ultrastructure of Candida albicans
Authors: Nishiyama Yayoi , Asagi Yukiyo , Hiratani Tamio , Yamaguchi Maki , Yamaguchi Hideyo , Yamaki Hiroshi
Published: June 2020

An amino acid antibiotic RI-331, (S)-2-amino-5-hydroxy-4-oxopentanoic acid, produced by a Streptomyces sp., is a novel antifungal agent active against a wide range of yeasts of medical importance including Candida albicans. This antibiotic was found out as one of those natural compounds which preferentially inhibited regeneration of C.albicans protoplasts to intact cells during our screening works. In the present study, the effect of RI-331 on C.albicans growing cells and protoplasts prepared therefrom were investigated morphologically by transmission electron microscopy.
The influence of lead at different concentration and pH on the ultrastructure of root cells from germinating soybean seeds
Authors: Crang Richard F. E. , Kuras Mieczyslaw , McQuattie Carolyn J.
Published: June 2020

Environmental pollution of lead in the USA has become less of a serious problem with the advent of non-leaded petroleum products. However, other sources of organic and inorganic compounds with lead content and, (in many parts of the world) leaded fuels, are still employed which create health and environmental problems. The conditions by which lead uptake occurs in plants, its translocation and toxic effects have been demonstrated by various investigators.

We have investigated the influence of lead on roots of germinating soybean (‘Williams’) seeds over the course of 48 hr treatments at atomic concentrations of 0.0, 250, 500 and 2,000 ppm. Imbibed seeds were placed on filter paper saturated with aqueous levels of PbCl2 adjusted to pH levels of 5.5, 3.5, and 2.5. Tissue slices about 1 mm behind the growing root tips were obtained and conventionally prepared for transmission electron microscopy. In most cases lead post-staining was not employed so as to not interfere with the presence of naturally-occurring lead deposits.
Characterization of beam-induced thinning and shrinkage of semi-thick sections in the HVEM
Authors: Kremer J.R. , O'Toole E.T. , Wray G.P. , Mastronarde D.M. , Mitchell S.J. , Mcintosh J.R.
Published: June 2020

It is well known that irradiation of plastic sections in a conventional transmission electron microscope (cTEM) causes specimen thinning and distortion. Thinning has been observed in the cTEM using several embedding media, using methods such as shrinkage of ordered paracrystalline structures, and shrinkage of sections coated with colloidal gold markers. The total thinning observed in the cTEM (80kev) is 30-50% for thin sections of epon araldite, but similar data do not exist for the HVEM at 1000 kev. Here we describe beam induced thinning and shrinkage of 0.2um sections in the HVEM.

Experiments were performed using 0.2um sections of EPOX 812/Araldite or LX112 with 15 nm and 30 nm gold particles affixed to either surface of the section. The sections were initially tilted to approximately 25° and irradiated with known dose rates. Micrographs were taken at different times between 0-20 minutes then the sections were tilted back to 0° for a reference micrograph.
Embedding of unfixed plant material in water-soluble resin: A simple preparation method for elemental analysis by ESI and EELS techniques
Authors: Lehmann Heiner , Kunz Ulrike
Published: June 2020

The localization of elements in cells and tissues by electron spectroscopic imaging (ESI) or electron energy loss spectroscopy (EELS) can be realized only in very thin specimens in order to avoid multiple scattering of the electrons. Therefore the biological material has to be cut in thin 30 nm sections.

The commonly used preparation procedure is a hydrous chemical fixation followed by dehydration, embedding in hydrophobic resin and ultrathin sectioning. The disadvantage of this procedure is the loss and/or dislocation of inorganic ions and other water-soluble substances. To overcome these difficulties the preferable preparation method is cryofixation in combination with cryosubstitution, but this is a very expensive and time-wasting procedure.

Meanwhile, we established a more simplified preparation technique, which shows reasonable results on plant cells without large central vacuoles: The living cells were directly put into freshly mixed Nanoplast FB 101, a water-soluble melanine resin, and stored for 24 h at 20° C in a silca gel containing exsiccator.
Detection of elements by ESI and analysis of EEL spectra in alveolar macrophages prepared by slam-freeze molecular distillation processing
Authors: Godleski John J. , Stearns Rebecca C. , Katler Marshall , Hastings Cindy L.
Published: June 2020

The capability to map the distribution of elements in relationship to ultrastructural cellular morphology has been a longstanding goal in anatomic research. With biologic materials, the technology for elemental detection is limited by the lack of retention of elements in ionic form during processing with liquid fixatives. Although we have been able to show that by selection of fixative and processing methods, 95% of cell associated iron remains with the cells after processing, we have not found an adequate general purpose liquid fixative to preserve ions in cells. Hence, we have turned to cryogenic methods which are more likely to retain intracellular ions.

Alveolar macrophages were obtained from hamsters by saline bronchoalveolar lavage. The cells were centrifuged and resuspended in medium RPMI 1640 at a concentration of 1×108/ml. A 5 μl aliquot of the cell suspension was placed on an agar mount and instantaneously frozen using a Life Cell® CF-100 unit for cryofixation.
High-performance combined freeze-dryer and sputter coater for cryosputtering of biological samples for Electron Microscopy
Authors: Fredriksson Bengt-Arne , Bell Paul , Lindroth Margaretha
Published: June 2020

Freeze-drying followed by sputter coating is the optimal method for preparing detergent-extracted cells for scanning electron microscopy. However, during transfer from the freeze dryer to the sputter coater, the cells are warmed to room temperature, exposed to the atmosphere, and returned to vacuum, which will cause “melting”, partial rehydration, and subsequent redrying of the sample. To avoid these potential sources of damage, we have developed an improved device that allows samples to be freeze-dried and then sputter coated while still frozen (cryosputtered) and without first exposing them to atmospheric moisture.

The construction is based on a Tis-U-Dry Histological Freeze dryer, model TFD-130 (FTS Systems, Inc., Stone Ridge, NY, USA). The walls of the cylindrical vacuum chamber are cooled to -130 °C by a three-stage refrigeration system and the circular platen in the bottom of the chamber is cooled to -110 °C by conduction. The chamber is evacuated with a turbomolecular pump (Balzer model TPH 330), backed by a rotary vane pump (Balzer model DUO 016B), giving a clean, high vacuum.
A polymer designed to optimize antigen localization in immunoelectron microscopy
Authors: Olesen J.B. , Heckman C.A. , Lukinius A.
Published: June 2020

We have developed a new polymer for the purpose of retaining the maximal antigenicity of proteins in samples embedded for electron microscopy. HACH is a mixture of hydroxyhexanedial, carbohydrazide and hydrazine that polymerizes at room temperature within 24 h. Tissues embedded in the polymer were thin sectioned and the sections, mounted on formvar films, were viewed with a Zeiss IOC transmission electron microscope. The ultrastructural features of cells in HACH-embedded tissues appeared similar to those previously found in GACH-embedded tissues.

The ability to preserve the antigenicity of a protein up to the polymerization stage has been tested in the following manner. Human red blood cells, from either Rh+ or Rh- donors, were first exposed to the HACH prepolymer and then rinsed and exposed to a commercially available anti-D antibody. After removal of the prepolymer, Rh+ red blood cells were clumped by anti-D antibody to the same extent as control, untreated cells (Fig. 1).
Contrast enhancement of pollen perforations for image analysis
Authors: Vezey Edward L.
Published: June 2020

Variation in pollen grain sculpturing constitutes one source of taxonomic evidence. With closely related species, this variation is primarily quantitative. Computerized image analysis produces accurate measurements of pollen surface features, thus making objective comparisons of quantitative sculpture variations possible. This study concerns manual methods that provide necessary contrast to features on reticulate or perforate pollen grain surfaces for image analysis. Repeatability of these methods was tested by statistically analyzing results of seven individuals. Image analysis was conducted using Kevex Feature Analysis software (Version 2.10) on a Delta class computer (Kevex Instruments, Inc., San Carlos, CA).

In image analysis, features are designated by “painting” specific regions of the gray scale. A prerequisite for “painting” is sufficient contrast between features and non-features. With pollen, this requirement is often not met because: (1) surface perforations or openings (called lumina if sculpture is reticulate) sometimes contain subsurface structural elements (Fig. 1); or (2) lumina cannot be differentiated based on contrast (Fig. 2).
EDX microanalysis of cellular components in the pollen of an angiosperm, Plumbago zeylanica
Authors: Russell S. D.
Published: June 2020

Pollen grains in flowering plants recognize compatible stigmas and transmit the sperm cells that they contain to the egg. The pollen grains bear specific proteins and lipids on their outer walls, which are the principal sources of both pollen-stigma recognition and human allergic response; they also contain the organelles required to form the pollen tube and the two sperm cells that participate in double fertilization. Although limited information is available about x-ray microanalysis of pollen grains, no information is available about the cellular distribution of elements.

The contents of pollen of Plumbago zeylanica L. were burst in double-distilled water onto formvar-coated Cu slot grids ( 1 × 2 mm) and air-dried at room temperature. Grids were then examined at 40 kV in a JEOL 2000 STEM (LaB6 e' source) equipped with a high-angle 10 mm2 Kevex Quantum detector and Delta analyzer. Spectra were collected for 100-500 livetime sec; digital x-ray maps were collected at resolutions of up to 128 × 256 (dwell time: 30 msec [10-15 collections], 15-30% deadtime at ca. 1,000 cps, adjusted using the free lens control).
Application of new cryo-SEM to prove rapid freezing survival of unprotected red blood cells
Authors: Fujikawa Seizo , Ueda Shinichi
Published: June 2020

It has been suggested that rapid freezing, by which vitrification or intracellular freezing with ice crystals less than lOnm in diameter can be achieved, may preserve not only ultrastructure but also cellular function, although the definite evidence, especially in unprotected and highly hydrated biological materials, is not yet obtained. In this study, new cryo-SEM which allows continuous observation of same frozen samples by means of cryo-SEM and then by means of freeze-replicas, was used to prove the possibility of cellular survival by rapid freezing.

Human red blood cells suspended in isotonic saline were used for materials. Samples with different thickness from 25μm to 200pm were loaded on holders and frozen by slamming against LHe cooled copper block by using metal contact machine (Meiwa Co. QF-5000). The entire frozen samples were carefully removed from the holder under liquid nitrogen. Some of the frozen samples were thawed rapidly and survival (% hemolysis) were determined.
Effect of amizet solution to the hepatic failure by endotoxin-treated rat
Authors: Hirohata Yasuhisa , Kobayashi Hiroshi , Pan Je , Zheng Heng , Aihara kaoru
Published: June 2020

Since endotoxin administration and also multiple organ failure(MOF) are accompanied by the acceleration of the proteolytic process although a sufficient energy source is given, it seems important to investigate the imbalance between consumption of branched-chain amino acids and increase of amino acid release in endotoxin treated liver. Therefore, authors have undertaken to investigate the ultrastructural alterations in endotoxin treated liver and explored the nutritional modification by branched-chain amino acid, Amizet.

Male and female Sprague-Dawley rats(200-300g) were housed on a 12hrs.-light/12hrs.- dark cycle with laminar flow. Experimental Protocol: Rats were divided at random into the following four groups.

Group 1: non-treated control.

Group 2: A new amino acids solution riche in branched-chain amino acids, Amizet, was intraperitoneally infused at three times a week for 8 weeks.

Group 3: received endotoxin(10mg/Kg, Difco Laboratories, Detroit) and observed for 8 weeks.

Group 4: received endotoxin(10mg/Kg), and Amizet was intraperitoneally infused in the same procedure as Group 2 and 3.
SEM and TEM evaluation of bacterial adhesion morphology to breast prosthetic biomaterials
Authors: Fassel Theresa A. , Van Over James E. , Hauser Christopher C. , Edminston Charles E. , Sanger James R.
Published: June 2020

Coagulase-negative Staphylococci have been implicated in late onset infection of breast prosthetic biomaterials used for augmentation and reconstruction. These prosthesis consist of either gel- or saline-filled smooth silicone rubber or textured polyurethane foam. The morphology of adhesion of staphylococci to the prosthesis is important in understanding their potential role in latent infection. Staphylococcus epidermidis rp62, a polysaccaride mucin producing strain, and Staphylococcus aureus strain 25923 are test organisms. A variety of fixative procedures employing en bloc reagents are compared in both SEM and TEM. Ruthenium red and alcian blue , uranyl acetate and ruthenium red with lysine have been used to preserve extracellular capsular/slime material in several bacterial species.

Specimens of prosthetic material are incubated with cells for 18 hrs at 35°C in tryptic soy broth. For SEM, specimens are fixed in 2.5% glutaraldehyde (GA) for 2 hrs and post-fixed in 1% OsO4 for 2.5 hrs. This is followed by a graded ethanol series, critical point drying with CO2 and coating with gold/palladium.
Image processing of immunogold-labeled titin filaments in skeletal muscle
Authors: Ramirez-Mitchell R. , Wright John , Wang Kuan
Published: June 2020

In our electron microscopic studies of the elastic behavior of titin filaments in skeletal muscle sarcomeres, immunogold techniques have been used to detect the elastic stretch-response of titin epitopes in the I-band region. The use of gold conjugates was essential, since in highly stretched muscles, distortion of the sarcomere led to the loss of lateral registration of epitopes. In such cases, antibody densities are smeared and difficult to detect. We observed, however, that gold beads sufficiently small ( < 5 nm ) to penetrate the I-band lattice were often obscured by electron dense stains. To overcome this technical problem, we developed a procedure using image processing techniques to differentiate and identify gold beads.

In the following example, titin filaments were rendered visible by stretching rabbit semitendinosus muscle using a split-fiber technique to a length where myosin and actin filaments no longer overlap. The titin filaments in the gap region were labeled by monoclonal anti-titin (RT13) followed by protein A-gold conjugates.
The identifying of intermediate filament-lamina-nuclear matrix system using immunogold labeling
Authors: Zhonghe Zhai , Renjie Jiao , Donglan Wu , Bo Zhang , Shutao Cai
Published: June 2020

The immuno — gold labelling technique was used in our work in combined with the use of monoclonal anti — keratin, vimentin, lamin A, and 298KD nuclear matrix protein antibodies to label the selective — extracted whole mount HeLa cells and BHK-21 cells.

The cells were grown on Formvar \carbon— coated nickle grids in Eagle’s MEM medium and extraction procedures was essentially according to Fey’s method with some modifications. Treatment first with 0.5 % Triton X — 100 to remove membrane system and some soluble proteins, then with 1 % Tween 40 and 0.5 % sodium deoxycholate to strip microtubules and microfilaments. DNase I and ammonium sulfate was added to remove chromatin and histone. For electron microscopy, above samples were prefixed in 0.25% glutaraldehyde, then incubated with monoclonal antibodies at 37 °C for 1hr, washed with PBS and then incubated with diluted protein A — gold solution at 37 °C for 1hr. The samples were conventionally fixed, dehydrated, dried through CO2 critical point.
Immunocytochemical methodology for the localization of neuronal antigens at the ultrastructural level
Authors: Pecci Saavedra Jorge , Connaughton Mark , José López Juan , Brusco Alicia
Published: June 2020

The use of antibodies as labels for the localization of specific molecules in the nervous systan has been extensively applied in recent years. Both monoand polyclonal antibodies or antisera have been employed. The knowledge of the organization of neuronal connectivities, gliovascular relationships, glioneuronal relationships and other features of nerve tissue has greatly increased.

A number of areas of the nervous systan have been analyzed in our laboratory, including the nuclei of the raphe system, the reticular formation, interpeduncular nucleus, substantia nigra, caudate nucleus, putamen, pallidum, spinal cord, pineal gland and others.

From a technical point of view, a number of variables needed to be taken into account in order to obtain reliable and reproducible results. The design of the optimal conditions of tissue fixation, embedding, sectioning, dilution of antibodies, and adaptation of Sternberger PAP technique were sane of the parameters taken into account to optimize the results. It is critical that each step of the technique be defined for each particular case.
High time resolution enzyme cytochemistry of rod outer segment
Authors: Saito Takuma , Takizawa Toshihiro
Published: June 2020

Cells and tissues live on a number of dynamic metabolic pathways, which are made up of sequential enzymatic cascades.

Recent biochemical and physiological studies of vision research showed the importance of cGMP metabolism in the rod outer segment of visual cell, indicat ing that the photon activated rhodopsin exerts activation effect on the GTP binding protein, transducin, and this act ivated transducin further activates phosphodiesterase (PDEase) to result in a rapid drop in cGMP concentration in the cytoplasm of rod outer segment. This rapid drop of cGMP concentration exerts to close the ion channel on the plasma membrane and to stop of inward current brings hyperpolarization and evokes an action potential.

These sequential change of enzyme activities, known as cGMP cascade, proceeds quite rapidly within msec order. Such a rapid change of enzyme activities, such as PDEase in rod outer segment, was not a matter of conventional histochemical invest igations.
An immunoelectron microscopic study of T-cell lymphomas, with special reference to Japanese adult T-cell lymphomas
Authors: Shamoto Mikihiro
Published: June 2020

A detailed ultrastructural morphology of antigen-antibody reactions using monoclonal antibodies is very valuable, not only for accurate diagnosis, but also for the understanding of the character of malignant lymphomas. This is a report of ultrastructural immuno-histochemical analysis of T-cell lymphomas, with special reference to Japanese T-cell lymphomas (ATL).

Biopsy specimens of lymph nodes from 30 patients with T-cell lymphomas, (including 20 patients with ATL) were used. Frozen sections of 10 u thickness were cut from specimens fixed in periodate-lysine-paraformaldehyde. The available antibodies were, OKT 3/Leu 4, OKT 4/Leu 3a, OKT 8, Tac, and FTF 148. The sections were placed on slides, and overlayed with appropriate diluted monoclonal antibodies and incubated for 2 hours. Then they were incubated with biotinylated horse anti-IgG for 2 hours, and in both an avidin-biotinylated peroxidase complex and a peroxidase mouse anti-peroxidase complex for 2 hours. They were fixed in 2% glutaraldehyde, then incubated with DAB hydrogen peroxide, and postfixed in 2% OsO4. The specimens were embedded in inverted gelatin capsules and cut with a diamond knife.
Immunolocalization of nuclear antigens in cryofixed cells which have been prepared by molecular distillation drying or freeze-substitution
Authors: Spector David L. , Derby Robert J.
Published: June 2020

Studies in our laboratory are involved in evaluating the structural and functional organization of the mammalian cell nucleus. Since several major classes (U1, U2, U4/U6, U5) of small nuclear ribonucleoprotein particles (snRNPs) play a crucial role in the processing of pre-mRNA molecules, we have been interested in the localization of these particles within the cell nucleus. Using pre-embedding immunoperoxidase labeling combined with 3-dimensional reconstruction, we have recently shown that nuclear regions enriched in snRNPs form a reticular network within the nucleoplasm which extends between the nucleolar surface and the nuclear envelope. In the present study we were inte rested in extending these nuclear localizations using cell preparation techniques which avoid slow penetration of fixatives, chemical crosslinking of potential antigens and solvent extraction. CHOC 400 cells were cryofixed using a CF 100 ultra rapid cooling device (LifeCell Corp.). After cryofixation cells were molecular distillation dried, vapor osmicated, in filtra ted in 100% Spurr resin in vacuo and polymerized in molds a t 60°C. Using this procedure we were able to evaluate the distribution of snRNPs in resin embedded cells which had not been chemically fixed, incubated in cryoprotectants or extracted with solvents.
A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology
Authors: Fritz Blayne , Naides Stanley J. , Moore Kenneth
Published: June 2020

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.

Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.
Immunocytochemical localization of xanthine oxidase in rat hepatocytes processed by quick freezing followed by freeze-substitution fixation
Authors: Ichikawa Misao , Ichikawa Atsushi , Nishino Takeshi , Nishino Tomako
Published: June 2020

Quick freezing of tissues, with freeze-substitution fixation, not only provides superior ultrastructural detail in cells and tissues but also retains soluble substances within their normal intracellular compartments. Combining this procedure with postembedding protein A-gold immunocytochemical technique permits precise localization of antigenic sites at the electron microscopic level.

Biochemical and cytochemical studies have demonstrated variously that xanthine oxidase, one of the flavoprotein enzymes involved in urate metabolism, is localized in cytozol, peroxisomal membrane, and/or peroxisomal crystalline cores of rat hepatocytes. This study is designed to determine the localization of xanthine oxidase in rat hepatocyte by high resolution immuno-electron microscopy.

Adult Wistar rats, 150-230 g, maintained on standard rat food pellets and water ad libitum were fasted for 12 h before sacrifice. To examine the presence of xanthine oxidase in peroxisomes in rat hepatocyte, some animals were treated with intramuscular injections of 250 mg (per day) of clofibrate, a hypolipidemic drug, for 10 days to generate peroxisome proliferation. Fresh tissue blocks were quickly frozen by the metal contact method, using liquid helium, and then freeze-substituted with either 2% OsO4 in acetone or 0.3% glutaraldehyde-acetone solution.
Cytochemical staining of connective tissue extracellular matrix with ruthenium hexammine trichloride (RHT)
Authors: Bray Douglas F.
Published: June 2020

The extracellular matrix (ECM) of fibrous connective tissue is a composite of protein- and carbohydrate-containing structures. Preservation of the protein components is achieved through fixation with glutaraldehyde and osmium tetroxide. Carbohydrates however are inadequately preserved and often lost using these fixatives. Cationic dyes such as ruthenium red, alcian blue, and several others have been used to stabilize and retain carbohydrates, particularly the proteoglycans (PGs), but usually with less than optimal results. The present study documents the improved preservation and staining of PGs and other carbohydratecontaining components of skin ECM using ruthenium hexammine trichloride (RHT).

Human neonatal foreskins were cut into 1 mm 3 blocks, fixed for 3 h in cacodylate buffered (0.1M, pH 7.2) 2.5% glutaraldehyde containing 1 mg/ml RHT (Polysciences, U.S.A.), postfixed 1 h in buffered 1% OsO4 also containing 1 mg/ml RHT, dehydrated in ethanol and embedded in Spurrs. In some cases the tissue was digested with chondroitinase ABC (Miles, U.S.A.) prior to fixation. Sections from tissue fixed without RHT were poststained in uranyl acetate and lead citrate. Figs. 2 - 5 are from 0.5 μm sections of tissue immunolabelled with monoclonal antibodies to type VI collagen.
Use of a novel colloidal gold probe to assess the distribution of Na,K-ATPase following covalent association in a variety of cell types
Authors: Bork Tracie , Mrsny Randall J.
Published: June 2020

Na,K-ATPase is an integral membrane protein complex capable of actively transporting Na+ and K+ ions. Function of this enzyme is essential for maintenance of cellular ion homeostasis and plasma membrane potential. Ouabain, a cardiac glycoside, has been demonstrated to selectively inhibits the transport ionic transport activity of Na,K-ATPase. Several soluble and membrane associated factors have been shown to modify the function of this transport enzyme by a variety of mechanisms. Recently, evidence has been obtained which suggests a movement of Na,K-ATPase from intracellular stores to the plasma membrane and back again, possibly in response to cellular events and in a fairly rapid time frame.

A number of methods including immunolabeling, tritiated ouabain autoradiography, and K+-dependent nitrophenylphosphate cytochemistry have been successfully employed to examine the distribution of Na,K-ATPase. We have used yet another approach, termed affinity-gold labeling, to localize the Na, K-ATPase of a variety of cell types. This approach, which employs a receptor-specific ligand and the potential for covalent association of the colloidal gold-containing probe to the receptor following association, has the added advantages of rapid identification of the labeled structure(s) and the potential for nearest-neighbor assessment for the receptor being labeled.
High-voltage cytochemistry for three-dimensional observation of nuclei and calculation of total numbers of nuclear pores in retinal Mueller glia
Authors: Ishigooka H. , Ueno S. , Hjelmeland L.M. , Landers M.B. , Ogawa K.
Published: June 2020

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.

Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).
Cell surface changes during aging of human bladder epithelium examined by lectin probes and SEM
Authors: Jacob J. , Ismail M.F.M.
Published: June 2020

Ultrastructural changes have been shown to occur in the urinary bladder epithelium (urothelium) during the life span of humans. With increasing age, the luminal surface becomes more flexible and develops simple microvilli-like processes. Furthermore, the specialised asymmetric structure of the luminal plasma membrane is relatively more prominent in the young than in the elderly. The nature of the changes at the luminal surface is now explored by lectin-mediated adsorption visualised by scanning electron microscopy (SEM).

Samples of young adult (21-31 y old) and elderly (58-82 y old) urothelia were fixed in buffered 2% glutaraldehyde for 10 m and washed with phosphate buffered saline (PBS) containing Ca++ and Mg++ at room temperature. They were incubated overnight at 4°C in 0.1 M ammonium chloride in PBS to block any remaining aldehyde groups. The samples were then allowed to stand in PBS at 37°C for 2 h before incubation at 37°C for 30 m with lectins. The lectins used were concanavalin A (Con A), wheat germ agglutinin (WGA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) at a concentration of 500 mg/ml in PBS at pH 7.A.
Analysis of Gene Expression Directed by a Thymic Locus Control in Transgenic Mice: Mechanisms and Application to Vectors for Gene Therapy
Authors: Aronow BJ , Ley CA , Ess KC , Witte DP
Published: June 2020

The formation of peripheral T cells from thymocyte progenitors is an intricate developmental process that requires the organized and coordinate expression of multiple genes. Adenosine deaminase (ADA) is an example of a gene that is subject to strong developmental regulation in T-cell precursors and is essential for the subsequent formation of T-cells in humans. We have sought to understand the mechanisms of ADA gene regulation from a basic point of view as well as to employ this to potential vectors for gene therapy.

Using transgenic mice have shown that the first intron of the ADA gene contains a powerful locus control region that directs high level gene expression within cortical thymocytes. Based on extensive mutational analysis of the regulatory region and analyses of gene expression that include quantitative gene expression, in situ hybridization, and biochemical characterizations of chromatin structure, we have demonstrated that the intronic locus control region (LCR) consists of a hierarchically structured 2300 base pairs of DNA sequence (Figure 1). The LCR is composed of a series of regulatory elements that include a centrally positioned 300 base pair classical enhancer domain within which there is a critical 30 base pair enhancer core. Within this core, there is a single binding site for the transcription factor c-Myb that is required for activity of the enhancer core, the enhancer, and the intact LCR. Beyond the 300 bp enhancer core on either side the LCR contains novel and puzzling 1 kb non-enhancer sequences that we have termed facilitators. These sequences enable gene copy proportional expression by facilitating the ability of the enhancer to function in chromatin. The effects of the facilitators are evidenced by their ability to allow for insertion-site-independent and gene-copy-proportional expression and they prevent variegated expression among similarly differentiated cell types (Figure 2). Thus, total gene expression does not indicate proper cell type specific expression. The facilitators also allow for the formation of a strong tissue and cell type specific DNAse I hypersensitive site at the enhancer. This suggests that the formation of a discrete organized chromatin structure as a function of developmental differentiation requires extensive DNA sequences, only some of which are of the enhancer type. The capabilities of the facilitators to activate a chromatin domain may also suggest their potential usefulness in vectors for gene therapy of both ADA deficiency and possibly other human genetic diseases. However, the distance and non-enhancer nature of the facilitators suggest that they may act differently than conventional regulatory elements. In support of this, the facilitators obey a strict position and orientation rules with respect to the enhancer.