bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlative transmission and high-resolution scanning electron microscopic studies on pituitary cells
Authors: Tai S.
Published: June 2020

Extensive cytological and histological research, correlated with physiological experimental analysis, have been done on the anterior pituitaries of many different vertebrates which have provided the knowledge to create the concept that specific cell types synthesize, store and release their specific hormones. These hormones are stored in or associated with granules. Nevertheless, there are still many doubts - that need further studies, specially on the ultrastructure and physiology of these endocrine cells during the process of synthesis, transport and secretion, whereas some new methods may provide the information about the intracellular structure and activity in detail.

In the present work, ultrastructural study of the hormone-secretory cells of chicken pituitaries have been done by using TEM as well as HR-SEM, to correlate the informations obtained from 2-dimensional TEM micrography with the 3-dimensional SEM topographic images, which have a continous surface with larger depth of field that - offers the adventage to interpretate some intracellular structures which were not possible to see using TEM.
Fixation and conductive staining by tannin-RuO4 for transmission and scanning electron microscopy
Authors: Takahashi Gen
Published: June 2020

Ruthenium tetroxide(RuO4) was first used as a stain in histology by Ranvier in 1887. Its use in TEM as a fixative or stain was reported by Gaylarde et al. However,the preservation of the cellular ultrastructure was very poor after RuO4 fixation alone, because RuO4 is a far more vigorous oxidant than osmium tetroxide. Peltari et al reported that a preceeding glutaraldehyde fixation helped to stabilize the ultrastructure, although the penetration of RuO4 into the tissue was poor.

In the present study, RuO4 has been found to be a useful fixative and staining reagent with the prerequisite of using RuO4 as a postfixative after prefixation with tannic acid (TA)-glutaraldehyde(GL) for thin-section TEM(TA-RuO4 method) or after preceeding osmication followed by TA mordanting for non-coating SEM(OsO4-TA-RuO4 method).

TA-RuO4 method for thin-section TEM
The polar development of membrane structure during spermiogenesis by freeze fracture and double replica technique
Authors: Liankai Dang
Published: June 2020

With the developing process from the spermatid to the sperm, the great changes of membrane structure are token place both in plasma membrane and nuclear membrane. This note will report the development of polarization in membrane and the distribution of nuclear pores and protein particles during spermiogenesis, and also compare the number of protein in P-face with that in E-face.

Mice, Rats, Rabbits and Dogs were operated by taking out the seminiferous tube. It was fixed in 2.5% glutaradehyde for 2 hrs 5 washed in cacodylated buffer, and then the sample was put in to 30% glycerol solution for 6-12hrs. Samples were put into liquid nitrogen rapidly and afterwards moved to BALZAR BAF-400D instrument to get Freeze-Fracture replica and double replica for observation with TEM.

The expression and the development of the polarization in membrane structure of the spermatid. The spermatid in different stepshas different characteristics and there is more polarization for membrane structure following spermiogenesis.
Sperm passage through the zona pellucida
Authors: Barros C. , Capote C. , Pérez C. , Crosby J. , Becker M.I. , De Ioannes A.
Published: June 2020

Sperm passage through the zona pellucida requires the occurrence of the acrosome reaction. This latter reaction, in turn, is supposedly needed to release the acrosomal enzymes, particularly acrosin which has been involved in the sperm binding to and penetration through the zona pellucida. The application of the immunogold technique using the monoclonal antiacrosin antibody ACR-C2E5 allowed us to identify acrosin in hamster, human and guinea-pig spermatozoa at the optic and scanning electron microscope, in which the immunogold labelling was silver enhanced. At the transmition electron microscope the monoclonal antibody was indirectly labelled with 10nm gold particles. In hamster spermatozoa there were three patterns of labelling: a) either no labelling, or an intense one over acrosomal region of apparently acrosome intact spermatozoa (Figures la,b, 2a & 3a); b) an intense labelling over the inner acrosomal surface and on the acrosomal cap of acrosome reacted spermatozoa (Figures lc,d, 2b & 3b); and c) either a slight labelling over the equatorial region or no labelling on acrosome reacted spermatozoa (Figure le,f,2c).
Crystalline-like structure in sperm membrane of Chinese Rhodeus sinensis, Acheilognathinae
Authors: Guan Ting-Lu , Yang Yi
Published: June 2020

It is well know that the spermatozoa are highly polarized cells. The membrane of mammals sperm have been studied in detail. The results have shown are markable diversity of structure with in an otherwise common place unit membrane. However, very few papers were dealt with teleost sperm membrane. Our privious report shows that Carassius auratus (Cyprinidae) sperm membrane have differentiated with regional specificity just like those in mammals. In general, intramembrane protein particles are highly organized at certain are as in the head plasma membrane forming crystalline-like structure, in contrast, rest areas of plasma membrane contain many random dispersed particles. The present study has shown the existence as well as the characteristics of crystalline-like structure in sperm membrane of Chinese Rhodeus sinensis, Acheilognathinae, Cyprinidae.

In spawing season the male fish are killed, apart of test is were took out and put into 2.5% glutaraldehyde buffered to pH 7.2 with 0.1mol/L sodium cacodylate.
Fine structure and possible functions of the hermaphrodite duct of some pulmonate snails (Mollusca: Gastropoda)
Authors: Hodgson Alan N.
Published: June 2020

The hermaphrodite duct of pulmonate snails connects the ovotestis to the fertilization pouch. The duct is typically divided into three zones; aproximal duct which leaves the ovotestis, the middle duct (seminal vesicle) and the distal ovotestis duct. The seminal vesicle forms the major portion of the duct and is thought to store sperm prior to copulation. In addition the duct may also play a role in sperm maturation and degredation. Although the structure of the seminal vesicle has been described for a number of snails at the light microscope level there appear to be only two descriptions of the ultrastructure of this tissue. Clearly if the role of the hermaphrodite duct in the reproductive biology of pulmonatesis to be understood, knowledge of its fine structure is required.

Hermaphrodite ducts, both containing and lacking sperm, of species of the terrestrial pulmonate genera Sphincterochila, Levantina, and Helix and the marine pulmonate genus Siphonaria were prepared for transmission electron microscopy by standard techniques.
Actin filaments in two spermatozoa of crustacea decapoda
Authors: Dupré E. , Schatten G.
Published: June 2020

Sperm of decapod crustaceans are formed by a round or cup-shaped body, a complex acrosome and one a few appendages emerging from the main body. Although this sperm does not have motility, it has some components of the cytoskeleton like microtubules, which are found inside the appendages. Actin filaments have been found in the spike of penaeidae sperms. The actual participation of the crustacean decapod sperm cytoskeleton during fertilization is not well understood. Actin is supposed to play an active role in drawing the penaeidae shrimp sperm closer to the egg after bending of the spike. The present study was aimed at the localization of actin filaments in sperm of the Robinson Crusoe island lobster, Jasus frontalis and in the crayfish Orconectes propincus, by fluorescent probes and low voltage scanning electron microscopy.
Electron microscope study of the effects of radiation on insect fertility
Authors: Ju-Hu Chiang Judy , Kuo-Cheng Chen Robert
Published: June 2020

Germ cells from the rice stem borer Chilo suppresalis, were examined by light and electron microscopy. Damages to organelles within the germ cells were observed. The mitochondria, which provide the cell with metabolic energy, were seen to disintegrate within the germ cell. Lysosomes within the germ cell were also seen to disintegrate. The subsequent release of hydrolytic enzymesmay be responsible for the destruction of organelles within the germ cell. Insect spermatozoa were seen to lose the ability to move because of radiation treatment. Damage to the centrioles, one of which is in contact with the tail, may be involved in causing sperm immobility.
The effect of freezing-thawing and washing on human spermatozoa: An Scanning and Transmission Electron Microscopy study
Authors: Ghoneim Shams , Moore Kenneth , Nessler Randy
Published: June 2020

Studies by different investigators have shown the difficulties in preserving the fine structure of human spermatozoa. Human semen, from fertile males, contains more abnormal forms than semen from other species. Artificial insemination has become widely accepted, and sperm banks are clinically established. It has been observed by researchers that, the quality and fertility of human semen is reduced following freezing and thawing. Malformed spermatozoa show either a single anomaly of each of their structural components-acrosome, nucleus and accessory structures-or a combination of these anomalies. Semen of infertile patients contains a higher percentage of abnormal forms than semen from fertile men. The examination of sperm cells in the SEM and TEM combined with other methods such as X-Ray microanalysis or immunocytochemical assays have not only led to a more accurate definition of the nature and intensity of the morphological defects, but also allows for detection of abnormalities unperceived with the light microscope.
Transmission Electron Microscopy of testes in mice treated with gossypol acetic acid
Authors: Majumdar S.K. , Khanna Amit
Published: June 2020

Recent studies have shown that gossypol acetic acid, a natural compound isolated from cotton seeds, is an effective and reversible male contraceptive in animals including humans. Gossypol has also demonstrated uncoupling activity in mitochondria and it is assumed that this is the major biochemical effect of the compound. Moreover, other studies have shown a variety of ultrastructural damage caused by gossypol in rat testes. Since little information on the ultrastructural effects of gossypol in mouse testes is available, this study was undertaken to evaluate the effects of the compound on testicular cells and sperm in DBA/2J mice.

Gossypol acetic acid purchased from the Sigma Chemical Co., St. Louis, Missouri, was used for this study. The compound was mixed in olive oil and was administered orally to four groups of mice. Each group received either 0 (control), 10, 20 or 40 mg/kg of the agent twice per week. The mice were sacrificed in pairs at 3, 6, 9 and 12 week intervals. Reversibility of gossypol was studied on mice allowed to recover for 15 weeks after the last treatment.
Ultrastructural analysis of collagen formation in the developing primate amnion
Authors: King Barry F. , Fry Grete N.
Published: June 2020

The amnion surrounding the mammalian embryo consists of the amniotic epithelium facing the amniotic cavity, a layer of extraembryonic mesoderm bordering the exocoelom and an intervening layer of extracellular matrix (Fig. 1). During gestation the amnion expands remarkably to acommodate the rapidly growing embryo. In this study we have examined the process of collagen fibril formation in the developing amnion of the rhesus monkey between 20 and 60 days of gestation.

Most cytological evidence of collagen fibril formation was observed in association with the extraembryonic mesodermal cells rather than the amniotic epithelium. The mesodermal cells h ad abundant cisternae of rough endoplasmic reticulum and a prominent Golgi apparatus. Elongated secretory vacuoles were associated with the Golgi apparatus and often contained parallel aggregates of fine filaments (Fig. 2). In some secretory vacuoles, periodic densities also were observed. Some striated collagen fibrils were observed in an apparent intracellular location in long, membrane-limited compartments (Fig. 3). Still other striated fibrils were observed in dense bodies, presumably lysosomes (Fig. 4).
STM of macromolecules
Authors: Allison D. P. , Thompson J. R. , Jacobson K. B. , Warmack R. J. , Ferrell T. L.
Published: June 2020

Beginning in 1982 when the first images were reported, the development of the scanning tunneling microscope (STM) has revolutionized microscopy in the 1980’s. Although the primary applications are for research on metal and semiconductor surfaces, success in imaging a number of biological samples, mounted on a variety of conductive surfaces, have established STM as a valuable emerging technology in biological research.

In 1987 we began our biological STM studies using tobacco mosaic virus (TMV), an easily identifiable rod shaped virus, to test the feasibility of using STM on naked biological samples. We deposited TMV by spraying an aqueous solution of the virus onto evaporated or sputter-coated palladium-gold (Pd/Au) films supported on flat mica surfaces. Although TMV could be clearly identified, its observed width was typically 70-100 nm instead of the known diameter of the virus, 18 nm. On evaporated Pd/Au substrates, tip traces revealed structures elevated above the substrate surface suggesting that the virus became coated with Pd/Au allowing normal conduction to occur. On sputter-coated substrates tip traces revealed depressed substructures.
Topological Locationzation of Epitopes on Hemocyanin by Molecular Immunoelectron Microscopy
Authors: Lamy Josette , Motta Geneviève , Boisset Nicolas , Taveau Jean-Christophe , Billiald Philippe , Lamy Jean
Published: June 2020

The intramolecular localization of an epitope on an antigen (AG) can be deduced from the architecture of the immunocomplex (IC) string. Indeed, the epitope is located in the surface portion of the AG in contact with the antibody (AB). The electron microscope is well adapted to the study of the IC architectures, and allows a direct topological localization of epitopes. In this work, the hemocyanin (Hc) of the scorpion Androctonus australis, a 1800 kDa protein comprising 24 polypeptide chains belonging to 8 different types, was used as the AG.

Sixteen epitopes were located on the Aa6 subunit using a double approach. First, the architecture of the IC strings composed of native 24-meric Hc and monoclonal antibodies (MABs) was studied by electron microscopy and image processing. Second, the relative positions of the epitopes were determined using IC containing the free subunit.

To determine the architecture of IC strings containing 24-meric Hc, whole Hemolecules were incubated with purified MABs specific for the Aa6 subunit, then the ICs were purified by gelfiltration on AcA 34, negatively stained with 2% uranylacetate, and observed in the electron microscope (Fig. 1).
Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles
Authors: Chestnut M. H. , Catrenich C. E.
Published: June 2020

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.
Scanning Electron Microscope Reveals Striking Differences in Cytopathic Effects of Equine Herpesvirus (EHV) Strains
Authors: Brako Emmanuel E. , Phaire-Washington L. , Brown Allison , Brako Norma
Published: June 2020

The phenomena of cytopathic effects (CPE) i.e., those virus-induced morphological changes in cultured cells observable at the level of light microscopy are commonly used in presumptive diagnosis of viral infections. The cytomorphological changes are similar for many viruses in the same Family or Genus. For example, in LLC-RK1 cell monolayers both EHV type 1-strain KyD (EHV-1) and EHV type 2-strain LK (EHV-2) cause focal cell rounding and polykaryocytosis which can be observed by phase-contrast microscopy (LM). To determine whether external and internal ultrastructural differences exist in LLC-RK1 cells (American Type Culture Collection # CCL 106) expressing CPE, cells were infected with each virus and processed for scanning electron microscopy (SEM).

Monolayer cultures of LLC-RK1 cells on 12 mm glass coverslips were inoculated with EHV-1 or EHV-2 at low input multiplicity of infection. After an hour’s adsorption at 37 C, residual inoculum was removed, and cultures were washed twice with serum-free medium and incubated in growth medium at 37 C.
Correlative Video Light, Immunofluorescence, and Electron Microscopy of Chromosome Attachment to the Forming Vertebrate Spindle: Implications for Force Production During Mitosis
Authors: Rieder Conly L. , Alexander Stephen P. , Hayden John , Bowser Samuel S.
Published: June 2020

This abstract summarizes a number of recently published observations from this laboratory on the formation of the mitotic spindle (1-3), and on the dynamic behavior of spindle microtubules (4).

Since chromosome movement during mitosis is, for the most part, directed towards a pole the emphasis of most mitosis research is to elucidate the molecular force-producing mechanism(s) behind this movement. Past studies reveal that poleward chromosome motion is dependent on the attachment of spindle microtubules (MTs) to the kinetochore of the chromosome. However, the origin of these MTs and their role in force production remain controversial. It is similarly unclear whether prometaphase and anaphase chromosomes are moved by the same, or a different, force-producing mechanism(s).

The mitotic spindle of cultured newt (T. granulosa) pneumocytes forms in an optically clear region of cytoplasm defined by a cage of keratin filaments (1). As in other cell types possessing an astral spindle, chromosomes in newt pneumocytes that are positioned closer to one pole at nuclear envelope breakdown initially form a monopolar attachment to, and move towards, that pole.
Visualization of the Cytoskeleton by Cryofixation, Immunoelectron Microscopy and 3-D Reconstruction Techniques
Authors: McDonald Kent
Published: June 2020

We are using various combinations of the following techniques to study cytoskeleton structure and function: cryofixation by high pressure freezing, double jet or plunge freezing; freeze substitution; cryosectioning of frozen hydrated or frozen substituted cells; EM immunolocalization; high voltage and conventional cryoelectron microscopy; serial sectioning (thick and/or thin); and computer-mediated 3-D reconstructions from serial sections.

One area where many of these tools and techniques are called into play is in the ultrastructural analysis of developmental mutant phenotypes in Drosophila. Staged embryos are ultrarapidly frozen by high pressure freezing then divided into three groups for further processing. One group is freeze-substituted in osmium-acetone then flat embedded and sectioned for examination by high voltage or conventional microscopy. This allows us to evaluate the quality of cryofixation and the percentage of well frozen embryos. From this material we will also cut serial sections which will be used to make 3-D models of the area of interest. The second group of frozen embryos will be put directly into a Reichert FC4 cryoultramicrotome and sectioned at -150 C. The cryosections are then examined in the cryostage of the high voltage EM. The third group is freeze-substituted in methanol plus aldehyde fixatives in preparation for immunological studies.
The Use of Noncryoprotected Frozen Tissue in Diagnostic Electron Microscopic Pathology
Authors: Minda J.M. , Lubensky I. , Pietra G.G.
Published: June 2020

Aldehyde fixatives are the reagents of choice to preserve human tissues for diagnostic electron microscopic examination. Since the need for ultrastructural studies is often unanticipated at the time of frozen sections, tissues are not routinely fixed in aldehydes. Alternatively, even if properly fixed, the sample may not contain representative lesions. This may require retrieval of tissue for electron microscopy from paraffin-embedded blocks or slides in which preservation of fine structure is markedly inadequate.

The present study was undertaken to evaluate the use of noncryoprotected frozen tissue for diagnostic electron microscopic evaluation. The tissues samples from the following lesions were available for study: bronchial carcinoid, duodenal somatostatinoma, granular cell tumor of breast, interstitial myocarditis (from an autopsy). The effect of the rapidity of freezing on ultrastructural preservation was studied in a normal rat liver. Each case had its own control in companion samples that had been routinely fixed by immersion in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate, pH = 7.4, (Karnovsky’s fixative).
Human Breast Carcinoma (Ultrastructural Observation of Ductal Carcinoma in 346 Patients)
Authors: Elliott Robert L. , Elliott Mary C. , Ledford Lindsay
Published: June 2020

Tissue from 346 patients with ductal carcinoma of the breast was fixed in Zambonis; post fixed in 1% OsO4; embedded in a Med Cast Resin; and thin sections were cut on an ultramicrotome. They were stained with uranyl acetate and lead citrate and examined in a Hitachi H-600 electron microscope.

Histologically, the tumors were intraductal and invasive. Ultrastructurally, lesions ranged from very well differentiated to extremely anaplastic with all gradations in between. Well differentiated tumors have large round nuclei with evenly dispersed chromatin and the cytoplasm contains tubular granular endoplasmic reticulum, free ribosomes and normal appearing ovoid and rod-shaped mitochondria (Fig. 1). The more anaplastic tumors have large irregularly shaped nuclei with clumped peripheral chromatin and very bizarre nucleoli. The cytoplasm has sparse endoplasmic reticulum and mitochondria are virtually absent, however, ferritin granules are abundant (Fig. 2 and 3). The anatomical presence of ferritin supports a biochemical study of tissue ferritin concentration in breast carcinoma.
Localization of Tumor-Associated Antigens C242 and C215 in Normal and Neoplastic Colon Mucosa
Authors: Sevéus Lahja , Ahlander Anders
Published: June 2020

Tumor-associated antigens are known as serum markers for clinical diagnosis of carcinomas. In particular, they are useful in the early discovery of relapse after chemotherapy or surgery. It is thought that antigens of this type could have prognostic significance as cell or tissue markers. However, knowledge on their distribution in cells and tissues is scanty. Therefore, we investigated tissue expression and subcellular distribution of two of these tumor-associated antigens by immunocytochemical techniques, using monoclonal antibodies (Mab C242 and Mab C215) raised in cell lines.

Colon tissue was obtained from 15 patients by surgical biopsy. The tissue was immediately fixed in 8% paraformaldehyde /0.2% glutaraldehyde in phosphate buffer pH 7.2. Subsequently the tissue was infused with 2.3 M sucrose and rapidly frozen in liquid nitrogen. Semi-thin (0.5 μm) and thin cryo-sections were cut on a cryoultramicrotome at -70°C and -110°C, respectively. We also used semi-thin and thin sections of tissue embedded in Lowicryl K4M at -35°C. Immunocytochemical labelling was carried out using 5 nm gold particles coupled to secondary antibodies (goat anti-mouse IgG). In the semi-thin sections, the gold label was enhanced with silver for light microscopical investigation.
Microvascular features that suggest effectors of blood-flow regulation in the brain: Evidence by SEM of corrosion casts of intracerebral vessels
Authors: Motti Enrico D.F. , Imhof Hans-Georg , Yasargil Gazi M.
Published: June 2020

Physiologists have devoted most attention in the cerebrovascular tree to the arterial side of the circulation which has been subdivided in three levels: 1) major brain arteries which keep microcirculation constant despite changes in perfusion pressure; 2) pial arteries supposed to be effectors regulating microcirculation; 3) intracerebral arteries supposed to be deprived of active cerebral blood flow regulating devices.

The morphological search for microvascular effectors in the cerebrovascular bed has been elusive. The opaque substance of the brain confines in vivo investigation to the superficial pial arteries. Most morphologists had to limit their observation to the random occurrence of a favorable site in the practically two-dimensional thickness of diaphanized histological sections. It is then not surprising most investigators of the cerebral microcirculation refer to an homogeneous network of microvessels interposed between arterioles and venules.

We have taken advantage of the excellent depth of focus afforded by the scanning electron microscope (SEM) to investigate corrosion casts obtained injecting a range of experimental animals with a modified Batson's acrylic mixture.
High-voltage electron microscopic studies of inflammatory cell attachment during blood-brain barrier inflammation
Authors: Lossinsky A.S. , Song M.J.
Published: June 2020

Previous studies have suggested the usefulness of high-voltage electron microscopy (HVEM) for investigating blood-bram barrier (BBB) injury and the mechanism of inflammatory-cell (IC) attachment. These studies indicated that, in evaluating standard conventional thin sections, one might miss cellular attachment sites of ICs in their process of attaching to the luminal endothelial cell (EC) surface of cerebral blood vessels. Our current studies in animals subjected to autoimmune disease suggest that HVEM may be useful in localizing precise receptor sites involved in early IC attachment.

Experimental autoimmune encephalomyelitis (EAE) was induced in mice and rats according to standard procedures. Tissue samples from cerebellum, thalamus or spinal cords were embedded in plastic following vascular perfusion with buffered aldehyde. Thick (0.5-0.7 μm) sections were cut on glass knives and collected on Formvar-coated slot grids stained with uranylacetate and lead citrate and examined with the AEI EM7 1.2 MV HVEM in Albany, NY at 1000 kV.
Potentiation of bromotrichloromethane hepatotoxicity in male rats exposed to 10 ppm dietary chlordecone: Electron Microscopic and biochemical study
Authors: Faroon O. M. , Henry R. W. , Soni M. G. , Mehendale H. M.
Published: June 2020

Previous work has shown that mirex undergoes photolytic dechlorination to chlordecone (CD) (KeponeR) in the environment. Much work has shown that prior exposure to nontoxic levels of CD causes potentiation of hepatotoxicity and lethality of CCl4, BrCCl3 and other halomethane compounds. Potentiation of bromotrichloromethane hepatotoxicity has been associated with compounds that stimulate the activity of hepatic mixed-function oxidase (MFO). An increase in the metabolism of halomethane by the MFO to a free radical initiates peroxidative decomposition of membranal lipids ending in massive cellular injury. However, not all MFO inducers potentiate BrCCl3 hepatotoxicity. Potentiation by much larger doses of phenobarbital is minimal and th at by a more potent inducer of MFO, mirex, is negligible at low doses. We suggest that the CD and bromotrichloromethane interaction results in a depletion of cellular energy and thereby reducing the cellular ability to undergo mitosis.
Ultrastructural changes in the respiratory system of mice after inhalation of mosquito-coil smoke
Authors: Yang V.C. , Lee H.R. , Chen C.S.
Published: June 2020

Mosquito coil is an anti-mosquito spiral commonly used in tropical and subtropical countries. Apart from the active gradient, bioallethrin, the major constituent of the coil is a mixture of organic compounds and wood dust. Combusion of these materials emits a very complex smoke which can reach the alveolar region of the lung easily. The biochemical and cellular changes in the respiratory system of rats after exposure to high dose of coil smoke have been reported. However, the low dose effects of the coil smoke have not been studied. In this experiment, asophisticated inhalation chamber for the simulation of room environment was designed to investigate the ultrastructural changes in the lung of mice afterexposure to low doses of mosquito coil smoke.

Male ICR mice aged 1 month were divided into 3 groups, an experimental, asham, and a non-treated control group, with 5-7 mice per group. The two former groups were exposed to the coils smoke, which lasted for 7 h daily, 6 days per week for 2 weeks. The inhalation chamber, measured 60 × 60 × 60 cm3, was installed with a ventilator. The speed of which was adjust to 0.6 m3/min to simulate the flow rate of air in an ordinary house. The control mice were housed in similar ventilation conditions.
Histogenesis of the mouse colonic mucosa with special reference to obliteration and re-opening of the colonic lumen
Authors: Chen Zhu-jun , Kataoka Katsuko
Published: June 2020

Temporary obliteration of the colonic lumen has been known prior to the formation of primary villi since an early light microscope (LM) study. We examined this process with a transmission electron microscope (TEM) using the descending colon of ICR mouse fetuses on days 13-18 of gestation (E13-E18, vaginal plug=E0). Pieces of the descending colon were doubly fixed in 2.5% glutaraldehyde and 1% OsO4 and embedded in an epoxy resin. Semi-thin sections stained with toluidine blue were examined by LM. Ultrathin sections doubly stained with uranyl acetate and lead citrate were examined by TEM.

On day E13,the colonic mucosa is lined with a single layer of undifferentiated epithelial cells (FIG. 1a), which were mutually joined by the primary junctional complex around the colonic lumen. Mitotic cells face the lumen and are connected with neighboring cells by the junctional complex. The epithelium looks stratified and the colonic lumen is extremely narrowed on days E15-E16 so that it cannot be identified by LM examination (FIG. 1b). A secondary junctional complex consisting of macula occludens with attaching microfilaments and desmosomes is formed between two epithelial cells apart from the colonic lumen. A cavity with microvilli is formed intracellularly and it subsequently opens with in the macula occludens of the secondary junctional complex to form the intra-epithelial cavity (FIG. 2, 3). A mitotic cell faces the intra-epithelial cavity and is joined with neighboring cells by these condary junctional complex (FIG. 4).
Purification of hepatitis C virus (HCV) using a new polycarbonate filter purification procedure
Authors: Miller M. F. , Lesniewski R. , Gagne G. D. , Peterson D. A.
Published: June 2020

For approximately 10 years we and others have repeatedly attempted to isolate and characterize the hepatitis C virus (HCV). Efforts to immune aggregate the putative virus, raise hyperimmune sera, or clone this illusive agent have been thwarted by the lack of availability of adequately purified virus. In this paper, we describe a new method for purifying HCV based on the sequential filtration of infectious chimpanzee blood plasma through a series of polycarbonate membrane filters of decreasing pore size (Nuclepore Corp., Pleasanton, Ca). Such filters have been used by others to size HCV and have previously been reported by us to be efficacious for purifying normal blood plasma seeded with known animal virus.

Chimpanzee blood plasma having an infectivity titer of 105 CID/ml (chimp infectious doses/milliliter) of HCV was purified as outlined in Fig. 1. Plasma was first clarified by low speed centrifugation for 20 minutes at 1000Xg in a tabletop centrifuge to remove large cellular debris and then sequentially pressure filtered through polycarbonate membrane filters having pore sizes of 3.0, 1.0, 0.8, 0.6, 0.4.and 0.2 um diameter.
Localization of constituent proteins of HIV Type 1 (HIV-1)
Authors: Goto Toshiyuki , Morita Chizuko , Ashina Taisuke , Ikuta Kazuyoshi , Kato Shiro , Nakal Masuyo
Published: June 2020

The localization of the constituent proteins of HIV-1 in the virions and HIV-1-infected cells was examined by indirect immuno-gold labeling with monoclonal antibodies (MoAb) to gag proteins (p18 and p24/p53), to elucidate viral formation of HIV.

Persistently HIV-1 (HTLV—IIIB,LAV-1)-infected MT-4 and MOLT-4 cell lines and their cloned cell lines were used as infected cells. Mouse MoAb against HIV-1 gag p18 (V17) and p24/p53 (V107) were used. The cells were fixed with 0.5-1% glutar-aldehyde in phosphate-buffered saline (pH 7.2), dehydrated in ethanol and embedded in epoxy (at 45° Cor 60°C) or Lowicryl K4M resin (at −30°C). The sections were incubated in MoAb at room temperature for 1 h and then incubated in anti-mouse goat IgG conjugated with gold (IgG-gold, 5 or 15 nm; Janssen) for 40 min. After being washed, the sections were stained with uranyl acetate and lead citrate, and were observed in an electron microscope with a tilting apparatus.

The spećific reactions with V17 and V107 were detected on HIV-1 particles and in the infected cells. No reactivity was noted between uninfected control cells and the MoAb, or between the infected cells and normal mouse serum. More than 97% of the HIV-1 particles embedded in epoxy resinat 60°C were labeled with gold after exclusion of the HIV particles that were attached to supporting film or completely embedded in the section (Fig. 1). Increased labeling was observed with Lowicryl (Fig. 2)and epoxy resin embedding at 45°C.
Immunogold localization of lactoferrin during neutrophil phagocytosis: A 3-D IVEM immunogold study on cryosections
Authors: Willard Andra S. , Lewis Jon C. , Thomas Michael J. , Taylor Richard G.
Published: June 2020

Colloidal gold-antibody conjugates have been used extensively as immunocytochemical markers for cell surface antigens and more recently for localization of intracellular antigens These latter applications have involved various pre- and post-sectioning procedures including etching of epon sections, use of water miscible resins, cell permeabilization and immunolocalization prior to embedding, and cryosectioning of frozen cells and tissues. For the most part, these techniques are limited to thin sections. Reported is the use of 3-D IVEM to localize the granule protein, lactoferrin, in thick cryosections using a modification of procedures originally reported for ultrathin plastic embedding. Human neutrophils, obtained from informed volunteers, were fixed for 90 min at room temperature either before or following a challenge with 0.8 μm diameter opsonized latex beads. The fixed cells were cryoprotected (2.3 M sucrose), frozen by immersion in liquid N2 and sectioned to 1.5 μm using a Reichert Ultracut E with FC4E cryo attachment. The sections were thawed, collected on carbon-stabilized, formvar-coated Ni grids.
Unique structure of rat lymph node HEV for the promotion of lymphocyte migration
Authors: Imada Masato , Dai Jibin , Sugiura Wataru , Yamashita Akira
Published: June 2020

Recently particular interest on the lymphocyte recirculation have been forcussed on the structural and functional natures of high-endothelial venules(HEV), in which large numbers of lymphocytes migrate physiologically across the vascular wall. In the present study, we have examined immunohistochemically and morphologically the microstructure of endothelium of HEV and the relationship between the endothelium and migrating lymphocytes, using thoratic duct lymphdrainaged rats which may permit us to investgate more easily their structures.

Male DA rats weighing between 160-240g were used. The animals were cannulated with polyethylene tubes thoratic duct using Gowans’s method, placed in the Bollman cages and the lymph drainage was maintained for 3days. For TEM, cervical lymph nodes were fixed, dehydrated through graded alcohols and embedded in Epoxy resin. Ultrathin sections were stained with aqueous uranyl acetate and lead citrate and examined on a JEOL JEM-200CX. For immunohistochemical study, frozen sections were cut and stained with the indirect immunoperoxidase technique using anti ICAM-1 (intercellular adhesion molecule) and anti HEV monoclonal antibodies (mAb).
The effects of dark rearing from birth until 30 days of age on the development of neurons and synapses in the superior colliculus and visual cortex of rats
Authors: Fukui Y. , Bedi K. S.
Published: June 2020

Rearing animals in dark conditions during early postnatal life has been shown to affect both the morphology and the normal functioning of the visual system. We have investigated the effects of rearing male rats in complete darkness from birth until 30 days of age, followed in some cases by a 35-day period of rehabilitation in “control” lighting conditions, on the synapse-to-neuron ratios in the superior colliculus and visual cortex. “Control” lighting conditions consisted of a room on a 12-hour red/12-hour light cycle. At 30 or 65 days of age, animals were killed, and the right superior colliculus and right visual cortex were dissected out and processed for embedding in Epon. The synapse-to-neuron ratios in the superior colliculus and visual cortex were estimated using a stereological technique, the “disector” method (Sterio, 1984). Neuronal nuclei were used as the counting unit for neurons and paramembranous densities for synapses.
Computer-assisted analysis of regenerating rabbit retinal pigment epithelium
Authors: Korte G. E. , Song M. J.
Published: June 2020

While examining thin sections of regenerating rabbit retinal pigment epithelium (RPE) we observed profound changes in ultrastructure as the cells matured. To assist in studying this problem serial thick sections (0.25 μm) through regenerating RPE cells were studied by highvoltage electron microscopy (HVEM) and a computer program (STERECON) used to generate stereo reconstructions.

Tissue was obtained from rabbits that received sodium iodate iv -- which destroys large expanses of RPE and permits examination of regeneration from surviving cells. One-two weeks after iodate administration the rabbits were euthanized, the eyes removed and tissue processed for transmission electron microscopy by routine procedures. Serial sections 0.25 μm thick were mounted on Formvar coated slot grids, stained with uranyl acetate and lead citrate and examined with an AEI EM7 1.2 MV HVEM at 1000 kV. Figure 1 illustrates observations made on six sections through the cell seen in Fig. 2. The cell was photographed at 5000x and printed as a montage at 15,000x.
An analytical determination and comparative synopsis of the vesicle content of the synaptic terminals in the dorsal lateral geniculate nucleus of the rabbit
Authors: Vázquez-Ortíz E. , Ríos A. , Caballero J.L.
Published: June 2020

We have determined the regression equations that rule the behaviour of the dependent variable, the area occupied by the synaptic vesicles, as a function of the independent variable, the area of the profile, for those profiles corresponding to the RLP, RSD, FI AND F2 synaptic terminals in the α sector of the dorsal lateral geniculate nucleus (dLGN) of the rabbit. The ultrastructural characteristics (Fig.1) are similar to those described by other authors in the cat and the monkey.

The rabbits used in this study were bred in captivity and were not albinos. After fixation by perfusion, the diencephalon was separated from the brain and cylinders were extracted with a 300 μm-diameter hypodermic needle in dorsoventral, anteroposterior and lateromedial directions. In this way we were able to obtain a set of samples distributed strategically throughout the α sector of the geniculate nucleus (α external dorsal, α external ventral, α external anterior, α external posterior, α external lateral and a internal regions, Fig. 2). The diencephalon and the extracted cylinders were treated in the normal ways for optical and electron microscopy, respectively.
Two subpopulations of ganglion cells in hydra are distinguished by an immunogold-double-labeling technique
Authors: Yu Cheng-Zhi , Westfall Jane A. , Grimmelikhuijzen C.J.P.
Published: June 2020

In the primitive nervous system of Hydra sensory cells and ganglion cells are easily distinguished by their location and distribution, but distinguishing subpopulations within these two groups of neurons is difficult. Both sensory cells and ganglion cells in Hydra and other coelenterates can be immunocytochemically stained with antisera against the molluscan cardio-excitatory neuropeptide FMRFamide as well as other peptides with the general structure < Glu... Arg-X-NH2, where X is an aromatic amino acid. Arg-Phe-amide (RFamide)-like peptides have be e n localized in densecored vesicles of ganglion cells in the peduncle of Hydra. but to date there is no ultrastructural evidence to distinguish between subpopulations of ganglion cells. Therefore, we used an immunogold-double-labeling procedure for electron microscopy with antisera to FMRFamide and RGamide (Arg-Gly-amide) to determine if these two peptides characterized different types of ganglion cells in the peduncle and lower part of the hypostome. In addition, we tested for colocalization of RFamide- and Antho-RFamide-like material in the same neuronal dense-cored vesicles.
The effect of a phorbol ester on gap junctions during amphibian myogenesis
Authors: Mi-bai (Tseng Ni-pai) Zeng , Mei-yun Zhou
Published: June 2020

It has been demonstrated that the treatment of a phorbol ester (TPA) interrupted the differentiation of amphibian muscle cells. In normal development concurrent with the differentiation changes from myoblasts to muscle cells, gap junctions also undergo a series of changes. The inhibitory effect of TPA on the intercellular commnication of cultured cells was reported by Yancey et al. among others. It is worth while to find out how TPA affect gap junctions in amphibian myogenesis.

Presumptive somite from mid-nurula of Cynops orientalis was extirpated and cultured in the medium used in our laboratory for 4 days, corresponding to the period when gap junctions were found to be at their highest incidence in normal development. For the experimental series, the explants were exposed to TPA (10 ng/ml) for the same length of time. Both the control and the TPA treated explants were fixed and processed for freeze-etching replicas. Careful searching for gap junctions were carried out.

Evident difference in the incidence of gap junctions were found between the control and the TPA treated series.
Organization of the cytoskeleton during cellularization in Drosophila melanogaster embryos: Confocal laser scanning and electron microscopy
Authors: Katoh Kazuo , Ishikawa Harunori
Published: June 2020

The three-dimensional organization of cytoskeletal components in the early Drosophila melanogaster embryos during cellularization was examined by confocal laser scanning microscopy of the whole embryos and thin section electron microscopy.

For confocal microscopy, Drosophila embryos at 2-3 hr after egg-laying were dechorionated and fixed with 8% paraformaldehyde-1% picric acid in 0.1M phosphate buffer (pH 7.2). Embryos were first blocked with normal goat serum, incubated with monoclonal antibody raised against Drosophila embryo α-tubulin for 4 hr, and then were incubated with FITC-conjugated anti-mouse IgG for 2 hr. Some embryos were stained with rhodamine-labeled phailoidin for F-actin visualization. After staining, the whole embryos were mounted on slide glass with an appropriate spacer and examined under the confocal microscope (Bio-Rad, Lasersharp MRC-500). For electron microscopy, dechorionated embryos were fixed with 1/2 Karnovsky's fixative followed by OsO4 fixation. To better preserve actin filaments, embryos were fixed with the same 1/2 Karnovsky's fixative containing 10 μM phailoidin and 0.1% saponin in 0.1M phosphate buffer, pH 7.2. Such fixed embryos were dehydrated and then embedded in Epoxy resin. Thin sections were cut and examined under a Hitachi H-800 type electron microscope.
The ultrastructure of the abdominal musculature of an orchestid amphipod
Authors: Levin Martin A. , Cale Lisa L. , Lynch-Holm Valerie
Published: June 2020

Orchestia is a genus of amphipod in the crustacean class Malacostraca. The order Amphipoda contains over 6000 species commonly called side swimmers, scuds and beach fleas(1). Most are marine bottom-dwellers utilizing their thoracic legs and posterior abdominal uropods for walking, crawling and swimming. However, some, like those in the genera Orchestia and Hyale are semiterrestrial. These amphipods, commonly referred to as “beach fleas,' “beach hoppers” or “sand fleas” can hop vigorously for great distances (up to 50 times their length) by extending their abdomens and telsons against the sand(2).

In our study, the ultrastructure of the dorsal muscle cord of Orchestia grillus was examined. Vogel(3) described the abdominal muscles of Orchestia cavimana as consisting of two groups of muscles: a strong, complex, dorsal muscle cord used mainly for hopping and a group of weaker, ventral, longitudinal and oblique muscles.

The specimens were collected in clumps of decaying seaweed and other detritus from the intertidal zone near the high water mark at Avery Point Beach, Connecticut.
The clathrin triskelion leg has an extensible proximal domain
Authors: Kocsis E. , Trus B.L. , Steer C.J. , Bisher M.E. , Steven A.C.
Published: June 2020

Clathrin is the major protein of coated membranes involved in receptor-mediated endocytosis and other membrane-trafficking reactions. The basic clathrin molecule, the triskelion, consisting of three heavy chains (190kDa) and three light chains (23-27kDa), polymerizes into a wide range of fenestrated polyhedral surface lattices. The capacity of triskelions to assemble into closed dodecahedral shells that are extravagantly polymorphic implies that certain degrees of freedom must be present in their molecular structure. To investigate this proposition, we have made a systematic study of the flexibility and the variability in leg-length exhibited by isolated triskelions.

Triskelions were isolated from bovine brain. Samples in 5mM Tris.HCl and 50% glycerol were nebulized on to freshly cleaved mica, rotary shadowed with platinum at an elevation angle of 8°, and lightly coated with carbon. The specimens were observed in a Philips EM400T electron microscope at a nominal magnification of 36,000x.