bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of spliceosomal UsnRNPs
Authors: Lührmann Reinhard , Behrens Sven-Erik , Kastner Berthold
Published: June 2020

The major snRNPs, Ul, U2, U4/U6 and U5, are essential trans-acting factors in the pre-mRNA splicing process. They assemble with a pre-mRNA and a number of other non-snRNP splicing factors prior to the splicing reaction to form an active spliceosome. We are interested in investigating the biochemical composition of UsnRNPs and their ultrastructure as well as their function in splicing. In HeLa cell nuclear extracts the spliceosomal UsnRNPs exhibit differential association behaviour depending on the salt concentration. Thus, at high salt (420 mM) the majority of the Ul, U2, U4/U6 snRNPs migrates on sucrose gradients at 10-12S, while U5 snRNP sediments at 20S. Under in vitro splicing conditions (i.e. at about 100 mM salt), U5 and U4/U6 snRNPs form a 25 S [U4/U6.U5]tri-snRNP-complex and U2 snRNPs sediment at about 17 S.

We have isolated the various types of UsnRNPs under native conditions using mainly immunoaffinity chromatography procedures. Today we can distinguish more than 35 distinct snRNP proteins. They can be grouped into two classes. The first class comprises eight common snRNP proteins which are present in each of the spliceosomal UsnRNPs. In addition, the individual snRNPs contain snRNP-specific proteins. These include three (70k, A, C) for the 12 S Ul snRNP, two (A′, B″ for the 12 S U2 snRNP, an additional eight for the 17 S U2 snRNP and eight for the 20 S U5 snRNP. The 25 S [U4/U6.U5]tri-snRNP-complex contains, in addition to the common proteins and the U5-specific proteins, a third group of six proteins which are essential for the stable formation of the tri-snRNP-complex. Thus, the different S-values of a particular snRNP particle result from differences in the population of snRNP-specific proteins associated with that particle.
Observation of neuronal activity using real-time voltage-sensitive dye imaging
Authors: Kauer John S. , Cinelli Angel , Wellis David , White Joel
Published: June 2020

Sensory systems are confronted with the problem of taking “information” in the outside world and encoding and manipulating it in forms that can be used in the neuronal world. A major challenge is to document how the transition between these worlds takes place (transduction) and, once it has taken place, how the data are manipulated by neural circuits (integration). Since the brain is an intrinsically parallel device, carrying out many functions simultaneously, it would appear as important to record brain activity in a similarly parallel manner as to record events in single cells and membranes. Optical recording of neuronal events offers a first step toward thing to observe events that are distributed among the cells and processes of a neuronal network.

In the sense of smell odors appear to be encoded by activity distributed across many neurons at each level of the system studied so far, from the sensory cells in the nose to the pyramidal cells in prepyriform cortex (for review see). Thus, to elucidate how the molecular properties of odorants are represented by neurons it is probably necessary to observe the patterns of distributed activation. The distribution of activity across many neuronal elements, in contrast to representing odor molecules by dedicated “labelled lines”, confers redundancy and fault tolerance on a system that is crucial for complex behaviors that underly survival for many animals species, as well as providing flexibility for being sensitive to large numbers of compounds.
Cell adhesion and cytoskeleton in the retinal pigmented epithelium
Authors: Opas Michal
Published: June 2020

The development and differentiation of tissues depend on the ability of cells to move and control their shape. In cell movement, a force generated by the contractile cytoplasmic machinery which includes the cytoskeletal microfilaments (MF) is, through cell-substratum adhesion, translated into traction. The chick embryonic retinal pigmented epithelium (RPE) differentiates in vitro, it can also be persuaded to transdifferentiate, and it displays differentiation dependent organization of the cytoskeleton, adhesiveness and ECM production.
High-voltage electron microscopy of subretinal scar formation
Authors: Korte G.E. , Marko M. , Hageman G.
Published: June 2020

Sodium iodate iv. damages the retinal pigment epithelium (RPE) in rabbits. Where RPE does not regenerate (e.g., 1,2) Muller glial cells (MC) forma subretinal scar that replaces RPE. The MC response was studied by HVEM in 3D computer reconstructions of serial thick sections, made using the STEREC0N program (3), and the HVEM at the NYS Dept. of Health in Albany, NY. Tissue was processed for HVEM or immunofluorescence localization of a monoclonal antibody recognizing MG microvilli (4).
Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex
Authors: Panté N. , Jarnik M. , Heitlinger E. , Aebi U.
Published: June 2020

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).
Visualisation of the interaction of a specific monoclonal antibody with the groel-like protein from Bordetella pertussis
Authors: Kessel M. , Gould-Kostka J.L. , Burns D.L.
Published: June 2020

We have characterised a protein from Bordetella pertussis, the whooping cough agent, and found it to be almost identical with the heat shock protein from E.coli, GroEL. These molecules, also termed chaperonins, are oligomeric proteins of approximately MW800.000 comprised of 14 subunits arranged in two rings of 7 subunits each, with a diameter of 15nm. When negatively stained, the GroEL-like molecules, examined in the electron microscope, are seen mainly in the top view clearly displaying the sevenfold symmetry, or as the less common side view with three characteristic electron dense striations separating four stain excluding regions (Fig. 1).
Linear and radial mass density of vitrified helical specimens determined by Scanning Transmission Electron Microscopy
Authors: Trachtenberg S. , Leonard K.R. , Tichelaar W.
Published: June 2020

We determined the linear and radial mass densities of vitrified bacterial flagella (Salmonella typhimurium), pill (Escherichia coli), Tobacco mosaic virus (TMV) and its RNA free coat protein (TMV-P) using STEM dark field images. Apart from their inherent importance and usefulness in the analysis of spherical particles, radial density functions may be potential substitutes for equatorial data in three-dimensional image reconstructions of helical particles. We explored this suitability, in terms of similarity between TMV STEM generated mass density functions and equators derived from X-ray diffraction.
Correction of the contrast transfer function to determine the radial density distribution of frozen-hydrated tobacco mosaic virus
Authors: Smith Michael F. , Langmore John P.
Published: June 2020

The purpose of image reconstruction is to determine the mass densities within molecules by analysis of the intensities within images. Cryo-EM offers this possibility by virtue of the excellent preservation of internal structure without heavy atom staining. Cryo-EM images, however, have low contrast because of the similarity between the density of biological material and the density of vitreous ice. The images also contain a high background of inelastic scattering. To overcome the low signal and high background, cryo-images are typically recorded 1-3 μm underfocus to maximize phase contrast. Under those conditions the image intensities bear little resemblance to the object, due to the dependence of the contrast transfer function (CTF) upon spatial frequency. Compensation (i.e., correction) for the CTF is theoretically possible, but implementation has been rare. Despite numerous studies of molecules in ice, there has never been a quantitative evaluation of compensated images of biological molecules of known structure.
Three-dimensional reconstruction of a mammalian Z-band from skeletal muscle
Authors: Schroeter J. P. , Goldstein M. A. , Bretaudiere J. P. , Sass R. L.
Published: June 2020

We have completed 3-d reconstructions of several regions of the Z-band in relaxed rat soleus muscle using the method of weighted back projection on a tilt series from two different longitudinal sections. Various displays of the reconstructions were interpreted after corrections for section shrinkage and comparisons to three dimensional models. Examination of cross-sections of the reconstructed Z-bands reveal that the lattice is in the small square form. We have previously shown that this form of the Z-band lattice is predominate in relaxed skeletal muscle. The reconstructions reveal that cross-connecting Z-filaments are arranged in opposing pairs along the axial filament. Successive pairs of filaments are rotated by ninety degrees about the axial filament, thus generating the four-fold appearance seen in the projected small square lattice.
Fluorescence in situ hybridization: Investigating the functional organization of genes and RNAs
Authors: Lawrence Jeanne Bentley , Carter Kenneth C. , Coleman John R. , Gerdes Michael , Johnson Carol V. , Xing Yigong
Published: June 2020

Developments in fluorescence in situ hybridization accrued over recent years now make it possible to detect and precisely localize nucleic acid sequences with a sensitivity sufficient to detect single copy genes or their primary transcripts (Reviewed in Ref.1). These advances have essentially revolutionized the field of molecular cytogenetics, providing not only a powerful approach to gene mapping and genetic diagnosis, but a new way to probe the higher level organization of the nucleus and chromosome. The methodology we and others primarily utilize is based on non-isotopic labelling of DNA probes with moieties such as biotin or digoxygenin, which are detected by fluorochrome-conjugated avidin or anti-digoxygenin antibodies. Alternatively, probes directly labeled with fluorochromes have recently become available. Appropriate modifications of protocols allow either cellular DNA or RNA to be targeted, and hybridization can be to intact cells, nuclei, or chromosomes.
Dynamic aspects of neuronal interphase nuclei: Visualization of subnuclear domains by phase contrast microscopy, confocal microscopy & ultrastructural cytochemistry
Authors: Boni Umberto De , Milankov Kosta , Amankwah Kwame S. , Park Paul C.
Published: June 2020

Neuronal chromatin moves, in a saltatory and periodic manner, within the 3-dimensional (3-D) space of interphase nuclei in vitro. This is generally known as nuclear rotation (NR) which has been proposed to function, during differentiation, in the transposition of specific chromatin domains into a cytotypic chromosome pattern, a pattern which, in part, may also be related to the functional state of the cell. Exposure of neurons in vitro to nerve growth factor or to neurotransmitters results in altered gene expression, in altered rates of NR, as well as in a reorganization of chromosome patterns. Moreover, long term potentiation, induced in neurons in hippocampal slices, reduces the number of detectable satellite DNA signals, possibly by increased clustering.
Light microscopy of living cells correlative to high-voltage EM and low-voltage SEM of cell cryo-whole-mounts
Authors: Malecki Marek
Published: June 2020

Analysis of motility phenomena in a living cell observed with light microscopy can be significantly enriched by preparing a whole-mount of this cell for high voltage electron microscopy (to reveal the intracellular organization) and for low voltage scanning electron microscopy (to reveal the surface topography). In earlier studies, cell whole-mount prepration by chemical fixation and drying was adequate for studies of slow cellular motions at the subcellular level (e.g. receptor movements). Fast cellular motions analysed at the supramolecular level (e.g. transmitter release, cytoskeleton reorganization) required development of much faster cryo-immobilization methods. However, in studies of cells grown on grids, these freezing methods involved time consuming transfer of these cells , from an incubator to a freezer, making impossible fine correlations between images of a living cell and its cryo-whole-mount. To overcome this constraint for correlative microscopical studies of neoplastic cell motility, I designed an instrument consisting of a freezer attached to a light microscope and allowing cryoimmobilization within miliseconds after recording. The main objective of the current project was refinement of an instrument and improvement of appropriate specimen cryo-preparation techniques.
Three-dimensional visualization of the eye
Authors: Masters Barry R.
Published: June 2020

The structure of the in situ rabbit cornea can be observed at high resolution and contrast with reflected light confocal microscopy. This paper describes the three dimensional reconstruction of the in situ cornea from an enucleated rabbit eye with confocal reflected light microscopy and volume rendering computer techniques. This paper shows the 3-D reconstruction of the full thickness of the cornea based on cubic voxels.
EM of human atherosclerosis: Aortic fatty streaks with transitional lesion features
Authors: Klemp K. Florian , Guyton J.R.
Published: June 2020

The earliest distinctive lesions in human atherosclerosis are fatty streaks (FS), characterized initially by lipid-laden foam cell formation. Fibrous plaques (FP), the clinically significant lesions, differ from FS in several respects. In addition to foam cells, the FP also exhibit fibromuscular proliferation and a necrotic core region rich in extracellular lipid. The possible transition of FS into mature FP has long been debated, however. A subset of FS described by Katz etal., was intermediate in lipid composition between ordinary FS and FP. We investigated this hypothesis by electron microscopic cytochemistry by employing a tissue processing technique previously described by our laboratory. Osmium-tannic acid-paraphenylenediamine (OTAP) tissue preparation enabled ultrastructural analysis of lipid deposits to discern features characteristic of mature fibrous plaques.
The distribution of “capsaicin-sensitive” nerve fibers in the lymphatic wall
Authors: Fruschelli C. , Guarna M. , Pucci A.M. , Alessandrini C. , Crestini F.
Published: June 2020

In a previous paper we have shown that guinea pig mesentery lymph collectors possess not only autonomic nerve fibers containing classical neurotransmitters but also peptidergic ones. These nerve fibers are sensitive to the depletory action of capsaicin, and are likely to be sensory in nature.

Therefore the aim of this study was to evaluate the effects of capsaicin of the nerve fibers of the lymphatic wall by ultrastructural methods and to study the distribution of the “capsaicin sensitive” nerve fibers in the lymphatic vessel wall.
Jansky-Bielschowsky syndrome, a lysosomal disease: First diagnosed cash in Oman
Authors: Buhl Line , Muirhead David
Published: June 2020

There are four lysosomal diseases of which the neuronal ceroid lipofuscinosis is the rarest. The clinical presentation and their characteric abnormal ultrastructure subdivide them into four types. These are known as the Infantile form (Santavuori-Haltia), Late infantile form (Jansky-Bielschowsky), Juvenile form (Batten-Spielmeyer-Voght) and the Adult form (Kuph's).

An 8 year old Omani girl presented wth myclonic jerks since the age of 4 years, with progressive encephalopathy, mental retardation, ataxia and loss of vision. An ophthalmoscopy was performed followed by rectal suction biopsies (fig. 1). A previous sibling had died of an undiagnosed neurological disorder with a similar clinical picture.
Ultrastructural evaluation of metastatic tumours in body fluid and fine needle aspiration biopsies
Authors: Winnett Marika
Published: June 2020

Metastatic tumours present a diagnostic problem especially when the primary site is unknown. In many cases it is not possible to determine the primary site by light microscopic examination of the cytology material (1,2). Ultrastructural evaluation can demonstrate special cell characteristic features which may help in providing an accurate and specific diagnosis of these tumours.
Silicone gel in biological systems
Authors: Greene William B. , Walsh Lyle G. , Silver Richard M. , Allen Joann , Maize John C.
Published: June 2020

Electron probe microanalysis of biopsies from two patients who had received silicone gel breast implants has revealed silicon (Si) in macrophages in an arthritic finger joint synovium (Fig. 1) and in a sclerodermatous skin lesion as well as in the fibrous capsule surrounding the implants in both patients (Fig. 2). The silastic envelope has been reported to be semipermeable with substances passing freely into and out of the implant. The polymer usually contains silica filler with a particle size of 30μm to impart added firmness, however, these sharp pointed crystals have not been fully characterized by Electron Microscopy. Silicone has been thought to be relatively inert, eliciting little or no tissue reaction. The substance has been injected or surgically placed into the human body as liquid, joints or in the form of breast augmentation prostheses. Recent reports have indicated that there is more than sufficient reason to change our thinking regarding this chemical and it's significance in biological reactions. There are 100,000 patients who undergo breast augmentation each year in the United States alone with over one million reported silicone implants. One clinical group reported that 4.4% of all new scleroderma patients had silicone breast implants. The patients reported in the study had implants from 2 to 21 years duration. The latency period may mean that scleroderma will increase parallel to the increase in breast augmentation over the last decade.
Substratum modification by endothelial cells (EC) in culture
Authors: Lewis J.C. , Hermanns I. , Grant K.W. , Evans S. , Gossen C. , Dekker A. , Kirkpatrick C.J.
Published: June 2020

Thrombosis resulting from blood interaction with prostheses is a concern following vascular grafting, and efforts have been made to endothelialize grafting materials and thereby reduce thrombogenicity. These efforts have focused upon altering polymer surfaces to enhance endothelial compatibility following surgery; however, using homologous EC attempts are being made to pre-seed grafting materials having defined surface characteristics. Such surfaces might be pre-absorbed with natural polymers as collagens, fibrin(ogen) or fibronectin. Irrespective of the starting character, these surfaces are rapidly altered following contact with tissues and body fluids, and as an extension by the EC themselves. To address this latter question, human umbilical vein endothelial cells (HUVEC) were seeded for whole-mouat microscopy onto carbon-stabilized, formvar-coated surfaces to which either collagen Type IV or its CB3 fragment had been adsorbed. Following culture for 3 or 24 hrs the cells were fixed in situ with glutaraldehyde, washed and processed for two-stage multiantigen immunogold microscopy. The first stage was comprised of rabbit anti-human fibronectin and mouse antihuman collagen Type IV. These were followed by second stage probes including goat anti-rabbit IgG (5nM gold), goat anti-mouse IgG (15 nM gold) and goat anti-human IgG (10 nM gold) as a control. HUVEC readily attached to and spread upon collagen IV and the CB3 fragment; the degree of spreading was much more pronounced with CB3 at both 3 and 24 hrs. The primary difference between the culture times was in cell density, with more cells at 24 than 3 hrs (Fig. 1). This difference could be explained by the additional generation time. The interaction of cells with the substratum was complex. This included deposition of proteins on the surface and extension of delicate macromolecular bridges from the cell periphery. Through the use of immunogold microscopy, fibronectin, collagen and immunoglobulin could be detected in all samples, including those plated in the absence of serum. Initially, collagen (IV and CB3) was more dense on the substrate than on the cells, and this was consistent with surface coating prior to cell seeding (Fig. 3). Fibronectin and IgG, on the other hand, were derived either from the medium or secreted from the cells, and each was associated with the cells or the cell edge (Fig. 4,5). Notably, detection of all three peptides was reduced over time, and this may be interpreted as secondary conditioning of the surface by HUVEC secreted products.
Dynamics of membranous organelles in the mitotic apparatus of two cell types: A live-cell, laser-scanning confocal study
Authors: Waterman-Storer Clare M. , Sanger Jean M. , Sanger Joseph W.
Published: June 2020

Membranous organelles comprise a large proportion of the living cell's cytoplasmic volume. In interphase, the endoplasmic reticulum (ER), mitochondria, and Golgi apparatus are characterized by a typical distribution in the cytoplasm. When cells enter M-phase, however, the cytoplasmic architecture undergoes dramatic rearrangements, as the nuclear envelope (NE) breaks down and the mitotic spindle assembles. During this process, the typical distribution of the membrane systems must be disrupted, so as to accommodate the cytoplasmic rearrangements. The role of membranous organelles within and around the mitotic apparatus has been a source of active investigation, as well as controversy. It is not known if membranes are an ubiquitous structural element of the spindle. Here, we present a laser-scanning confocal study of membrane dynamics in living mitotic cells' demonstrated by the fluorescent vital membrane dye 3,3″-dihexyloacarbocyanine iodide (DiOC6 (3)). By exploiting the thin focal planes achieved by the confocal microscope, the precise distribution of membranes within the spindle was attained in the two epithelial cell lines; PtK2 and LLC-PK. This study demonstrates that the distribution and quantity of cytoplasmic organelles within the spindle of living cells is cell-type specific.
Interrelationship of the cellular distribution and secretion of regular structure (RS) protein in Bacillus licheniformis nm 105
Authors: Zhu Xiaorong , McVeigh Richard , Ghosh Bijan K.
Published: June 2020

A mutant of Bacillus licheniformis 749/C, NM 105 exhibits some notable properties, e.g., arrest of alkaline phosphatase secretion and overexpression and hypersecretion of RS protein. Although RS is known to be widely distributed in many microbes, it is rarely found, with a few exceptions, in laboratory cultures of microorganisms. RS protein is a structural protein and has the unusual properties to form aggregate. This characteristic may have been responsible for the self assembly of RS into regular tetragonal structures. Another uncommon characteristic of RS is that enhanced synthesis and secretion which occurs when the cells cease to grow. Assembled RS protein with a tetragonal structure is not seen inside cells at any stage of cell growth including cells in the stationary phase of growth. Gel electrophoresis of the culture supernatant shows a very large amount of RS protein in the stationary culture of the B. licheniformis. It seems, Therefore, that the RS protein is cotranslationally secreted and self assembled on the envelope surface.
Ultrastructural evidence for a parasitophorous duct in human erythrocytes infected with malaria parasites
Authors: Schneider Timothy , Pouvelle Bruno , Taraschi Theodore F.
Published: June 2020

The human malaria parasite, Plasmodium falciparum, spends all but a few minutes of its 48 hour asexual life cycle in host erythrocytes (RBC). Models of the host-parasite complex, obtained by transmission and freeze-fracture electron microscopy, suggest that the intraerythrocytic parasite is contained in a continous, sealed vacuole (PVM). The parasite has an obligate nutritional requirement for serum fatty acids, lipids and proteins. Since mature erythrocytes do not endocytose, a long standing enigma has been the mechanism by which intracellular parasites obtain access to these (macro)molecules. Using a variety of fluorescent labeled macromolecules and confocal fluorescence microscopy (CFIM), we demonstrated that macromolecules in serum did not cross the erythrocyte or PVM, but rather gained direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Being limited by the resolution of the light microscope, we initiated TEM investigations to identify the duct at the ultrastructural level.
Improved preservation of freeze-dried biological specimens
Authors: Simon Martha N. , Lin Beth Y. , Wall Joseph S.
Published: June 2020

Specimens prepared by the wet-film technique (injecting unstained biological specimens into a drop of buffer on a thin carbon substrate which has never seen air, washing extensively, blotting to a thin layer of liquid, plunging the grid into nitrogen slush, and freeze-drying overnight) then visualized in the scanning transmission electron microscope (STEM) usually have reasonably wel1-preserved structures. However, there is a certain variability from day to day and sometimes even from one area to another on a given grid. This can occur for different reasons which may be inextricably related. The thin carbon film can be non-uniform at the molecular level with hot spots for strong attachment of some specimens, a part of a biological specimen may attach strongly while the rest of it thrashes about in Brownian motion ruining any perceivable structure, and the final thickness of liquid before freeze-drying may vary slightly which may affect the preservation of the structure.
Some quantitative applications of vascular corrosion casting
Authors: Hossler Fred E.
Published: June 2020

Preparation of replicas of the complex arrangement of blood vessels in various organs and tissues has been accomplished by infusing low viscosity resins into the vasculature. Subsequent removal of the surrounding tissue by maceration leaves a model of the intricate three-dimensional anatomy of the blood vessels of the tissue not obtainable by any other procedure. When applied with care, the vascular corrosion casting technique can reveal fine details of the microvasculature including endothelial nuclear orientation and distribution (Fig. 1), locations of arteriolar sphincters (Fig. 2), venous valve anatomy (Fig. 3), and vessel size, density, and branching patterns. Because casts faithfully replicate tissue vasculature, they can be used for quantitative measurements of that vasculature. The purpose of this report is to summarize and highlight some quantitative applications of vascular corrosion casting. In each example, casts were prepared by infusing Mercox, a methyl-methacrylate resin, and macerating the tissue with 20% KOH. Casts were either mounted for conventional scanning electron microscopy, or sliced for viewing with a confocal laser microscope.
In situcryofixation of lung tissue using the PS1000, a hand-held metal mirror cryofixation device
Authors: Godleski John J. , Stearns Rebecca C. , Katler Marshall , Rufner Robyn , Sweeney Theresa D. , Lightfoot Fred G.
Published: June 2020

We have previously reported methods to cryofix the extracellular lining layers of the large airways and trachea. We seek to study the extracellular lining layers of both airways and alveoliin situbecause it is the interaction of these lining materials with inhaled particles that determines the subsequent responses of phagocytic cells to the particles. Cryopreservation of the lining fluids of lung parenchyma is difficult because of the complexity of distal lung structure, and the need to fix the lung in its natural state, i.e., filled with air. Any method used must be able to obtain the frozen specimen from the inflated lung. This requirement precludes the possibility of having a cryogen come into primary contact with the internal surface of an alveolus. However, if optimal fixation through the pleura could be attained, then the lining layers of subjacent alveoli could be studied. In this report, we describe a new method to cryopreserve distal lung tissue for optimal study of the extracellular lining layers of the alveolus. Alveolar tissue is sufficiently preserved in the inflated state to maintain itsin vivocharacteristics.
Alternatives to traditional epoxy embedding “kits”: Novel choices
Authors: Mascorro J.A. , Kirby G.S.
Published: June 2020

Epoxy resin embedding “kits” invariably are offered as one epoxy resin coupled with the hardners dodecynyl succinic anhydride (DDSA), nadic methyl anhydride (NMA) and the amine catalyst 2,4,6-Tri(dimethylaminomethyl)phenol (DMP-30). This well-known mixture has been used widely for many years, perhaps in part because it is offered in the form of a convenient kit. Microscopists should be aware that packaged kits, eventhough they produce successful results, possess a never-changing high viscous character that could compromise optimum infiltration or damage delicate specimens. Past studies have shown clearly that the kit compnents DDSA and DMP-30 are particularly viscous compounds. Replacing DDSA with the less viscous nonenyl succinic anhydride (NSA), and substituting either dimethylaminoethanol (DMAE) or benzyldimethylamine (BDMA) as the catalyst of choice in place of DMP-30 will significantly reduce the viscosity of the overall mixture while producing easily manageable media that maintain fluidity longer, insure optimum infiltration, and ultimately produce results equal to those obtained with traditional kits.
High-pressure freezing and freeze substitution of plant tissue
Authors: Sharp William P. , Roberson Robert W.
Published: June 2020

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.
Use of the tannic acid/para-phenylenediamine lipid cytochemical technique for SEM of atherosclerotic endothelium
Authors: Robinson K.A. , Apkarian R.P.
Published: June 2020

A cytochemical technique for the preservation and visualization of neutral lipid in atherosclerotic tissue was developed by Guyton and Klemp.This tannic acid/para-phenylenediamine (TA-PDA) procedure prevented the extraction of extracellular lipid by dehydrating solvent, which permitted it to be imaged in the aortic subendothelium by TEM. We applied the technique to enable documentation by SEM of lipid particles associated with the luminal surface of aortic endothelium during experimental atherogenesis. Preliminary results in a porcine model had suggested that a lipid exocytic, or retro-endocytic, process might occur in chronic hyperlipidemia.
TEM of the catalytic activity of adenosine monophosphate deaminase in murine lung
Authors: Bielat K. , Tritsch G.
Published: June 2020

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The chloro-analog of AMP, i.e., 6 Chloropurine riboside 5’ monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which 1iberates Cl− which is precipitated with added Ag+, and, after exposure to light electron dense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10μM pentostatin (deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this enzyme. Because many cells have ecto 5’-nucleotidase activity, pentostatin was added to prevent manifestation of adenosine deaminase activity of dechlorination of any nucleoside that might be formed.
Role of PKC isozyme III (γ) in water transport in amphibian urinary bladder
Authors: Mia A.J , Oakford L.X. , Thompson P.D. , Ning Z.H. , Yorio T.
Published: June 2020

Vasopressin stimulated water flow across renal epithelia is thought to occur through a V2 receptor coupled to adenylcyclase. The increase in water flow occurs as a result of a fusion of water channels with the apical membrane and is indicative of an increase in membrane capacitance following hormone addition.

What controls the cycling of water channels and their insertion into the membrane is uncertain. Our laboratory has demonstrated that renal epithelia as well as amphibian urinary bladder membranes, contain a vasopressin V1 receptor which upon activation results in the breakdown of phosphoinositide and the formation of inositol triphosphate and diacylglycerol, the latter an activator of protein kinase C (PKC). The initiation of transepithelial water flow also appears to involve V1 receptors and possibly activation of PKC. To test this hypothesis, we have been using activators of PKC, such as phorbol esters and mezerein, as pharmacological tools to determine if PKC activation results in similar physiological responses as the hormone. Several PKC isozymes, upon activation, are known to be translocated to the apical membrane as visualized by FITC immunofluorescence. Previously, we reported co-localization of PKC subtypes I (γ) and II (β) in toad urinary bladders using monoclonal antibodies and protein A-gold probes. This report includes the localization of PKC subtype III (α) and its distribution pattern using immunogold labeling.
EM localization of chromaffin cell ATPase
Authors: Kriho V. , Pappas G. D.
Published: June 2020

During exocytosis of the chromaffin granules, ATP is released. ATP can then be hydrolyzed by the ecto-ATPases of the plasma membrane to provide adenosine for reuptake or for activation of P1purinoceptors. Chromaffin granule membranes also possess ATPase activity. This activity is linked to the uptake of catecholamines from the cytoplasm into the membrane-bound granule compartment.

In this report we combine EM cytochemistry and immunogold labelling to provide further evidence for the presence of ATPase on both the plasma membrane and granule membranes of bovine chromaffin cells in culture.
Detection of 1-nm immunogold labels with a field-emission TEM/STEM
Authors: Otten Max T. , Stierhof York-Dieter , Humbei Bruno M.
Published: June 2020

The size of the gold labels is known to be an important factor affecting immunogold labelling efficiency. In many cases it has been found that the smaller the label, the better the efficiency. Ultra-small gold colloids were therefore introduced to increase the sensitivity of the immuno-gold labelling technique. Such ultra-small labels are difficult to detect in the regular TEM image and for efficient detection silver enhancement is needed to enlarge the gold labels. Unfortunately, the reliability of the various silver-enhancement methods is poorly known. Possible problems are the lack of control over reproducibility, homogeneity and efficiency of the enhancement and the possibility of spontaneous nucleation of silver particles without a gold precursor (non-specific silver precipitation) and merging of several gold labels into a single silver particle. An assessment of these factors requires a technique that allows visualisation of the unenhanced ultra-small gold labels as well as silver-enhanced ones, prefereably in relation to the tissue morphology.
Time-lapse x ray microscope movie of the germinating garden pea seed
Authors: Newberry Sterling , Vozzo J. A. , Marko Michael
Published: June 2020

The plant embryo is relatively insensitive to x-rays. One should like to take advantage of this property to follow the early processes of seed germination by time-lapse x-ray microscopy. Preliminary work has shown that radiation exposure can be reduced by two orders of magnitude if a minimum number of frames are exposed and the movie then generated by slowly dissolving one frame into the next. Image processing also helps the problem of low contrast in the living image. Present work has been directed against the problem that geotropism makes the embryo grow out of the plane of the picture (the instrument does not operate horizontally). The approach taken should also simplify the problems of maintaining a suitable environment for the seed and make the instrument available between exposures when working with seeds which have long germination times. The garden pea was chosen as a test material because of its environmental tolerance, short maturation cycle, and similarity to pine seeds in size and radiation tolerance.
Localization of silica in Equisetum arvense (horsetail)
Authors: Reine Barbara A.
Published: June 2020

Silica is deposited as amorphous silica gel in the shoots (as well as in leaves, roots and reproductive structures) of many vascular plants such as grasses and cereals (notably rice and oat), hemp, pea, nettle, palms, sedges, and horsetails. Of this group, the horsetails, grasses and sedges, exhibit an especially pronounced capacity to take up large amounts of silicon as silicic acid from the soil and polymerize it as hydrated silica in various tissue locations and sometimes specialized cells (3). The application of this naturally produced silica and other minerals to technology, has stimulated a renewed and refocused interest in biomineralization (2). Historically, the silica-impregnated shoots of horsetails (Equiserum) were used by pioneer settlers for scouring dirty dishes and as a substitute for sandpaper. Today, rice hulls, whose silica content can exceed 20 wt.%, are pyrolyzed commercially to produce silicon carbide whiskers and platelets for use in composites. Rice hulls are also used as fuel in co-generation plants in rice-growing regions of the country but if combustion takes place in certain oxygen concentration and temperature regimes, crystalline “free silica” forms to a substantial extent and. if discharged as respirable dust, may present a significant public health risk.
Electron microscopy and laser scanning microscopy of high-pressure frozen freeze-substituted samples of Sordaria humana
Authors: Czymmek Kirk J.
Published: June 2020

The process of spore formation (ascosporogenesis) has not been clearly defined in ascomycete fungi. Many conflicting reports exist concerning details of ascospore initial development. The relatively slow fixation (up to several minutes) of conventional chemical techniques does not adequately preserve many structures for which ultrarapid freezing techniques (within milliseconds) are particularly suited. High-pressure frozen freeze-substituted samples of the ascomycete Sordaria humana were thick-sectioned for laser scanning microscopy (LSM) and thin-sectioned for transmission electron microscopy (TEM). Perithecia were too large (up to 500μm in diameter) to freeze adequately using plunge or propane-jet freezing techniques. The LSM was used to examine portions of the perithecia which were too large for viewing by TEM without taking numerous low magnification micrographs.
Calcium uptake and changes in membrane morphology of CL. sporogenes bacterial spores
Authors: Panessa-Warren B.J. , Tortora G.T. , Warren J.B.
Published: June 2020

The bacteria comprising the genus, Clostridium, cause many severe infections in humans (tetanus, gas gangrene, botulism), and are particularly virulent and difficult to kill. Like the other spore forming bacteria, Clostridial endospores have a high calcium content (1-15% by wt), which is believed to play a role in the resistivity of this spore to sporocides. This paper presents a series of experiments which compare normal clostridial spore morphology and ultrastructure, to the morphology of identical spores treated to reveal sites of calcium deposition; study the effects of calcium depletion; and determine by TEM and SEM if the spores have the capability to alter their shape or respond to their environment to facilitate infection.
Multiple mineral precipitations inside magnetotactic microorganisms
Authors: Farina M. , Lins U. , de Barros H.G.P. Lins , Broderick R.
Published: June 2020

Magnetotactic microorganisms have been described mainly as swimming magnetic bacteria (MB) from natural waters which produce magnetite crystals (1) or as the magnetotactic multicelluar aggregate (MMA) producing the iron sulphides greigite, pyrite (2) and possibly pyrrhotite(3). Also regions containlng Iron and oxygen were described in MMA but with no complete analytical information (3). We present here, some new aspects on mineral precipitations inside both MB and MWAs.
3-D analysis of cellular lipid metabolism: Reconstruction of the tubular network
Authors: Jerome W.G. , McEwen B.F. , Minor L.K. , Rothblat G.H. , Glick J.M.
Published: June 2020

A predominant feature of early atherosclerotic lesions is the presence of macrophages and smooth muscle cells in which large quantities of lipid have accumulated. This lipid occurs within both small, cytoplasmic, cholesterol ester droplets and large, lipid-swollen secondary lysosomes. Time resolved studies suggest lysosomal engorgement promotes the disease progression. Using cultured cells we have shown that lysosomal engorgement in smooth muscle cells is accompanied by the elaboration of an extensive network of tubules. The tubules are rich in acid hydrolases and appear to be an extension of the trans Golgi network. As part of our effort to understand the causes and significance of lysosomal lipid accumulation we have undertaken to characterize the extent and development of the tubular network. The tortuous nature of the network makes it difficult to visualize by conventional thin section electron microscopy. Thus, intermediate voltage electron microscopy of thick (1-3 um) sections in which the tubules have been contrasted using acid phosphatase cytochemistry has been essential to these studies. Cultured rabbit smooth muscle cells were incubated with sonicated lipid droplets composed of 72% cholesteryl oleate, 25% triolein, and 3% phospholipid. Prior to incubation, cells had no visible lipid stores and 92% of cells showed no acid phosphatase-containing tubules. In cells with tubules, the tubules occupied less than 0.1% of the cell volume. In contrast, after 4 h of exposure to droplets 81% of cells showed lysosomal lipid accumulation with 67% exhibiting extensive, interconnecting, acid phosphatase positive tubular networks. Although stereo-pair images provided much information about the extent of the network (figure 1), the exact relationship of the network to lipid-engorged lysosomes was somewhat ambiguous due to superposition of objects. In order to clarify the relationship of network to lysosome, a tomographic reconstruction was computed from an IVEM tilt series encompassing +/− 60° in 2° increments. Images were digitized using a DAGE model 81 video camera. Rotational alignment using fiduciary markers was accomplished using the algorithm described by Lawrence. Translational alignment and weighted back-projection reconstruction were carried out as described by McEwen. Volumes were rendered using a PIXAR computer graphics workstation. The reconstruction (figure 2) revealed that extensions of the network made contact with the lipid-swollen lysosomes suggesting that the network might transfer hydrolases directly to secondary lysosomes.
Temporal and spatial interrelationships of confronting cisternae to nuclear envelope
Authors: Palisano John R.
Published: June 2020

Although confronting cistemae (CC) have been observed in a variety of tumor cells and normal fetal rat, mouse, and human epithelial tissues, little is known about their origin or role in mitotic cells. While several investigators have suggested that CC arise from nuclear envelope (NE) folding back on itself during prophase, others have suggested that CC arise when fragments of NE pair with endoplasmic reticulum. An electron microscopic investigation of 0.25 um thick serial sections was undertaken to examine the origin of CC in HeLa cells.
Convergent-beam electron diffraction studies of domains in tetragonal phase of lead zirconate titanate ceramics
Authors: Dass M.L.A.
Published: June 2020

Lead zirconate titanate Pb(ZrxTi(1-x))O3(PZT) ceramics are ferroelectrics formed as solid solutions between PbTiO3and PbZrO3. Among the different phases in the ferroelectric state, the primary ones are the Ti+4rich tetragonal phase and the Zr+4rich rhombohedral phase. The coexistence of both T and R phases at the boundary composition has been reported using the convergent beam electron diffraction method. In an attempt to characterize the ferroelectric domains in the different phases, a study on the tetragonal phase is reported here, as such an analysis is useful in identifying the phases at the phase boundary.

The ceramic used in this study was prepared by conventional ceramic processing and the composition of the sample examined was Pb(Zr0.55Ti0.45)O3. The structure has been found to be tetragonal with lattice parameters a=4.0155Å and c=4.1033Å using X-ray diffraction studies of powder samples.