bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquisition and analysis of fluorescence ratio images using a multispectral microanalytical imaging program
Authors: Freudenrich Craig C. , Boyle Robert T. , Kopf David A. , Ingram Peter , LeFurgey Ann
Published: June 2020

Any study regarding the influence of physiological, pathological, or pharmacological/toxicological factors on cellular ion homeostasis requires knowledge of multiple parameters including cytoplasmic ion concentrations, ion fluxes, and subcellular elemental distribution so that an accurate assessment of the state of ion homeostasis can be obtained. These multiple parameters are often obtained by using different types of microscopy and microanalysis. For example, cytoplasmic ion concentrations are measured by fluorescence digital imaging microscopy using various ion indicators, while cellular and subcellular element contents are determined by electron probe X-ray microanalysis (EPXMA) digital imaging. For correlating information from various microscopies, it would be advantageous to have one software package and image format that operates on the same computer platform. Here, we describe the modification of an existing software (ImagNSpect) developed for EPXMA digital imaging for use in analyzing fluorescence digital images.

Fluorescence images (490 and 440 nm excitation, 540 nm emission) of BCECF-loaded cultured rat hepatocytes (Fig. 1a, b) were obtained on an inverted fluorescence microscope (Carl Zeiss, Inc., Thorn wood, NY) equipped with a rotating excitation filter wheel, a CCD camera (Photometries, Ltd., Tucson, AZ), a controller box, and a Macintosh Quadra 800 computer (Apple Computer Corp., Cupertino, CA).
Light and Electron Microscopic localization of inducible nitric oxide synthase in the heart of rats with myocarditis
Authors: Nishikawa T. , Ishiyama S. , Takeda K. , Shimojo T. , Hiroe M. , Kasajima T.
Published: June 2020

Nitric oxide synthase (NOS) is an enzyme involved in the synthesis of nitric oxide (NO). The inducible form of NOS (iNOS) is induced in the presence of cytokines and produces massive amounts of NO which may be harmful to living tissue. In acute myocarditis, a serious disease which can be fatal, extensive myocardial cell damage associated with massive inflammatory cell infiltration is usually observed and large amounts of cytokines are released from inflammatory cells. However, the mechanism of the heart tissue injury is still unclear. In this study, we investigated the role of iNOS in the myocardial tissue injury in acute myocarditis.

The heart tissue specimens were taken from Lewis rats with experimentally induced autoimmune myocarditis. An immunohistochemical study was performed using iNOS antibody raised in New Zealand white rabbits immunized with synthetic peptides (17 amino acids) corresponding to the C-terminal of macrophage iNOS. The specificity of the antibody was confirmed by Western blot analysis. Immunoelectron microscopic studies were carried out by the gold-labeled IgG method using ultrathin sections of LR white resin-embedded material.
The molecular structure of thiol-monolayers on gold imaged with an Atomic Force Microscope
Authors: Jaschke M. , Wolf H. , Schönherr H , Ringsdorf H. , Bamberg E. , Butt H.-J.
Published: June 2020

Thiols spontaneously form monolayers on gold. An important question is which factors determine the structure of the thiols. The two extreme positions are: (a) the structure of the underlying gold determines the packing of the thiol molecules; (b) the geometry of the thiol molecules and the interaction between thiols determine their two-dimensional structure.

To help answer this question two series of thiol molecules on gold (111) were imaged by atomic force microscopy. In order to reduce the force between tip and sample, and to prevent distortion of the thiol monolayer, all images were taken in ethanol at forces between 0.5 nN and 3 nN.

The first series consisted of several azobenzene-thiols:

Although these molecules possess a terminal alkyl chain, their lattice is different from the well known hexagonal (√3 × √3) R 30° lattice (with respect to the underlying gold (111) lattice) of alkane-thiols. Instead, we found two stable rectangular lattices which could both be described by lattice constants of 0.61 nm and 0.79 nm, and an angle of 89°. These structures were also found on polycrystalline gold. Also, varying the length of the alkyl chain (molecules (i) and (ii)) or using a disulfide instead of a thiol (molecules (ii) and (iii)) did not change the lattice constants. Thus we conclude that the lattice for azobenzenethiols is end-group dominated.
Measuring forces between biological macromolecules with the Atomic Force Microscope: characterization and applications
Authors: Lee Gil U , Chrisey Linda , Colton Richard J.
Published: June 2020

Structure and function in biological macromolecular systems such as proteins and polynucleotides are based on intermolecular interactions that are short ranged and chemically specific. Our knowledge of these molecular interactions results from indirect physical and thermodynamic measurements such as x-ray crystallography, light scattering and nuclear magnetic resonance spectroscopy. Direct measurement of molecular interaction forces requires that the state of a system be monitored with near atomic resolution while an independent force is applied to the system of 10−12 to 10−9 Newton magnitude. The atomic force microscope (AFM) has recently been applied to the study of single molecular interactions. The microfabricated cantilever of the AFM, a force transducer of small yet variable stiffness and high resonance frequency, produces a transducer of 10−15 N/Hz1/2 force sensitivities and 0.01 nm position accuracy.

This presentation describes the AFM measurement of the molecular interaction forces in the model ligand-receptor system streptavidin-biotin and between complementary strands of DNA.
Tomographic reconstruction of mitochondria
Authors: Mannella C.A. , Marko M. , Buttle K. , Penczek P. , Leith A. , Frank J.
Published: June 2020

During oxidative phosphorylation, free energy of substrate oxidation is used to generate electrochemical gradients across the mitochondrial inner membrane. Full understanding of this process requires knowing the pathways for internal diffusion of ions and metabolites inside the organelle. This, in turn, requires detailed information about the organization of the membranes that compartmentalize the mitochondrion and the distribution of transport proteins (proton pumps, ion channels, metabolite carriers) on these membranes.

We have undertaken the study of the compartmentation of rat-liver mitochondria (conventionally fixed and plastic-embedded) using electron microscopic tomography. Reconstructions of isolated mitochondria have been computed using projection images collected on the Albany high-voltage electron microscope from sections (0.5-1-μm thick) tilted around one axis over +/− 70° at 2° increments. A problem encountered with this approach is the directional loss of resolution due to the “missing wedge” of information in Fourier space in the direction parallel to the tilt axis. To overcome this problem, we have developed an alignment algorithm that allows projections collected over two or more tilting directions to be used in the same modified back-projection calculation.
Practical electron tomography
Authors: Woodcock C.L.
Published: June 2020

Despite the potential of the technique, electron tomography has yet to be widely used by biologists. This is in part related to the rather daunting list of equipment and expertise that are required. Thanks to continuing advances in theory and instrumentation, tomography is now more feasible for the non-specialist. One barrier that has essentially disappeared is the expense of computational resources. In view of this progress, it is time to give more attention to practical issues that need to be considered when embarking on a tomographic project. The following recommendations and comments are derived from experience gained during two long-term collaborative projects.

Tomographic reconstruction results in a three dimensional description of an individual EM specimen, most commonly a section, and is therefore applicable to problems in which ultrastructural details within the thickness of the specimen are obscured in single micrographs. Information that can be recovered using tomography includes the 3D shape of particles, and the arrangement and dispostion of overlapping fibrous and membranous structures.
Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos
Authors: Marshall W.F. , Dernburg A.F. , Harmon B. , Sedat J.W.
Published: June 2020

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.

Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.
Near-field microscopy of membrane domains
Authors: Hwang J. , Betzig E. , Edidin M.
Published: June 2020

Results from several different methods for probing the lateral organization of cell surface membranes indicate that these membranes are patchy, divided into domains. The data suggest that on average these domains are 0.1-1 μm across and that they persist for 10’s to 1000’s of seconds. At least some domains in this size range, when labeled by fluorescent proteins or lipids ought to be detectable by conventional, far-field, fluorescence microscopy. However, though some images are consistent with a domain structure for membranes, most far-field images of fluorescent cell surfaces lack the detail necessary to define domains.

We have used near-field scanning optical microscopy, NSOM, of fluorescent-labeled cells to visualize membrane patchiness on the nanometer scale. This method yields images with resolutions of 50 nm or less. In our near-field microscope the labeled sample is illuminated by a optical fiber probe, with an aperture of 50-80nm. The probe is scanned over the cell surface at a distance of ˜ 10 nm from the surface. Only surface fluorescence is excited by the scanned probe.
Confocal microscopy of single living cells
Authors: Lemasters John J.
Published: June 2020

The advent of laser scanning confocal microscopy solves the dilemma of studying thick specimens with optical microscopy by creating optical slices less than 1 μm in thickness. Increasingly, confocal microscopy is an essential analytical tool for studying the structure and physiology of living cells. Because confocal microscopy collects light from only a fraction of the specimen volume, greater illumination is required. Consequently, photodamage and photobleaching are greater considerations, especially for study for living cells where repeated measurements over time are desired. To minimize photodamage, laser intensity should be attenuated by 100-1000 fold, photomultiplier circuits should be operated at highest sensitivity, and stable fluorophores should be used. When these conditions are met, literally hundreds of high resolution confocal images can be obtained from single cells loaded with parameter sensitive fluorophores.

The number of parameter-specific fluorophores useful for observing single living cells by confocal microscopy is large and increasing. By labeling with calcein and collecting serial images, the volume, shape and surface topography of single living cells are reconstructed with results rivaling scanning electron micrographs.
Surgical repair of long bone defects or trauma by guided tissue engineering with composite implants of porous bioactive glass-ceramic (A-WGC) particles bound by a plaster/CaO/P2O5cement contained by a resorbable vicryl™ mesh
Authors: Hanker J.S. , Hanker L.C. , Giammara B.L.
Published: June 2020

One of the biggest problems associated with the repair of trauma in long bones is their requirement to withstand greater mechanical stress than any other bones of the body. After the success we achieved with composite hydroxylapatite (HA)/plaster(PP) in craniofacial bone repair in approximately 200 animals (cats and rats) and 300 humans in our laboratory, clinics and surgeries, it was felt that an area which could perhaps benefit from our experience in bone repair might be orthopedic surgery. When first informed of our desire to apply our biomaterials interest and experience to long bone trauma patients, others expected little success with these biomaterials that were so successful in craniofacial surgery.

We felt, however, that success might be achieved for long bone repair with plaster of Paris (CaSO4·½H2O) and/or calcium phosphate. The feeling of my laboratory received some support from Larry Hench's 1988 article. In this article he pointed out that bioactive ceramics such as hydroxylapatite (HA) possibly could bond to bone.
Immunocytochemical double staining with the ELF method
Authors: Baskin D. G. , Vathanaprida U C. , Stahl W. L.
Published: June 2020

The reagent 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone has recently become commercially available as the ELF (enzyme-labeled-fluorescence) phosphatase substrate. The ELF method is basically an alkaline phosphatase enzyme immunostaining procedure in which the reaction product is fluorescent instead of chromogenic. The ELF substrate produces a photostable, bright yellow-green fluorescence (500-580 nm) with low background. The use of the ELF substrate as a green label may have advantages over FITC in double-labeling immunocytochemical staining procedures that employ Cy3, which frequently shows residual orange-red fluorescence when viewed with conventional FITC filter sets.

To evaluate the suitability of ELF for dual immunofluorescence with Cy3-labeled second antisera, we used paraffin sections of rat pancreas fixed in 4% paraformaldehyde. Primary antibodies were an anti-insulin monoclonal antibody (1:50) (Biogenex) and anti-glucagon polyclonal antibody (1:1K)2, in Tris-HCL, 1% BSA, 0.1% TX-100, pH 7.4. The insulin antibody was applied overnight at 6 °C, followed by goat anti-mouse IgGCy3 (Jackson Immunochemical Research) for 60 min. at room temperature (RT).
Immunoelectron microscopy of the connective tissue matrix following cryotechnical processing
Authors: Keene D.R. , McDonald K. , Ridgway C.C.
Published: June 2020

Our understanding of the microarchitecture of connective tissues is based primarily on micrographs taken of tissues stabilized by immersion fixation. Considerable extraction of tissue components, particularly small matrix molecules, occurs even during fixation in glutaraldehyde. An alternative to immersion fixation is the technique of cryostabilization whereby tissue components are instantaneously immobilized and surrounded by non-crystaline (amorphous) or microcrystaline ice. The ice is replaced at low temperature (-100 to -85°C) by acetone containing fixative, and the tissue finally embedded in epoxy. A comparison of micrographs taken from tissue embedded following immersion fixation and those taken following cryotechnical processing reveals striking differences in the structure of cells, basement membranes, and matrix (Fig. 1a and 2a).

Our recent objective has been to utilize sections from samples which have been stabilized by high pressure freezing for immunocytochemical labeling experiments with the expectation that such studies would result in a more precise localization of matrix components. The results to date suggest that many of the difficulties normally experienced in section surface labeling technique are still a problem: Antigens may be affected by the chemistry of freeze-substitution and choice of embedding media.
Fresnel interference in point-projection imaging of purple membrane
Authors: Zhang X. , Spence J. , Qian W. , Taylor D. , Taylor K.
Published: June 2020

Experimental point-projection shadow microscope (PPM) images of uncoated, unstained purple membrane (PM, bacteriorhodopsin, a membrane protein from Halobacterium holobium) were obtained recently using 100 volt electrons. The membrane thickness is about 5 nm and the hexagonal unit cell dimension 6 nm. The images show contrast around the edges of small holes, as shown in figure 1. The interior of the film is opaque. Since the inelastic mean free path for 100V electrons in carbon (about 6 Å) is much less than the sample thickness, the question arises that how much, if any, transmission of elastically scattered electrons occurs. A large inelastic contribution is also expected, attenuated by the reduced detection efficiency of the channel plate at low energies. Quantitative experiments using an energy-loss spectrometer are planned. Recently Shedd has shown that at about 100V contrast in PPM images of thin gold films can be explained as Fresnel interference effects between different pinholes in the film, separated by less than the coherence width.
Effects of the pseudomonad phytotoxin coronatine on tomato leaf structure and ultrastructure
Authors: Palmer D.A. , Bender C.L.
Published: June 2020

Coronatine is a non-host-specific phytotoxin produced by several members of the Pseudomonas syringae group of pathovars. The toxin acts as a virulence factor in P. syringae pv. tomato, allowing the organism to multiply to a higher population density and develop larger lesions than mutant strains unable to produce the toxin. The most prominent symptom observed in leaf tissue treated with coronatine is an intense spreading chlorosis; this has been attributed to a loss of chlorophylls a and b in tobacco. Coronatine's effects on membrane integrity and cell ultrastructure have not been previously investigated. The present study describes changes in tomato leaves in response to treatment with purified coronatine, infection by a coronatine-producing strain of P. syringae pv. tomato, and infection by a cor" mutant.

In contrast to H2O-treated tissue, coronatine-treated tissue showed a diffuse chlorosis extending approximately 5 mm from the inoculation site. Leaf thickness, cell number, and cell dimensions were similar for both healthy and coronatine-treated, chlorotic tissue; however, the epidermal cell walls were consistently thicker in coronatine-treated leaves (Figs, la and lb).
Metallothionein expression during wool follicle development
Authors: Nishimura N. , Nishimura H. , Tohyama C. , Cam G.R. , Adelson D.L.
Published: June 2020

This study investigated metallothionein(MT) expression in proliferating or differentiating cells of developing wool follicles. Dividing cells in foetal sheep skin were identified by incorporated BrdU, which was injected into the jugular vein of a ewe before sacrifice. MT gene expression was assayed using a digoxigenin-labelled sheep MT-1b riboprobe as described previously. Immunohistochemical staining for MT protein and BrdU was carried out using rabbit-anti rat MT-1 serum and anti-BrdU, as described elsewhere. Foetal sheep skin was obtained from 60 to 130 days of gestation, fixed in HistoChoice or Bouin's and embedded in paraffin.

At 71 days of gestation, MT-1b mRNA was detected in the epidermis(EP), primarily in the spinous cells (Fig. la). At 90 days, MT-1b mRNA was concentrated at the tip of the follicle rudiment(F), but not in the presumptive dermal papilla(DP) (Fig. 1b). At 112 days, a symmetric distribution of MT-1b mRNA was observed in the follicle bulb matrix (Fig. 1c). However, very little MT-1b mRNA was detected in the matrix cell layers adjacent to DP. At 130 days, strong MT-lb gene expression was localized to the cytoplasm of cells in the follicle bulb(B) and keratogenous zone(K)(Fig. Id).
Substructurel features of LLC-PK1porcine kidney cells grown on Anocell filter supports
Authors: Mia A.J. , Johnson S. , Franklin J. , Johnson J.F. , Puttaparthi K. , Oakford L.X. , Yorio T.
Published: June 2020

LLC-PK1cells, derived from porcine kidney cortex, have been used for a variety of studies including sodium-coupled hexose transport, vasopressin/calcitonin sensitive adenylate cyclase(1) and vasopressin V1and V2receptor-mediated endocytosis involving clathrin coated pits and vesicles(2,3,4,5). Recently, we reported on the morphological profile of these cells using the techniques of scanning electron microscopy (SEM). SEM studies of confluent LLC-PK1cells exhibited surface morphology similar to intact tissue. These cells also responded to ADH (antidiuretic hormone, vasopressin) or MZ (mezerein), a non-phorbol activator of protein kinase C (PKC) with a propagation of numerous microvilli over the apical plasma membrane typical of ADH responsive tissue. The current study evaluates the cytomorphological features of cultured LLC-PK1cells following hormone addition.

LLC-PK1cells were grown on Anocell filter supports using DMEM media in a 5% CO2/air environment in an incubator at 37°C. Upon confluency, cultured cells were washed in buffer and then treated with 100 mU/ml ADH or MZ10-6M for 15 min. Control and stimulated tissues were fixed in 2% glutaraldehyde in PIPES with a postfixation in 1% osmium tetroxide for 1 hr for either SEM or TEM.
Continued expression of neuronal traits in chromaffin cells of the adult bovine adrenal gland
Authors: Kriho V. , Yang H.-Y. , Lieska N. , Kriho N. , Pappas G. D.
Published: June 2020

Intermediate filaments (IF) are a major constituent of the cytoskeleton of most eukaryotic cells.While there are five major classes of IF, amino acid sequence and secondary structure homologies suggest that all IFs evolved from a single precursor gene early in the history of life. Characteristically, IF are 10 nm filaments whose distribution, in most cases, is tissue-type specific. For example, the IFs of muscle cells contain the muscle-specific IF protein desmin, and those of epithelial cells contain subunits of the keratin family. Neurofilaments (NF) are a class of IFs expressed in most neuronal cells and are composed of three polypeptide subunits with apparent molecular weights of 68,000 (NF-L), 150,000 (NF-M) and 200,000 (NF-H). Interestingly, NFs are present in chromaffin cells cultured from bovine adrenal medulla. Chromaffin cells are derived from the neural crest, and the presence of NFs confirms their relatedness to neurons. However, the presence of NFs in vivo has been the subject of debate.
FT-IR microspectroscopic analysis of diseased white matter brain tissues
Authors: Le Vine Steven M. , Wetzel David L.
Published: June 2020

In situ FT-IR microspectroscopy has allowed spatially resolved interrogation of different parts of brain tissue. In previous work the spectrrscopic features of normal barin tissue were characterized. The white matter, gray matter and basal ganglia were mapped from appropriate peak area measurements from spectra obtained in a grid pattern. Bands prevalent in white matter were mostly associated with the lipid. These included 2927 and 1469 cm-1 due to CH2 as well as carbonyl at 1740 cm-1. Also 1235 and 1085 cm-1 due to phospholipid and galactocerebroside, respectively (Figs 1and2). Localized chemical changes in the white matter as a result of white matter diseases have been studied. This involved the documentation of localized chemical evidence of demyelination in shiverer mice in which the spectra of white matter lacked the marked contrast between it and gray matter exhibited in the white matter of normal mice (Fig. 3).

The twitcher mouse, a model of Krabbe’s desease, was also studied. The purpose in this case was to look for a localized build-up of psychosine in the white matter caused by deficiencies in the enzyme responsible for its breakdown under normal conditions.
Alterations in the number of motoneurons containing immunoreactive calcitonin gene-related peptide(CGRP)& choline acetyltransferase(ChAT) in the cervical spinal cord of the wobbler mouse during the development of the motoneuron disease
Authors: Vacca-Galloway L. , Zhang Y.Q. , Bose P. , Zhang S.H.
Published: June 2020

The Wobbler mouse (wr) has been studied as a model for inherited human motoneuron diseases (MNDs). Using behavioral tests for forelimb power, walking, climbing, and the “clasp-like reflex” response, the progress of the MND can be categorized into early (Stage 1, age 21 days) and late (Stage 4, age 3 months) stages. Age-and sex-matched normal phenotype littermates (NFR/wr) were used as controls (Stage 0), as well as mice from two related wild-type mouse strains: NFR/N and a C57BI/6N. Using behavioral tests, we also detected pre-symptomatic Wobblers at postnatal ages 7 and 14 days. The mice were anesthetized and perfusion-fixed for immunocytochemical (ICC) of CGRP and ChAT in the spinal cord (C3 to C5).

Using computerized morphomety (Vidas, Zeiss), the numbers of IR-CGRP labelled motoneurons were significantly lower in 14 day old Wobbler specimens compared with the controls (Fig. 1). The same trend was observed at 21 days (Stage 1) and 3 months (Stage 4). The IR-CGRP-containing motoneurons in the Wobbler specimens declined progressively with age.
Fungal infection of seed: a source of cotton textile imperfections
Authors: Goynes W. R. , Ingber B. F. , Thibodeaux D. P.
Published: June 2020

Cotton seed develop within a thick-walled boll that is usually divided into three or more compartments called locules. The seed and fibers that grow within a locule are called a lock. An unopen boll is shown in figure 1. The cotton fiber is a single cell and develops from the epidermis of the seed. The cell wall, or primary wall, of the fiber, a complex mixture of cellulose, protein, waxes, pectins, and other plant related materials, elongates for approximately 17-25 days. Completion of this elongation is overlapped by the beginning of secondary wall synthesis which deposits successive layers of cellulose inside the primary wall. This main body of the fiber is composed almost entirely of cellulose. Production of a commercially useful fiber depends on the completion of this secondary wall development since it provides both fiber strength and dyeability. If this growth process is inhibited at any stage, less mature fibers with thin secondary walls are produced, and if it is interrupted before secondary wall production begins, undeveloped fibers are produced that can cause problems during processing into textiles.
EM and microspectrophotometric analysis of biological delignification of plants
Authors: Akin D. E. , Rigsby L. L. , Morrison W. H. , Sethuraman A. , Eriksson K.-E. L.
Published: June 2020

Aromatic constituents such as lignin bind to carbohydrates within plant cell walls and thus render the plant carbohydrates less utilizable as food and energy. Chemical methods used to upgrade the quality of plant biomass are costly, expensive, and unsafe. White rot fungi, which are the only known microorganisms that, to any extent, can remove lignin from plant cell walls, offer a biological solution to upgrading plant quality. Microscopic analyses provide information on the site of delignification that is strategically important in improving use of plant biomass.

Stems of grasses and a legume were treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus for 6 weeks. Treated residues were analyzed for structural modifications using scanning and transmission electron microscopy and for aromatic constituents using ultraviolet (UV) absorption microspectrophotometry and gas chromatography. These modifications were related to improved utilization of cell walls by rumen microorganisms.

UV absorption microspectrophotometry, in conjunction with gas-chromatography of alkali-treated plants, indicated that ester-linked ferulic and ρ-coumaric acids were particularly susceptible to removal by both fungal species.
Ultrastructural characterization of an in vitro model of human epidermis: its potential for sulfur mustard study
Authors: Petrali J.P. , Lopolito P.T. , Hamilton T.A. , Oglesby-Megee S.B. , Rhoads L.S.
Published: June 2020

Morphological correlates of sulfur mustard toxicity continue to be studied in in vivo models such as the haired and hairless guinea pig and the domestic weanling pig. These animal models provide useful evidence for the mechanisms of sulfur mustard-induced lesions, however the correlation with human skin is not exact and the availability of a continuing supply of any animal model is problematic. In vitro studies have largely been performed with cultured monotypic human cells such keratinocytes and lymphocytes. Although monotypic cellular models have added data to the mechanisms of the observed cytopathology, the acclimatized nature of media-nourished cells may present a different phenotype for study than in vivo. To bridge the information gap between monolayers of cells and in vivo studies, in vitro skin equivalents have been proposed as suitable models. We have used several commercially available human skin equivalents in the past, however the lack of basement membrane components, the absence or paucity of hemidesmosomes, general problems with replication and inconsistences of availability, question the usefulness of commercial preparations at this time.
A SEM study of atrazine effects on red shiner (Cyprinella lutrensis)
Authors: Messaad Ibrahim A. , Peters Edward J. , Rogers Douglas G. , Lee Kit W.
Published: June 2020

Atrazine is widely used in agriculture to control weeds. However, little is known about the effects of long-term exposure in fish. Normal fish gill morphology and ultrastructure have been studied using scanning electron microscopy (SEM). Pollutants including pesticides can cause lesions in gills, which ultimately affect osmoregulation and oxygen consumption.

Specimens of red shiner (Cyprinella lutrensis) were collected from the Platte River, Nebraska, by seining and treated with sodium chloride and malachite green to reduce infections. Fish were randomly distributed to aerated glass aquaria. Biological sponge filters were used to keep aquaria clean during the 14 day bioassay. Fish were maintained at a 22°C on a 12:12 photoperiod, and fed a commercial fish food once daily. Atrazine concentrations of 0, 10, 100, and 1000 μg L-1 were used. For scanning electron microscopy, fish were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer. Then, gills were removed and post-fixed in 1% OsO4.
ultrastructural process of intestinal wound healing
Authors: Vaughn Rick L. , Saxena Shailendra K. , Sharp John G.
Published: June 2020

We have developed an intestinal wound model that includes surgical construction of an ileo-cecal patch to study the complex process of intestinal wound healing. This allows approximation of ileal mucosa to the cecal serosa and facilitates regeneration of ileal mucosa onto the serosal surface of the cecum. The regeneration of ileal mucosa can then be evaluated at different times. The wound model also allows us to determine the rate of intestinal regeneration for a known size of intestinal wound and can be compared in different situations (e.g. with and without EGF and Peyer’s patches).

At the light microscopic level it appeared that epithelial cells involved in regeneration of ileal mucosa originated from the enlarged crypts adjacent to the intestinal wound and migrated in an orderly fashion onto the serosal surface of the cecum. The migrating epithelial cells later formed crypts and villi by the process of invagination and evagination respectively. There were also signs of proliferation of smooth muscles underneath the migratory epithelial cells.
Baculovirus ACMNPV as well as RNA- and protein-synthesis inhibitors induce apoptosis in a continuous midgut cell line, FPMI-CF-203, of Choristonuera fumiferana (Lepitoptera: tortricidae)
Authors: Brownwright A.J. , Palli S.R. , Caputo G.F. , Sohi S.S.
Published: June 2020

Apoptosis is an active cellular self-destruction regulated by expression or repression of certain genes. Apoptosis can be caused by a variety of both external and internal stimuli. Whether these different stimuli that can cause apoptosis converge into a final pathway that leads to self-destruction is not known. This paper compares apoptosis caused by a baculovirus, an RNA-synthesis inhibitor and a protein-synthesis inhibitor.

Inoculation of IPLB-SF-21 (SF-21) and FPMI-CF-203 (CF-203, Fig. 1, ) cells with Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV), respectively, results in successful infection as visualized by formation of occlusion bodies (OBs). However, inoculation of CF-203 cells with AcMNPV, or SF-21 cells with CFMNPV, is unsuccessful and no OBs are seen. Inoculation of CF-203 cells with AcMNPV results in premature lysis of cells beginning at 12 hr post-inoculation (pi) and most of the cells are lysed by 48 hr pi.
Ultrastructure of human herpesvirus 7
Authors: Goldsmith C. S. , Burns D. , Pellett P. E. , Zaki S. R. , Black J. B.
Published: June 2020

Human herpesvirus 7 (HHV-7) was originally isolated in 1989 from the peripheral blood lymphocytes of a healthy individual. Most children between 2 and 5 years of age develop antibodies against HHV-7, and the virus has recently been implicated as the causative agent in secondary episodes of the childhood disease roseola infantum. Approximately 90% of the general population is seropositive, and virus can be isolated from the saliva of up to 75% of healthy adults. This study describes the ultrastructural growth properties of an HHV-7 isolate obtained from a saliva specimen.

Cord blood lymphocytes (CBLs) were inoculated with cell-free virus and harvested at days 1, 3, 4, 5, and 7. The growth curve for HHV-7 was determined by examining the ultrastructure of the cells for signs of viral production and by monitoring the cells with an anti-complement immunofluorescence assay for the presence of viral proteins.

The nuclei of HHV-7-infected CBLs typically had viral nucleocapsids scattered throughout, although the capsids would occasionally converge into organized structures (Fig. 1).
Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane
Authors: Arora Krishan K. , Decker Glenn L. , Pedersen Peter L.
Published: June 2020

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Aroraet al(4)].

In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.
Microvasculature and structure of hemal lymph nodes in the wall of the rabbit bladder: a vascular corrosion casting and EM study
Authors: Hossler F.E. , McKamey M.I. , Monson F.C.
Published: June 2020

A comprehensive study of the microvasculature of the normal rabbit bladder, revealed unusual "capillary glomeruli" along the lateral walls. Here they are characterized as hemal lymph nodes using light microscopy, SEM, TEM, ink injection, and vascular casting.

Bladders were perfused via a cannula placed in the abdominal aorta with either 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for fixation, 10% India ink in 0.9% saline and 0.1M phosphate (pH 7.4) for vessel tracing, or resin (Mercoximethylmethacrylate: catalyst, 4:1:0.3; Ladd Research Industries) for vascular corrosion casting. Infusion pressure was 100mm Hg. Fixed tissue was sectioned from epon-araldyte resin, and stained with toluidine blue for light microscopy, and lead and uranium for TEM. Ink injected tissue was photographed directly from saline-filled bladders illuminated from below. Resin-filled tissue was macerated in 5% KOH and distilled water. Casts were critical point dried, sputter coated with goldpalladium, and examined by routine SEM at 10 KV.
Methods for preparation of cribellate spider silk fibrils for Transmission Electron Microscopy
Authors: Cutler B.
Published: June 2020

Spider silk has been examined by transmission electron microscopy (TEM) for over 50 years. A later, more detailed study produced higher resolution images and included a discussion of fixation with osmium tetroxide (OSO4) vapor. The fixation resulted in only minor effects compared to unfixed specimens. Cribellate spiders produce a nonviscous adhesive silk that has as a significant component very fine fibrils produced by a structure called a cribellum. An opportunity to study cribellate silk in a spider family not previously investigated also led to a study of different fixation methods. Immature specimens of Titanoeca nigrella (Chamberlin) (Araneae, Titanoecidae) were collected under rocks at the Cimarron National Grasslands, Morton Co., Kansas. Spiders were kept in the laboratory and spun normal appearing webs. Copper and gold (for OSO4 treatments) 200 m grids were dragged through the webs. All combinations of treatments were used, but formaldehyde exposure always preceded OSO4 exposure which preceded sputter coating. Grids were examined with a JEOL 1200 EXII TEM. An accelerating voltage of 60kv and 100 um objective aperture were used for imaging.
Cluster labels - Au11 and W11
Authors: Hainfeld James F.
Published: June 2020

The undecagold (Au11) complex has previously been described and was used to label the biotin binding site an ovidin to ∼10 Å resolution. This is the first time this high a resolution has been obtained with a label on single isolated molecules. The gold complex was also used to mark carbohydrate moieties on a glycoprotein.

A W11 complex linked to a long hydrophobic tail was used to label lipid bilayers. An improved Au11 complex which is monofunctional has now been synthesized by two independent routes, one by Frey and one by Safer.
Ultrasturcture of cerebellar cells and neuropil in rats subjected to partial global cerebral ischemia
Authors: Dimlich R.V.W. , Biros M.H.
Published: June 2020

Although a previous study in this laboratory determined that Purkinje cells of the rat cerebellum did not appear to be damaged following 30 min of forebrain ischemia followed by 30 min of reperfusion, it was suggested that an increase in rough endoplasmic reticulum (RER) and/or polysomes had occurred in these cells. The primary objective of the present study was to morphometrically determine whether or not this increase had occurred. In addition, since there is substantial evidence that glial cells may be affected by ischemia earlier than other cell types, glial cells also were examined. To ascertain possible effects on other cerebellar components, granule cells and neuropil near Purkinje cells as well as neuropil in the molecular layer also were evaluated in this investigation.
The staining pattern of the tropomyosin sequence is displayed in a new paracrystal
Authors: Lamvik M.K. , Klatt L.L.
Published: June 2020

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.
Texture details in experimental lattice images from a monolamellar paraffin crystal
Authors: Dorset D. L. , Zemlin F. , Reuber E. , Beckmann E. , Zeitler E.
Published: June 2020

The direct visualization of crystal structure at "molecular" (ca 3Å) resolution has become a routine procedure in electron microscopy in the last few years for organic materials which are resistant to electron beam damage by virtue of π-electron derealization or electrical conductivity. More recently, similar images from an aliphatic material, i.e. the paraffin n-tetratetracontane, were published based on work with an electron microscope equipped with a He-cooled superconducting objective lens. Correlation-averaged electron images at 2.5A resolution were shown to correspond well to a theoretical image based on a multislice calculation for the known crystal structure and produced at the phase contrast transfer function of the electron microscope objective lens for the defocus value used in the experiment.
Low-temperature scanning electron microscopy (ltsem) of frozen-hydrated lung
Authors: Bastacky J. , Hook G. R. , Finch G. L. , Hayes T. L.
Published: June 2020

The air-liquid interface of the airways and alveoli in the lung produces functionally significant surface forces. The configuration of this interface, however, is in question since electron microscope sample preparation methods generally remove tissue water. We have developed techniques to study the normal structure of mammalian lung in the frozen-hydrated state with water preserved as ice utilizing the low-temperature scanning electron microscope (LTSEM) of Pawley and Norton
Secondary and backscatter electron imaging of silver stained ciliated protozoa
Authors: Paulin Jerome J.
Published: June 2020

The use of secondary electron (SE) signals in the scanning electron microscope provides a wealth of information on the cortical topography of the ciliated protozoa. Surface manifestations of such morphogenetic events as cell division and regeneration can easily be visualized in the SE mode of the SEM, but concomitant alterations occurring below the surface of the cell can not be visualized. Ciliatologists have used the "Protargol" (silver protein) method to study the cortical features of the ciliates at the light microscopical level. The protein silver stain clearly reveals cilia, microtubules, kinetosomes and nuclei. Small et al. have demonstrated the usefulness of this technique in conjunction with backscatter electron (BSE) detection in the SEM. A strong BSE signal can be obtained from argentophilic structures within the specimen due to the atomic number contrast between the deposited silver and the low atomic number elements normally found in cell cytoplasm. Therefore, the use of the SE and BSE signals with good spatial resolution should clearly demonstrate the relationship between surface and subsurface features.
Identification of hepatitis a virus in cell cultures by solid phase immune electron microscopy
Authors: Humphrey C.D. , Cromeans T.L. , Cook E.H. , Bradley D.W.
Published: June 2020

There is a variety of methods available for the rapid detection and identification of viruses by electron microscopy as described in several reviews. The predominant techniques are classified as direct electron microscopy (DEM), immune electron microscopy (IEM), liquid phase immune electron microscopy (LPIEM) and solid phase immune electron microscopy (SPIEM). Each technique has inherent strengths and weaknesses. However, in recent years, the most progress for identifying viruses has been realized by the utilization of SPIEM.
Identification of maize mosaic virus by immunoelectron microscopy
Authors: Bradfute O. E.
Published: June 2020

Maize mosaic virus (MMV) causes a severe disease of Zea mays in many tropical and subtropical regions of the world, including the southern U.S. (1-3). Fig. 1 shows internal cross striations of helical nucleoprotein and bounding membrane with surface projections typical of many plant rhabdovirus particles including MMV (3). Immunoelectron microscopy (IEM) was investigated as a method for identifying MMV. Antiserum to MMV was supplied by Ramon Lastra (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela).
Interspecific comparison of elutable cell wall extensin precursors by transmission electron microscopy
Authors: Heckman J. W. , Kielszewski M. J. , Lamport D. T. A. , Muldoon E. P. , Terhune B. T. , Willard J. J.
Published: June 2020

In addition to cellulose microfibrils, the primary cell wall of many higher plants contains extensin, a class of hydroxyproline-rich glycoprotein (HRGP). Despite its predominately hydrophilic amino acid composition, most cell wall HRGP remains insoluble even after complete deglycosylation with anhydrous HF, suggesting a covalently linked macromolecular network. This led to the development of the "warp-weft" cell wall model, based on an extensin network ("weft") penetrated by cellulose microfibrils (the "warp")(FIG. 1). Extensin precursors elute rapidly from intact cells and cell wall preparations of tomato, carrot, and cucumber, with mild salt solutions. Tomato precursors, at least, are block copolymers of a few repeating sequences. Gel filtration data and immuno-crossreactivity suggest that tomato P2 and cucumber callus precursor are similar. TEM confirms the rod-like structure and the lengths of these molecules.
Phase contrast and scanning electron microscopy of a soil amoeba, Hartmannella glebae
Authors: Upadhyay J. , Schurr M. J. , R. J. Lesseps
Published: June 2020

The genera Hartmannella and Acanthamoeba have been controversial. Some species of Acanthamoeba have been cited as species of Acanthamoeba, Mayorella, or Amoeba and a confusing lack of agreement persists in current literature. The genus Hartmannella was redefined by Page in 1967 to include those species which assumed a simple monopodial, limax-like form during locomotion and, where known, the cysts were smooth-walled and rounded with no opercula. The genus Acanthamoeba, on the other hand, possessed tapering, hyaline pseudopods (acanthopodia) and a cyst made up of an ectocyst and a polyhedral or stellate endocyst, with excystment by removal of opercula.
Transmission Electron Microscopy, diffraction and electron probe X-Ray microanalysis of senile scleral plaques of the eye
Authors: Tousimis A. J.
Published: June 2020

Scleral plaques of the human eye are small, oval, grayish lesions located 1 to 2 mm in front of the insertion of the lateral rectus muscle. They are common in individuals over 50. Roper's review attributes them to "dehydration, sclerosis, and hyalinization of the connective tissue." Calcium carbonate was reported by Klein-Moncrieff and calcium sulfate and apatite mixed with gypsum by Cogan et al. In the present study light and electron microscopy along with electron diffraction and electron probe x-ray microanalysis (both wave length and energy dispersive) were used to examine the morphology and elemental composition of 12 eyes with the majority of them with unilateral and some bilateral plaques.