bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamics of calcium control in local compartments
Authors: Guthrie P. B. 
Published: June 2020

Neurons undergo a number of morphological changes during the establishment, maintenance, and restructuring of neuronal circuits. During these morphological transformations, different portions of a single neuron may simultaneously exhibit markedly different behaviors. Calcium has been strongly implicated as a regulator of morphological plasticity. We have investigated the extent of autonomous calcium regulation within plastic structures during various stages of development and remodelling. We have found that these plastic structures have highly compartmentalized calcium regulation.During neurite pathfinding, growth cones encounter many different guidance cues. We have found that the autonomous calcium regulation capabilities of specific compartments can also be influenced by intercellular interactions. In the developing grasshopper limb, the pioneer neurons extend processes form transient electrical connections appropriate guidepost cells (Fig 1). These electrical connections allow the guidepost cells to locally reduce calcium concentrations within specific regions of the pioneer neurite, creating local regions of autonomous calcium regulation.We have used calcium ionophores to demonstrate the existence of local sub-cortical calcium compartments, and at least one mechanism of their maintenance.
Otoconia perfect/imperfect crystals
Authors: Fermin César D. , Martin Dale 
Published: June 2020

Otoconia of higher vertebrates are interesting biological crystals that display the diffraction patterns of perfect crystals (e.g., calcite for birds and mammal) when intact, but fail to produce a regular crystallographic pattern when fixed. Image processing of the fixed crystal matrix, which resembles the organic templates of teeth and bone, failed to clarify a paradox of biomineralization described by Mann. Recently, we suggested that inner ear otoconia crystals contain growth plates that run in different directions, and that the arrangement of the plates may contribute to the turning angles seen at the hexagonal faces of the crystals.Using image processing algorithms described earlier, and Fourier Transform function (2FFT) of BioScan Optimas®, we evaluated the patterns in the packing of the otoconia fibrils of newly hatched chicks (Gallus domesticus) inner ears. Animals were fixed in situ by perfusion of 1% phosphotungstic acid (PTA) at room temperature through the left ventricle, after intraperitoneal Nembutal (35mg/Kg) deep anesthesia. Negatives were made with a Hitachi H-7100 TEM at 50K-400K magnifications. The negatives were then placed on a light box, where images were filtered and transferred to a 35 mm camera as described.
Geochemical applications of synchrotron x-ray microspectroscopy
Authors: Rivers M. L. , Sutton S. R. , Bajt S. , Delaney J. S. 
Published: June 2020

The synchrotron x-ray fluorescence microprobe at bending magnet beamline X-26A at the National Synchrotron Light Source has been used for a number of years for geochemical trace element microanalysis using collimated white radiation. More recently an incident beam monochromator and 8:1 focusing mirror have been added. These optics permit the formation of a small (30-100 micron) intense beam of monochromatic radiation suitable for use in x-ray absorption spectroscopy, including both near edge (XANES) and extended fine structure (EXAFS) techniques. This system has been used to study a number of problems in the earth and environmental sciences, including: oxidation state of uranium in contaminated waste sites; oxidation state of Cr in olivine inclusions in diamonds from the Earth's mantle; oxidation state of Mn in wheat roots infected with the take-all disease. We report here some recent results obtained on studies of the oxidation state of Fe in cosmochemical systems.Oxygen fugacity is one of the most important parameters in determining the cosmochemical and geochemical evolution of a system.
Monochromatic X-ray microscopy in the water window with a compact laser system
Authors: Kado M. , Richardson M. , Gabel K. , Jin F. 
Published: June 2020

It is a dream of biologist to observe the intact structure of a biological specimen at high resolution without any artifacts. The development of high resolution x-ray microscopy is a new technique well suited to this purpose. The advantages of x-ray microscopy are seen to be: (i) the contrast can be provided by its components, thereby avoiding possible artifacts caused by the staining and fixation of specimens; (ii) x-ray microscopy is capable of observing thicker specimens (up to a few (μm)than electron microscopy with less damage; (iii) the actual location of the element can be visualizedwhen the proper x-ray wavelength is chosen. (iv) three-dimensional observation may be possible with asingle exposure of x rays. X-ray contact microscopy is presendy the most suitable method by which to evaluate these advantages.It is generally accepted that the highest contrast in x-ray images of in-vitro biological specimens will be obtained with radiation have a wavelength in the so-called “water window”, (2.3-4.4 nm). Both laser plasmas and synchrotrons are bright source of radiation in this region.
Bridging the resolution gap between x-ray crystallography and electron microscopy
Authors: Stewart P. L. , Fuller S. D. , Burnett R. M. 
Published: June 2020

While x-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy can provide complementary structural information on larger assemblies. A significant computational challenge is faced in bridging the resolution gap between the two techniques. X-ray crystallographic data is collected in the range of 2-10 Å, while image reconstructions from electron micrographs are at a resolution of 25-35 Å. A further problem is that density derived from cryo-electron micrographs is distorted by the contrast transfer function of the microscope, whichaccentuates certain resolution bands.A novel combination of electron microscopy and x-ray crystallography has revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus (Fig. 1), derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions (Fig. 2). A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution (Fig. 3).
3-D structure of single filaments from the acrosomal bundle of Limulus sperm by 400 kV cryoelectron microscopy: Scruin binds to homologous actin subdomains
Authors: Agris Jacob , Schmid Michael F. , Jakana Joanita , Matsudaira Paul , Chiu Wah 
Published: June 2020

Scruin (102 kDa), an actin binding and bundling protein, is found in a 1:1 complex with actin in the acrosomal process of Limulus sperm. The protein sequence of scruin is unrelated to any known actin binding proteins but reveals a tandem pair of homologous domains (Way and Matsudaira, manuscript submitted). Sequence comparisons reveal homology in each domain with kelch, a gene in Drosophila that is important in nutrient transport into the oocyte and in the maintenance of a cellular structure called the ring canal, and with MIPP, a mouse placental protein of unknown function. Their similarity with scruin suggests a cytoskeletal function and they may form a new family of actin crosslinking proteins. The actin binding domains in this family of proteins have not been identified.To understand the structural basis of actin crosslinking and bundling, we have been studying the acrosomal bundle by cryoelectron microscopy. The actin bundle of the Limulus acrosomal process is structurally unique because it can be considered as a single 3-dimensional crystal in space group PI, with 28 actin subunits and scruin molecules per unit cell.
A comparison of three cryotechniques (freeze-drying, cryosubstitution, cryosectioning) of specimen preparation for immunolabelling of bacterial antigens ofCoxiella burnetii
Authors: McCaul T. F. , Gould R. J. 
Published: June 2020

Immuno-electron microscopy has allowed the selective localisation of molecules with high resolution and high specificity. Cryopreparatory methods have provided better retention of antigenicity suitable for precise immunolabelling together with optimal structural preservation of cellular components. Cryosubstitution and cryoultramicrotomy have widely been exploited for immunolabelling. Molecular Distillation Dryer (MDD), a form of freeze-drying technique, has recently been used for immunolabelling of Plasmodium falciparum stress proteins and nuclear ribonucleoprotein particles in cultured cells. In the present study, we report the comparison of all three cryotechniques in the immunolabelling of bacterial antigens ofCoxiella burnetii.The highly infectiousC. burnetiiwas prefixed in 3% glutaraldehyde (66 mM cacodylate buffer, pH 6.8 ). The cells were then pre-embedded in 2% low-temperature agarose on Durapore hydrophilic membrane prior to cryofixation using a LifeCell CF100 metal-mirror system. A 1% glutaraldehyde in 100% methanol was used as a medium for cryosubstitution in a Reichert CS Auto Cryosubstitution apparatus.
Synaptic organization of the gustatory NST
Authors: Whitehead M. C. 
Published: June 2020

A fundamental problem in taste research is to determine how gustatory signals are processed and disseminated in the mammalian central nervous system. An important first step toward understanding information processing is the identification of cell types in the nucleus of the solitary tract (NST) and their synaptic relationships with oral primary afferent terminals. Facial and glossopharyngeal (LIX) terminals in the hamster were labelled with HRP, examined with EM, and characterized as containing moderate concentrations of medium-sized round vesicles, and engaging in asymmetrical synaptic junctions. Ultrastructurally the endings resemble excitatory synapses in other brain regions.Labelled facial afferent endings in the RC subdivision synapse almost exclusively with distal dendrites and dendritic spines of NST cells. Most synaptic relationships between the facial synapses and the dendrites are simple. However, 40% of facial endings engage in complex synaptic relationships within glomeruli containing unlabelled axon endings particularly ones termed "SP" endings. SP endings are densely packed with small, pleomorphic vesicles and synapse with both the facial endings and their postsynaptic dendrites by means of nearly symmetrical junctions.
Combined fluorescent and gold nucleic acid probes
Authors: Powell Richard D. , Hainfeld James F. , Churchill Mair E. A. , Belmont Andrew S. 
Published: June 2020

Site-specific probes which incorporate both fluorophores and the 1.4 nm “Nanogold” gold particle have been prepared (Fig. 1). Specimens labeled with these probes may be imaged by fluorescence microscopy, then examined at higher resolution in the electron microscope without an additional gold-labeling step: this allows better specimen preservation and unprecedented correlation between the two sets of complementary data. In addition, the covalent gold attachment procedure allows the preparation of electron microscopy probes using small molecules, which cannot be coupled to colloidal gold.Two types of probes have been prepared. In the first, the Nanogold particle was covalently attached, via peripheral primary amine groups, to Hoescht-33258, a fluorescent dye which is specific for AT-rich regions of double-stranded DNA. Excess Hoescht-33258 was removed by membrane separation. The resulting reagent was tested in Chinese hamster ovary cells: results are shown in Figs. 2 and 3. Specific staining of the nucleus was observed both by fluorescence microscopy and in the light microscope, using silver enhancement (Danscher's procedure) to visualize the gold-labeled regions.
HVEM tomography of the Golgi apparatus: A structural study of the trans-cisternae and trans-Golgi network
Authors: Ladinsky Mark S. , Kremer James R. , Furcinitti Paul S. , Howell Kathryn E. , McIntosh J.Richard 
Published: June 2020

The Golgi apparatus is a membranous organelle that plays central roles in the secretory processes of eukaryotic cells. The apparatus consists of multiple convoluted cisternae, some of which are connected to a tubulo-vesicular system known as the trans-Golgi network (TGN). The TGN is thought to be the primary site for sorting and targeting of lipids and proteins to other cellular locations. Only a few studies have addressed the 3-D structure of the Golgi apparatus, and fewer still have focused on the structure of the TGN.We have employed high voltage electron microscopy (HVEM) and computer axial tomography to study the TGN in 3-D. For these experiments, the trans-most cisternae and TGN of normal rat kidney (NRK) cells were labeled by photoconversion of internalized BODIPY-ceramide. Cells were then postfixed and embedded in Epon-Araldite. Semithick (250nm) sections were cut, post-stained with uranyl acetate and lead citrate, and surface-labeled with 15nm colloidal gold to provide fiducial markers for image alignment. Sections were viewed at 18,300x in a JEM-1000 HVEM operating at 1MeV.
Optimizing immunohistochemical staining for the HGF receptor c-MET in formalin-fixed paraffin-embedded archival human tissue
Authors: Evans B.A. , Song K.C. , Strom S.C. , Zarnegar R. , Bahnson R. , Bisceglia M.H. , Michalopoulos G.K. , Becich M.J. 
Published: June 2020

Frozen tissue immunohistochemistry is a useful technique employed in diagnostic and research investigations and is often used despite a loss of morphological detail. Formalin fixation yields much better tissue preservation, but may mask the antigen sites. We have optimized a technique that allows us to study the distribution of the HGF receptor, c-MET, in formalin fixed, paraffin embedded tissue using commercially available polyclonal antisera in which we unmasked antigens sites with protease digestion. The technique we use is detailed below.Sections are incubated in the following:1)4 μm paraffin sections are deparaffinized and hydrated2)0.03% protease (Type XXIV: Sigma) for 2 min at RT3)0.5% H2O2in methanol for 30 min at RT4)Protein Blocking Agent (Lipshaw/Immunon) for 1 hr at RT5)1° antibody ( Santa Cruz Biotech, Santa Cruz, CA) for 2 hr at RTa.)c-MET (c-12) rabbit polyclonal IgG, 1:200 dilution in PBSb.)h-MET (c-28) rabbit polyclonal IgG, 1:100 dilution in PBSc.)m-MET (c-21) rabbit polyclonal IgG, 1:100 dilution in PBS
Ultrastructural morphology of Pneumocystis carinii exposed to (β-1-3 glucan synthase inhibitors LY 302146 and LY 307853
Authors: Goheen M. P. , Bartlett M.S. , Current W.L. , Shaw M.M. , Smith J.W. 
Published: June 2020

Pneumocystis carinii (PC) is an extracellular pathogen which frequently causes pneumonia in immunocompromised patients and is a significant cause of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). Trimethoprim plus sulphamethoxazole and pentamidine are the most often prescribed treatments for PC induced pneumonia but these drugs often cause severe side-effects in individuals with AIDS and are less effective if relapse occurs. For these reasons the search for more effective compounds continues.The cell walls of PC cysts are rich in glucosyl/mannosyl/and N-acetyl-D-glucosamine residues and are digested by treatment with Zymolyase a β-1-3 glucanase. Therefore, compounds which are inhibitors of β-1-3 glucan synthase may be effective against PC. Immunosupressed mice infected with PC have been treated sucessfully with compounds LY302146 and LY307853 sodium salt both of which are inhibitors of β-1-3 glucan synthase. To help delineate their mode of action, electron microscopic evaluation of samples of PC grown in cultures containing these compounds was undertaken.
Structural analysis of the E. coli RUVB branch migration protein by EM and image analysis
Authors: Yu X. , Benson K. , Stasiak A. , Tsaneva I. , West S. , Egelman E. H. 
Published: June 2020

We have been interested in the structure and function of proteins involved in genetic recombinaton. The ruv locus on the E. coli chromosome contains three genes (ruvA, ruvB and ruvC) that are important for genetic recombination and DNA repair. The ruvA and ruvB genes form part of the SOS response to DNA damage and encode the RuvA and RuvB proteins. Together, RuvA and RuvB promote the branch migration of Holliday junctions in a reaction that requires ATP hydrolysis. Each protein plays a defined role, with RuvA responsible for DNA binding (and, in particular, junction recognition), whereas the RuvB ATPase provides the motor for branch migration. Sequence analysis has identified RuvB as a member of a superfamily of helicases, and experimentally it has been shown that RuvB, in the presence of RuvA, acts as an ATP-dependent helicase.When purified RuvB protein was incubated (in the presence of the ATP analog, ATP-γ-S) with covalently closed, relaxed dsDNA, double-ringed structures were observed on the DNA in the electron microscope (Fig. 1). The DNA must be passing through the center of these rings, since the rings are always aligned along a common axis.
Comparison of hydathodes and stomata in Ficus formosana maxim
Authors: Chen C. C. , Chen Y. R. 
Published: June 2020

Two major ways of water discharge from plant leaves are vapour transpiration and liquid guttation through stomata and hydathodes, resptectively. However, besides pure water, guttation fluid contains various salts, sugars and other organic substances which cause injury to plants through their accumulation on leaf surface. Functionally, hydathodes not only play an important role on water excretion but also on retrieval of minerals and their structures are different from those of stomata, even though both have same phylogenic origin. In the present study, the distribution, strcuture, and cytology of hydathodes and stomata of Fisuc formosana Maxim were reported.Leaf blades of fully expanded leaf were cut with a razor blade into small cubes in fixation buffer containing 2.5% glutaraldehyde. These were transfered to fresh fixation buffer for 2 h, postfixed in 1% osmium tetraoxide for 4 h, dehydrated through an ethanol series, and then infiltrated and embedded in Spurr's resin. Sections in golden color were collected on grids, doubly stained with uranyl acetate and lead citrate, and observed with a Hitachi H-600 transmission electron microscope (TEM).
The use of fractal analysis to describe in vitro drug effects: Effect of lovastatin on CaCo-2 cell differentiation
Authors: Maleeff Beverly E. , Hart Timothy K. , Klinkner Anne M. , Waites C. Robbie , Bugelski Peter J. , Kerns William D. 
Published: June 2020

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoAR) have been shown to inhibit cell growth and to affect cell differentiation in a variety of in vitro systems. The human colon carcinoma cell line CaCo-2, when grown on permeable membranes, undergoes high order differentiation into a well-organized monolayer of columnar enterocytes. However, in the presence of the HMG CoAR inhibitor lovastatin, these cells fail to differentiate, lose contact inhibition and overgrow the culture support. Changes in cell differentiation can be described by measurements of mean cell height (MCH) or mean monolayer height (MMH), and are illustrated with representative micrographs. In this study, we have applied fractal geometry in addition to traditional morphometry to quantitatively describe the effect of lovastatin on CaCo-2 cell differentiation in vitro.CaCo-2 cells were plated at sub-confluency (∽ 60,000 cells/cm2) on collagen-coated Transwell™ permeable polycarbonate membrane supports (Costar Corp., Cambridge, MA) in culture dishes and allowed to attach for at least 24 hours.
Microscopic investigations of novel intracellular bodies of a Nocardia SP
Authors: Stites J. L. 
Published: June 2020

A Nocardia sp.was found during an initial transmission electron microscopic (TEM) examination to have unusual intracellular bodies (ICB's) which do not appear to have been described previously in the literature. Most intracellular structures within bacteria have been classified as storage granules, a product of membrane invagination (i.e. mesosomes), or vacuoles. In bacteria there are no known intracellular membrane-bound organelles, and all internal membranes are invaginations of the unit membrane. Several microscopic-level examinations of the Nocardia sp. ICB's were initiated in order to determine their overall structure, classification, and internal constitution.Different TEM staining procedures were performed to determine possible molecular components of the ICB. In all of the staining protocols the ICB's showed a lack of electron density similar to the cell wall. Because the ICB's showed no affinity to any stain, it appeared they do not have strong positive charge (phosphotungstic acid), are not protein rich (en bloc uranyl acetate), lack glycogen and are not phosphate or sulphur rich (lead citrate), nor do they contain lipids or ribonucleic acids (osmium tetroxide).
X-ray analysis of materials: Avoiding the pits and other practical hints
Authors: Sandborg Alan 
Published: June 2020

Introduction:EDS analysis of samples in a SEM or TEM is usually portrayed as a very simple process; just image your specimen, turn on the x-ray analyzer and all will be revealed about the chemistry of your sample. Unfortunately, that approach can be very misleading at best, and disastrous at worst. The analyst should get used to preparing himself for EDS x-ray analysis just like he or she does for any other process. A set of preparation steps should be followed until they become an unconscious method to be used before accepting the information coming from the EDS x-ray spectrum. After proper basic preparation, interpretation of the results and the selection of operating parameters is still very important due to the detailed nature of the specimen.Preparation:The most basic need is for the EDS system to be calibrated. This can be a difficult or simple task depending on the age of the system. Next, the basic geometry of the beam, sample and EDS detector needs to be considered. Before starting to analyze a specimen, the operator has probably put a good deal of effort into obtaining an optimum image. But the optimum parameters for imaging and x-ray analysis are rarely the same.
Managing digital images with SEM viewer™
Authors: Denault Lauraine , de la Parra Robin , von Roesgen Claude 
Published: June 2020

In 1992 Millipore's Corporate SEM Lab made the transition from conventional analog recording of images to digital acquisition and archival. Commonly requested images could be easily and reliably retrieved, without significant loss of image quality or resolution.In theory, digital images could be sent over a network to desktop PC's for review, (rather than producing hard copy of all images). Using established wide area network system, the scope of incorporating digital images significantly broadened. A significant barrier appeared in that the images were acquired using two different hardware/software packages, by two different SEMs (ISI DS130-C and ElectroScan ESEM). Although both generate TIFF images the "flavors" are somewhat different. The TIFF formats were incompatible because the tags used for magnification, micron bar, and descriptor line data were different. As a result, only the image itself generated from one program could be read by the other program, as with all available TIFF readers. Our dilemma was how to enable a single program to recognize and display all necessary information i.e., magnification, descriptor line, and the micron bar with the image.