bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation Of Dna Organization In Spermatozoa Using High Resolution Scanning Electron Microscopy
Authors: Klaus Angela V , Steven Ward W 
Published: July 2020

Abstract
The average somatic nucleus is only 10 microns in diameter, yet contains 2 meters of DNA coiled within its volume. This compaction must be accomplished in such a way so that the DNA remains accessible to the machinery of transcription, replication and repair. Sperm nuclei also have compacted DNA; however, structurally and functionally the arrangement of DNA in sperm is vastly different from that of somatic DNA. Somatic DNA is initially compacted by coiling around histone octamers, while sperm DNA is bound to elongate, highly basic proteins called protamines. Protamines bind the major groove of the double helix and allow adjacent DNA strands to come in close contact by neutralizing the negative charge of the sugar-phosphate backbone. Figure 1 illustrates the two most widely accepted models of DNA compaction in both somatic and sperm nuclei.
The arrangement of DNA within sperm nuclei is difficult to study because it is so highly compressed.
Light Microscopy, Confocal Laser Scanning Microscopy And Scanning And Transmission Electron Microscopy Of Cerebellar Golgi Cells.
Authors: Castejόn O , Sims P 
Published: July 2020

Abstract
The cerebellar cortex of albino mice, hamsters, teleost fishes, primates and human have been examined by correlative microscopy to study the Golgi cell soma, dendritic processes, axonal plexus and synaptic connections in the granular and molecular layers. For light microscopy (LM) toluidinc blue stained-plastic embedded scmithin sections and Golgi light microscopy preparations were used. For confocal laser scanning microscopy (CLSM) of hamster cerebellum the FM4-64 fluorescent stain was used as intracellular tracer (1). Conventional and high resolution scanning electron microscopy (SEM) of teleost fishes, primates and human were coated with gold-palladium and chromium (2). I Transmission electron microscopy (TEM). either by ullrathin sections or frccze-clching replicas, were examined to characterize synaptic connections in the granular and molecular layers. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform microncurons. 10-25 μm in maximal dimension, surrounded by the granule cell groups. Golgi light microscopy.
Incorporation Of Membrane Proteins Into Lipid Bilayers For Scanning Transmission Electron Microscopy And Single Particle Reconstruction
Authors: McAlduff DE , Heng YM , Ottensmeyer FP 
Published: July 2020

Abstract
The experimental approaches available to study the structure of most membrane proteins are limited. X-ray crystallography or electron crystallography rely on the availability or creation of 3D or 2D crystals to obtain high resolution structural information of membrane proteins. Single particleimaging of detergent solubilized protein is also possible, but removes the protein from its native environment. Torecreate suchan environment, we have developed a method which permits membrane proteins to be visualized in a lipid bilayer support.
A copper grid overlaid with a holey plastic film is vertically submerged into a lipid monolayer (Fig.l). This action creates planar lipid bilayers spanning 1 to 10 μm holes of the plastic film. These synthetic membranes have a thickness of approximately 40 Å, comparable to literature values for various cell membranes and are reasonably stable under a defocused beam in a transmission electron microscope (TEM)(Fig.2). Proteoliposomes with appropriate proteins in their membrane are added to the still-wet fresh lipid bilayers such that the liposomes fuse with the planar membrane (Fig.3).
Potassium Permanganate Staining Allows For The Visualization Of An Outer Surface Component Of Selected Ehrlichiae
Authors: Hayes SF , GMunderloh U , Goodman JL , Kocan KM 
Published: July 2020

Abstract
The Ehrlichia spp. are tick-borne pathogens that infect leucocytes and erythrocytes of human beings and animals (1-4). Three ehrlichial organisms, E. chaffeensis, E. equi, HGE (human granulocytic ehrlichiosis agent, equivalent to E. phagocytophila and closely related to E. equi), and Anaplasma marginale were studied with respect to their morphology after using a combined fixative/potassium permanganate staining protocol (5). Cell cultures infected with the respective organisms were fixed and treated as follows: Tissues were initially fixed with Karnovsky's fixative [glutaraldehydeparaformaldehyde, 2.5/4.0% respectively], containing 0.1% tannic acid, in a Sorenson's phosphate buffer with 0.1M sucrose. Post fixation with OsO4 or without it occurred in some samples. En bloc staining with 1% aqueous uranyl acetate was always done. Dehydration was in a graded ethanol series followed by embedding in Spurr's™ resin. Sample sections were stained for 30-60 minutes at RT with 1% potassium permanganate. These were then washed for 5-15 seconds with 0.025-0.05% sodium citrate, jet washed with dH20, dried and examined (6).
Visualization Of Uptake Of High Density Lipoprotein By Rat Aortic Endothelial Cells In Vitro
Authors: Chao W T , Yang V C 
Published: July 2020

Abstract
The high concentration of low-density lipoprotein in the plasma is the major risk factor of atherosclerosis. On the other hand, another plasma lipoprotein—high-density lipoprotein (HDL) — is inversely correlated with atherosclerosis. Recent studies have demonstrated that HDL mediates the transport of cholesterol from peripheral tissues to the liver. However there is considerable debate about the mechanisms by which high density lipoprotein removes excess cholesterol from cells. Two different pathway were suggested: (i) a docking receptor promoting cholesterol translocation, or (ii) a receptor mediated intracellular endosomal pathway termed “retroendocytosis”. In the present study, we performed immunofluorescence and electron microscopy to directly visualize the uptake of HDL using cholesterol-load rat aortic endothelial and smooth muscle cells.
Endothelial cells were obtained from rat aorta and cultured in medium under 5% CO2 / 95% air atmosphere. Then confluent monolayers of endothelial cells were incubated in cholesterol for 48 hr. The cells were precooled for 2 hr at 4°C with PBS containing HDL-Dil or HDL-gold (10 nm) at a concentration of 80 j± g protein per ml PBS. Internalization experiments were carried out by incubation at 37°C for 0, 5, 15, and 30 min, followed by three washes with PBS (pH 7.4). Then the cells were processed for fluorescence and transmission electron microscopy.
Subcellular Distribution Of Basic Fibroblast Growth Factor-Like Molecules In Fibroblasts And Extracellular Matrix
Authors: Aktas R G , Kayton R J 
Published: July 2020

Abstract
Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts. To some degree bFGF regulates proliferation and extracellular matrix (ECM) production by these cells. In this study we investigate (i) the distribution of bFGF in fibroblasts and ECM of supporting tissues (ii) the subcellular location of this protein in vivo and, (iii) mechanisms for release of this growth factor from fibroblasts.
Spurr's resin embedded ultrathin sections from adult rat sciatic nerve, adrenal gland, kidney; and from normal human lung and skin were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics Inc.) specific for bFGF using a two-step immunogold labeling method. Controls were incubated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, anti-Histamine)(Fig. 4).
A Post-Embedding Colloidal Gold Immunocytochemical Approach To The Study Of Matrix Accumulation In Glomerular Basement Membrane.
Authors: Miller Caroline A , Cosgrove Dominic 
Published: July 2020

Abstract
Alport renal disease pathogenesis is characterized by a progressive irregular thickening, thinning, and splitting of the glomerular basement membrane (GBM), which culminates in a focal and segmental glomerulonephritis and progressive loss of glomerular filtration, leading to uremia and death. A mouse model for this disease was produced using a gene targeting approach (Cosgrove et al., 1996). The resulting model displays renal pathology that is very similar to that observed in humans. As matrix accumulation has long been associated with the thickened regions of the GBM, this model provided a means to study the molecular composition and ultrastructural localization of matrix in these rarefied regions of the GBM in the Alport mouse.
We examined three matrix molecules based on preliminary data; type IV collagen α l and α2 chains, laminin-1 and fibronectin. Immunohistochemical analysis showed that while all three of these molecules localize primarily to the mesangial matrix of normal mouse glomeruli, in the Alport glomeruli these molecules seem to be heavily deposited in the GBM.
The Primary Cilium of MDCK Cells is a Flow Sensor
Authors: Praetorius H A , Spring K R 
Published: July 2020

Abstract
The primary cilium, a solitary non-motile structure projecting from the centriole of mammalian cells, has been a subject of interest of anatomists since 1898. Surprisingly, the function of the primary cilium in mammalian cells is completely unknown. Virtually all of the epithelial cells of the mammalian kidney, with the exception of the intercalated cells of the collecting duct, express a single primary cilium on their apical (lumenal) surface. These structures were ignored by physiologists until recently when the mechanical properties of the primary cilia of cultured cells of renal origin were measured and it was proposed that they could serve as flow sensors. The proposal stemmed from the observation that flow rates comparable to those observed in renal tubules resulted in a deflection of the cilium of a few microns and that the stiffness of the cilium was commensurate with mechanosensing.
The primary cilia of renal tubular cells are generally about 2 - 3 μm long whereas those of cultured renal cells become much longer as the cells age and may extend 50 μm or more into the medium. MDCK cells, a cultured cell line derived from the collecting duct of the canine kidney, exhibit two cell types analogous to the principal and intercalated cells of the collecting duct. MDCK principal cells express a primary cilium that is visible by high-resolution DIC microscopy. About one week after splitting, the primary cilium of MDCK principal cells is about 8 μm long and is well suited for testing the hypothesis that the cilium serves as a mechanosensor of fluid flow.
Fluorescent Speckle Microscopy in Studies of Cytoskeletal Dynamics During Cell Motility
Authors: Waterman-Storer CM , Salmon W C 
Published: July 2020

Abstract
We have discovered a new method, Fluorescent Speckle Microscopy (FSM), for analyzing the dynamic movement and turnover of macromolecular protein assemblies, such as the cytoskeleton, in living cells (Waterman-Storer et al., 1998). FSM compliments or replaces the techniques of fluorescence recovery after photobleaching or photoactivation of fluorescence for measuring protein dynamics in vivo. For FSM, cells are microinjected with a very low fraction of fluorescently labeled subunits that co-assemble with unlabeled subunits to give a structure with a fluorescent speckled appearance in diffraction-limited wide-field or confocal digital fluorescence images. At low fractions of fluorescent subunits relative to unlabeled subunits, fluorescent speckles vary randomly in intensity according to the number of fluorescent subunits within a diffraction-limited region. in time-lapse FSM image series, movement of the fluorescent speckle pattern indicates translocation of structures, while changes in speckle intensity indicate subunit turnover. We have used FSM to study microtubule and actin behavior in interphase and mitotic cells. We use kymograph analysis to quantitate the movement of speckled structures (Fig 1) and are currently developing analysis procedures to quantify subunit turnover in structures.
We have applied these methods to the study of microtubule and actin cytoskeletal dynamics in migrating vertebrate cells in culture. Interactions between the microtubule and actin cytoskeletons underlie fundamental cellular processes such as cytokinesis and cell locomotion, but are poorly understood.
A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells
Authors: Maddox Paul , Canman Julie , Grego Sonia , Salmon Wendy , Waterman-Storer Clare , Salmon ED 
Published: July 2020

Abstract
High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.
Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.
Surfing the Conformational Wave of Acetylcholine Receptor Channel Gating
Authors: Cymes Gisela , Grosman Claudio , Auerbach Anthony 
Published: July 2020

Abstract
The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.
Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.
Design of a Microscopy System for Quantitative Spatial and Temporal Analysis of Multicellular Interactions
Authors: Sudar D , Callahan D , Parvin B , Knowles D , Ortiz de Solorzano C , Barcellos Hoff MH 
Published: July 2020

Abstract
The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture.
Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.
Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy
Authors: Toomre Derek , Keller Patrick , Diaz Elena , White Jamie , Simons Kai 
Published: July 2020

Abstract
Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (&lt;100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).
Self-Interference Fluorescent Emission Microscopy for Biological Imaging
Authors: Swan AK , Tong Y , Moiseev L , Ünlü M S , Goldberg BB 
Published: July 2020

Abstract
We present a new method of fluorescence imaging, which yields an unprecedented nm scale vertical resolution. The method uses the unique spectral signature of the fluorescent emission intensity above a reflecting surface to determine vertical position unambiguously. Emission from several heights could be resolved by deconvoluting the spectrum, so that three dimensional imaging is possible. The related technique of standing wave microscopy uses the spatial intensity variation for increased resolution. Applications of this technique include intracellular imaging and screening for specific bacteria, virus or proteins, where discrimination between raised fluorescently labeled specifically bound markers from non-specific binding is crucial.
The emission spectrum of fluorescent markers is modified in the presence of a reflecting surface. Within the wavelength range of the fluorescent emission, the selfinterference of the emitted photons from the direct and reflected path results in enhanced or suppressed emission (constructive or destructive interference) depending on the height and wavelength.
Application of Correlative Microscopy to Genetic Therapy Research of Cystic Fibrosis and Other Human Diseases
Authors: Moore Kenneth C , Wang Guoshen , McCray Paul , Shao Jian , Moninger Tom , Welsh Michael , Nessler Randy 
Published: July 2020

Abstract
Microscopy is an essential tool for developing strategies to use genetic therapy in the treatment of diseases. Negative staining (fig. 1) and cryo-electron microscopy are critical in understanding the structure of native and hybrid vectors to be used as transfer agents. Enzyme cytochemistry is widely used for the detection of markers that aid in determining the rate of gene transfer to cells and tissues. Immunocytochemistry is necessary to identify new gene products. Light, confocal and electron microscopy are used to evaluate pathogenesis, inflammation and cellular changes. For much of the past decade, we have been working to identify the specific gene and cell structure (chloride channel) that is compromised in Cystic Fibrosis patients. Subsequently, appropriate vectors had to be developed for treatment. A significant amount of time and effort has been expended to identify effective viral and other vectors to be used in the transfer of the normal gene construct.
In order to facilitate experimentation, we have developed a cell culture system utilizing normal and cystic fibrosis airway epithelia. These cultures are maintained at an air interface and the cells differentiate into the same types found in vivo.
Study of Distribution and Transport Events of the GluR1 AMPA Receptor: Combination of Genetically Modified Receptors and Multi-Resolution Microscopy
Authors: Stelljes A , Bushong EA , Martone ME , Wiseman PW , Hood KL , Mayford M , Ellisman MH 
Published: July 2020

Abstract
The formation of memory and the process of learning are believed to be regulated, at least in part, by the expression, distribution, and redistribution of glutamate receptors. The expression of these receptors at synaptic sites has a major impact on the strength of synaptic connections, and the AMPA receptor subunit GluR1 appears to play a key role within this system. Increasing evidence suggests that previously silent synapses become activated through rapid AMPA receptor insertion upon appropriate stimulation, and thus the trafficking of this receptor subunit from cellular stores to the synapse is of prime interest.
We are using a transgenic mouse expressing a GFP-tagged form of GluR1 (GluR1-GFP) in order to study the dynamic changes in GluR1 expression and distribution occurring during brain development and following induction of long-term potentiation (LTP). The fusion protein is transcribed under the control of the CaMKIIα promoter, which restricts the localization to forebrain neurons and is coupled to a tetrepressible system, thus allowing for the control of transcription with doxycycline. The anatomical distribution of GluR1-GFP on the light level is consistent with that of wild-type GluR1 (Fig. 1).
We are combining molecular biology with quantitative 3D image analysis on the light and electron microscopic level. The highly fluorescent polar tracer Alexa Fluor 568 (Molecular Probes Inc.) is being used to fill GluR1-GFP expressing pyramidal neurons in tissue slices fixed with aldehydes