bluuebunny/crossref_metadata_2025 huggingface dataset:
Towards EM tomography of large cellular volumes
Authors: Delaney A. , Bresler Y. , Belmont A.S.
Published: June 2020

Three-dimensional reconstructions of large cellular subvolumes would be quite valuable in addressing a number of basic questions related to cell ultrastructure. More specifically, we are interested in obtaining reconstructions with several nm resolution over projected areas of 5-50 um diameter and 1-10 um depth. Recently, EM tomography has been combined with serial sectioning as a means of accomplishing this goal. In practice, however, a number of technical problems must still be solved to realize the full potential of this approach, and most applications of EM tomography to date have been limited to high resolution reconstructions over much smaller volumes or lower resolution reconstructions over larger volumes.

A major limitation is the limited depth of focus of the electron microscope. Although this depth of focus usually exceeds the section thickness, at very high tilt the range of underfocus through overfocus in the image will approach the diameter of the reconstructed area.
Same-cell correlative video light microscopy/Electron Microscopy tomography: An approach to understanding kinetochore behavior during mitosis
Authors: McEwen B.F. , Osorio G. , Cole R. , Rieder C.L.
Published: June 2020

The goal of structural biology is to determine structure, structural relationships, and changes in these parameters in an effort to comprehend mechanism of function. In this context same-cell correlative LM/EM provides a powerful approach for elucidating the mechanisms responsible for the behavior of cell components. In this method the event of interest is followed in vivo by video-LM, and the cell then fixed at a critical time during the observational period for a subsequent 3D EM analysis. In this manner the history of a particular event or response can be correlated with the 3D ultrastructure underlying the event.

Biologists have long sought to elucidate the mechanism(s) that generate, control and coordinate the poleward and away-from-pole motion of sister kinetochores on each chromosome during mitosis. These “congression” movements require the association of microtubules with each kinetochore (kMTs), and ultimately align the chromosome on the spindle equator. Motion in each direction is distinct because the slow growing (−) ends of kMTs end near the spindle pole while the rapidly growing (+) ends terminate in the kinetochore plate.
Histochemical aspects of nucleic acid detection
Authors: Hamkalo B. A. , Unger Elizabeth R.
Published: June 2020

This symposium brings together several approaches for the detection of specific nucleic acid sequences that have potential applications at the histochemical level.

Trask et al. report on the use of fluorescence in situ hybridization (FISH) techniques to study the arrangement of DNA sequences in normal and diseaserelated chromosomes. The sites of specific DNA sequences can be fluorescently tagged. Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using fluorescence microscopy. The distances between points on the same or different chromosomes can be determined in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed.

Hamkalo and co-workers have used non-radioactive methods at the EM level for the detection of nucleic acid sequences by in situ hybridization. Analysis of metaphase chromosomes by electron microscopy allows for high resolution mapping of chromosomes. A variety of labelling procedures have been employed to illustrate the utility of high resolution nucleic acid sequence mapping in these preparations.
Analysis of DNA in archival tissues: comparison of in situ hybridization and polymerase chain reaction for detection of human papillomaviruses
Authors: Unger E.R. , Vernon S. D. , Lee D.R. , Miller D. , Reeves W.C.
Published: June 2020

Surgical pathology tissue archives have been discovered to be a valuable resource for investigators seeking to study molecular markers in well characterized patient material. The potential to analyze patient material stored for several years means that correlation of results of testing with disease outcome can be accomplished rapidly. The use of archival tissue is also important for relatively uncommon diseases which are difficult to obtain in a prospective fashion. The accuracy of molecular tests in formalin-fixed paraffin-embedded tissues can be compromised by variations in fixation and processing as well as by time of storage and storage conditions. To be reliable, molecular assays must be optimized to compensate for variations in tissue preservation. Both in situ hybridization (ISH) and polymerase chain reaction (PCR) assays have been designed to study DNA in archival tissues. The advantage of ISH is that the analysis is placed within a morphological context. This allows exclusion of cases where the marker is present in nonlesional tissue and allows detection of sub-populations of cells within the lesion.
Subcellular organization of snRNPS in mammalian cells
Authors: Matera A. Gregory
Published: June 2020

Our laboratory is interested in aspects of eukaryotic gene expression that may be regulated at the level of nuclear architecture and/or localization. Understanding the spatial organization of genes and gene products within mammalian nuclei will provide valuable insight into how the various metabolic processes such as replication, transcription, processing and transport are orchestrated. In order to begin to address these questions, we have chosen to study the cell biology of RNA processing. Small RNA-protein complexes known as ribonucleoproteins (RNPs) are central factors in numerous RNA processing reactions. Much of what is currently known about the subcellular organization of RNPs is based upon the locations of the protein components of RNPs, not the RNA components. Furthermore, since many RNAs share common protein subunits it is essential to develop probes which are specific to the individual RNAs. We utilize highly specific antisense oligonucleotide probes and fluorescence in situ hybridization (FISH) to ascertain the organization of RNPs and their associations with particular subdomains within mammalian cells.
Membrane microdomains: lessons from microscopy
Authors: Robinson J.M. , Oliver J.M
Published: June 2020

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.

This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.
Segregation of cell adhesion molecules into membrane microdomains facilitates leukocyte-endothelial interactions
Authors: Erlandsen S.L.
Published: June 2020

Cells interact with their extracellular environments by means of a variety of cellular adhesion molecules (CAM) and surface ligands. In many instances, CAMs interact in a sequential temporal fashion which suggests that these adhesion molecules may occupy or be polarized to various membrane microdomains on the cell surface. Detection of CAMs can be accomplished by a variety of methods including immunofluorescent microscopy and flow cytometry, and by the use of immunocytochemical markers (i.e. colloidal gold) in electron microscopy. The development of high resolution field emission SEM in the mid 1980's and the Autrata modification of the YAG detector for backscatter electron detection at low voltage has greatly facilitated the recognition of colloidal gold probes for detection of surface CAMs. Low voltage FESEM with Bse imaging provides increased resolution of cell surface topography (~3nm at 3-4 keV) which can be observed in 3-dimensions, and simultaneously permits detection/high spatial resolution of immunogold label by atomic number contrast.
Quantifying IgE receptor aggregation from SEM-immunocytology
Authors: Wofsy Carla , Sanders Margaret L. , Donohoe Gregory W. , Pujol Matthew , Oliver Janet M.
Published: June 2020

Improved procedures for imaging immunogold markers on scanning electron micrographs, new procedures for extracting gold particle coordinates from the images, and both new and standard statistical methods for analyzing spatial point patterns make it possible to study quantitatively the aggregation of cell surface molecules and their localization in specialized membrane domains. The imaging procedures have been described previously. Here we present the methods we have developed or adapted for image processing and for statistical analysis of the immunogold point patterns. We apply the methods to study the aggregation of the high affinity receptor for IgE (Fc∊RI) on rat basophilic leukemia (RBL) cells.

The aggregation of cell surface receptors is a necessary step in most known receptor-mediated cellular processes. For example, activation of the intrinsic kinase activity of growth factor receptors requires receptor aggregation, as does the initiation of a variety of antigen-induced responses in the immune system. In the most common form of allergic reaction.
Compartmentalized cell signaling in caveolae
Authors: Anderson Richard G.W.
Published: June 2020

Caveola undergo an internalization cyclic that begins with the conversion of a flat segment of membrane into a vesicle and ends when that vesicle returns to the cell surface. During this cycle, resident molecules are sequestered in the interior of the vesicle where they perform special functions. Previously we showed that the internalization step is interrupted by activators of PKC such as phorbol-12,13-dibutyrate (PDBu). Recently we found that caveolae contain a resident population of PKCα molecules that phosphorylate a 90-kDa protein during invagination. In unstimulated cells, virtually all of the detectable PKCα in plasma membranes is in caveolae. Treatment of the cells with PDBu or agents that raise the concentration of diacylglycerol (DAG) in the cell displaced PKCα from caveolae, which blocked the phosphorylation of the 90-kDa protein and membrane invagination. Caveolae also contain a protein phosphatase that dephosphorylated the 90-kDa protein once PKCα was gone or inactive. PKCα was also dissociated from caveolae and invagination was inhibited after cells were incubated in the presence of histamine. This effect was blocked by pyrilamine but not cimetidine, suggesting that histamine was acting through H1 receptors.
High-resolution light microscopy of living organs in situ
Authors: McCuskey R. S.
Published: June 2020

Most organs in anesthetized small laboratory animals can be studied in vivo by light microscopy of relatively thin (3-5mm), transilluminated areas of the organ. Thicker areas of the organs in these species, as well as thicker organs of larger animals can be examined only by epi-ilumination. However, the resolution obtainable with epi-ilumination usually is inferior to that realized with transillumination. This paper reviews these methods using the liver as an example of the organ of study. A standard compound trinocular microscope is used which is modified for in vivo microscopy and is equipped for both transillumination and epi-illumination. After the animal is anesthetized, the liver is gently exteriorized through a subcostal, abdominal incision and positioned over a window of optical grade mica or glass on a specially designed, heated microscope stage having provisions for draining irrigation fluids. The window overlies a long working distance condenser. The liver is covered by a piece of Saran or Mylar film which holds it in position and limits movements induced by respiration, the heart and the intestines.
Predicting spatial activity patterns in a neural map from a probabilistic atlas of 3-D reconstructed neurons
Authors: Jacobs G. , Theunissen F.
Published: June 2020

In order to understand how the algorithms underlying neural computation are implemented within any neural system, it is necessary to understand details of the anatomy, physiology and global organization of the neurons from which the system is constructed. Information is represented in neural systems by patterns of activity that vary in both their spatial extent and in the time domain. One of the great challenges to microscopists is to devise methods for imaging these patterns of activity and to correlate them with the underlying neuroanatomy and physiology. We have addressed this problem by using a combination of three dimensional reconstruction techniques, quantitative analysis and computer visualization techniques to build a probabilistic atlas of a neural map in an insect sensory system. The principal goal of this study was to derive a quantitative representation of the map, based on a uniform sample of afferents that was of sufficient size to allow statistically meaningful analyses of the relationships between structure and function.
Characteristic feature on structure analyzed by high-resolution electron crystallography
Authors: Fujiyoshi Y. , Mitsuoka K. , Hirai T. , Murata K. , Miyazawa A. , Kimura Y.
Published: June 2020

The structure of bacteriorhodopsin (bR), which was already analyzed by Henderson et al., is studied by our new electron cryo-microscope equipped with Field Emission Gun (FEG) and Slow Scan CCD camera (SSCCD), because our system together with ice embedding technique enable us to solve the structure of bR at various pH conditions between pH 4.0 and 10.0. Ionization of amino acid is naturally closely related to the translocation of proton and then the function of the proton pump of bR. Therefore, observation of translocation of proton in bR is very important, if possible. Both ice embedding and high resolution techniques are essential to achieve this intention. Therefore, we intended to develop an electron cryo-microscope fit to these techniques and recently we had succeeded it.

We collected whole sets of diffraction patterns for bR up to 70 degree tilt at pH 5.5 by using SSCCD, and merged these data of 300 diffraction patterns.
Structure design of osmo-regulated H2O-channel inferred from high-resolution projection map obtained by electron crystallography
Authors: Jap Bing K. , Li Huilin
Published: June 2020

The aquaporins (AQP) are a class of water channel proteins belonging to the major intrinsic protein (MIP) superfamily. This class of proteins includes the osmo-regulated H20-channel protein AQP-CHJP, which was first identified in erythrocyte membranes and later found to be present in a variety of tissues from organs such as the kidney, gall bladder, lung, intestine and eyes. These channels are believed to be osmotically regulated and to transport water across a number of endothelial and epithelial cell layers during fluid secretion and absorption. AQP-CHJP is extremely specific for the transport of water; small solutes such as urea and ions have been shown to be impermeable to the channels.

We have chromatographically purified AQP-CHJP from bovine red blood cell membranes to homogeneity with the use of octylglucoside as the detergent. We have also reconstituted the purified protein with dimyristoyl phosphatidylcholine, yielding crystalline ordered arrays. To increase the percentage of highly coherent crystalline patches, the reconstituted membrane patches were further treated with phospholipase A2 to remove excess lipids.
Low-dose energy-filtered TEM of frozen hydrated virus particles using a post-column energy filter
Authors: Grimm R. , Baumeister W.
Published: June 2020

Zero-loss filtered imaging of frozen hydrated macromolecular structures has become a commonly applied method over the last ten years, Instruments with in-column filters have been dominant due to their commercial availability (maximum voltage 120 keV), and because they allow the microscope to be used as for standard imaging, with essentially no modification in the procedure for low-dose microscopy Post-column filters have been used mainly in materials science and for intermediate and high voltage microscopes.

At the MPI in Martinsried we have recently added a Gatan post-column imaging filter (model 678) to and existing CM 12 electron microscope (120 keV, LaB6 cathode). The lowest magnification of the microscope with the objective lens excited is 600x at the viewing screen (lowered from standard 3000× by free lens control). The imaging filter uses a central portion of about 2 mm diameter of the image or the screen, and adds an additional magnification of 18x.
High magnification in-lens field emission SE-I SEM of carbon based styrenes, diamonds, and endothelial cell membranes
Authors: Apkarian Robert P. , Tarr Jonathan A. , Kaufmann Myron J. , Menger Fredric M.
Published: June 2020

High resolution topographic imaging of carbon based samples have been problematic primarily due to the low secondary electron-I yield from specimens of low atomic mass. Both cold cathode and Schottky field emission (SFE) in-lens SEMs produce small beam diameters (5-10 Å) and achieve their highest resolution when operated at high (20-30 keV) accelerating voltages. The Topcon in-lens SFE-SEM/STEM has been in service at Emory U. for four years, and has generated quality images of carbon based materials and biological membranes. Enrichment of the SE-I signal from hydrocarbon based specimens was achieved by the application of a 1 run thick, fine grain Cr film and provided images of 1-2 nm particulate features without decoration or enlargement. Specimen beam interaction and the resultant particle contrast in a high magnification analog image recording was compared for graphitic carbon, diamond, and chromium coated polyvinylstyrene and fenestrated endothelial cell membranes (FECM).

Chemical vapor deposition (CVD) was used to deposit various forms of carbon on Si (111) wafers.
Human T-lymphocytes (Jurkat cells) expressing TAT-72: LM and TEM
Authors: Gilloteaux J. , Koch J. , Jamison J. M. , Summerst J.L.
Published: June 2020

The human immunodeficiency virus (HIV-1) is dependent upon the synthesis of TAT for efficient transcriptional activation and replication1. TAT serves to transactivate the HIV-LTR which leads to an increased transcriptional activation of approximately 1000 fold.

We have transfected human T-lymphocytes with an expression vector containing the HIV-1 LTR driving the synthesis of TAT-72. A stable cell line was generated by selection with G418.

Cells were centrifuged with a cytospin and stained for nucleic acids with methyl green and pyronin Y in order to visualize and to localize DNA and RNA respectively in each preparation examined by light microscopy (LM). In addition, cells were fixed with a 3.5% glutaraldehyde buffered solution (0.1M Na cacodylate) for 5 min at room temperature, 35 min at 4°C, centrifuged in a microfuge (400 rpm, 3 min) to obtain a cellular pellet. Cell pellets were washed in a buffer-sucrose solution and postfixed in a 1.5% aqueous solution of OsO4. Dehydration and epoxy embedding was followed by sectioning for TEM, following treatment with uranyl and lead salts.
SEM of the tongue and related vomeronasal structures in Coluber constrictor and Uta stansburiana
Authors: Froerer J. , Gardner J. S.
Published: June 2020

The forked tongue in snakes and lizards serves to follow the pheromone trails of both prey and conspecifics. The three phases of the tongue flicking cycle are interrelated with three functional structures involved with the transportation of stimulus particles from the environment to the vomeronasal organ (VNO). (1) The sample phase involves protrusion of the tongue, collection, and transfer of stimulus particles to the anterior processes. (2) A transfer phase in conjunction with the anterior processes serves to facilitate the collection and localization of stimulus particles from the tongue. (3) The delivery phase includes anterior process elevation and the delivery of particles to the VNO. The VNO simultaneously deciphers the chemical concentrations, enabling the animal to follow pheromone trails in relation to the most volatile side, right or left. The morphology of the tongue, anterior processes, and VNO of a previously unevaluated snake and lizard are presented here.

Snake species Coluber constrictor and lizard species Uta stansburiana were used in this study. Snakes were collected in Northern Utah and lizards were collected in Southern Utah. All specimens were prepared for scanning electron microscopy using standard techniques.
Molecular chaperones HSP-70 and BiP are present in the secretory pathway of exocrine glands
Authors: Menini T. , Bendayan M.
Published: June 2020

In the past few years the study of a new group of proteins, the molecular chaperones, has demonstrated their relevant roles in cell biology. These proteins have been extremely well conserved during evolution, with percentages of homology reaching 50% between members of the same family (l).

The heat shock 70 kDa family of proteins belonging to the molecular chaperone group includes at least 4 members which are permanent residents of several cellular compartments. Besides being induced by stress, these proteins fulfil many important functions such as collaborating with the proper folding and assembly of newly synthesized proteins and facilitating their transport and translocation into organelles (2).

Different studies have demonstrated the involvement of these proteins in cell secretion in bacteria and yeast (3). Few data are however available for mammalian cells (4). In this regard, we have set out a study to demonstrate the presence and localization of two members of this family of proteins, the HSP-70 and the BiP, on several exocrine glands.
Recognition-alignment and adhesion in myoblast fusion
Authors: González Santander R. , Toledo Lobo M.V. , Martínez Alonso F.J. , Martínez Cuadrado G. , González-Santander Martínez M , Monteagudo M.
Published: June 2020

Myoblast fusion results from a sequence of different stages, previously demonstrated “in vitro”. After withdrawal from the cell cycle, myoblasts align forming long chains, in a process termed “recognition-alignment”. This stage is extracellular Ca2+ and N-Cadherin dependent. Alignment is followed by adhesion, defined as the stage prior to membrane fusion when aggegates are resistant to dispersal by EDTA. Adhesion is extracellular Ca2+-independent and N-CAM-dependent. Membrane fusion originates multinucleate myotubes.

We have studied these stages of myoblast fusion at the brachial myotome of chick embryo from 51 to 105 h. of incubation. Samples were obtained by embryo microdissection and included the neural tube, the notochord and the brachial somites. These samples were embedded in araldite by conventional methods. Some samples were embedded in Unicryl, a recently formulated GMA derived resin.

The first myoblasts clusters were observed in the ventral-lateral region of the brachial myotome in 22-24 Hamburger and Hamilton stages embryos. Clusters of pre-fusion myoblasts are usually surrounded by “electrondense blast cells” within a basal lamina in process of formation.
Cytochemical study of glycogen in myoblasts of the chick embryo myotome
Authors: González Santander R. , Toledo Lobo M.V. , Martínez Alonso F.J. , Martinez Cuadrado G. , González-Santander Martínez M. , Monteagudo M.
Published: June 2020

Potassium ferrocyanide-reduced osmium has been used as secondary fixative to study the presence of glycogen in different tissues. However, this method has not been previously applied to skeletal muscle development research. This study reports the use of this method to analyze the structure and cytoplasmic distribution of glycogen particles in the myoblasts of the brachial myotome of chicken embryos at 51-105 h. of incubation (Hamburger and Hamilton stages 16 to 25).

In those sections post-fixed with potassium-ferrocyanide reduced osmium it is observed that glycogen particles stain in contrast to the rest of the structures of the cell (Figs. 1,2). In those sections processed for T.E.M., following conventional methods, glycogen particles are occasionally observed in less contrast and number that those with potassium-ferrocyanide reduced osmium. An explanation for this differences might be in the effects of potassium-ferrocyanide reduced osmium fixation preventing the loss of glycogen during dehydration and staining with uranyl acetate. We conclude that this cytochemical method may be specific for glycogen detection.
A method for encystation of Entamoeba invadens
Authors: Morales-Vallarta M. , Villarreal-Treviño L. , Ramíez-Bon E. , Said-Fernández S. , Mata-Cárdenas B.
Published: June 2020

Some axenic encystaticn media for Entamoeba invadens have been described for different authors: Rengpien and Bailey medium (AEM (1), Vázquezdelara-Cisneros and Arroyo-Begovich (low glucose medium) (2) and Das et al. medium (3). The first medium must aging for one month for reach maturity, moreover its efficiency is also variable, on the other hand, the three media require cells at middle log-phase for good results. In this paper we report not a medium but a method for encystation of Entamoeba invadens.

Trophozoites of Entamoeba invadens IP-1 strain were incubated with 103 cells/ml in 16 × 150 mm screw capped tubes, each containing 10 ml of culture medium (TP-S-1 (4) or TYI-S-33 (5) or FEHPS (6)) without vitamines (except FEHPS medium), and gassed with CO2 (0.2 l/min/2 min) through a pasteur pi _ pette with a cotton filter and incubated to 25°C. After 4 days the culture medium is eliminated and sustituted with 10 ml of the same medium but without glucose, newly gassed as above mentioned and incu bated to 25°C. After 4-5 days, formation of cystic structures reach a maximum efficiency of 70-80%.
What microscopic studies of confronting cisternae and annulate lamellae reveal about their similarities and differences
Authors: Palisano John R. , Renzaglia Karen S.
Published: June 2020

Knowledge of the origin and function of confronting cisternae (CC) and annulate lamellae (AL) has been limited because they are so infrequently observed. CC were first described by Porter in 1955 and have since been observed in a variety of rapidly proliferating tumor cells and selected fetal tissues. AL were first reported in 1952 by McCullough in sea urchin eggs. Although AL have since been observed in a variety of cells, they have been predominately found in oocytes, spermatocytes, and tumor cells. A microscopic study was initiated to gain a better understanding of the origin and role of these two membranous organelles that are frequently found in rapidly dividing cells that lose the ability to express these organelles once their rapid proliferative stage ceases. Because it has proven difficult to interpret the flow of membrane in the mitotic cell by electron microscopy alone, data obtained by electron microscopy was correlated with fluorescence microscopy wherever possible.
Rabbit extrasinovial regenerating tendon: an experimental study
Authors: González Santander R. , Plasencia Arriba M.A. , Lopez Alonso A. , Toledo Lobo M.V. , Martínez Cuadrado G. , Martínez Alonso F.J. , González-Santander Martínez M. , Monteagudo M.
Published: June 2020

The roles of the extrasynovial tendon cells (Achilles tendon) throughout the repairing process following injury are controversial. This study was performed to investigate the ultrastructural features of activated tenocytes and collagen synthesis experienced while healing after partial tenotomy and repair.

Adult rabbit Achilles tendons were partially tenotomized. Before the suture of the paratenon, the borders of the surgical dissection were treated with different substances copolimerized with hydrogel. Hydrogel non-treated, tenotomized tendons were considered as controls. The rest of the samples were treated with hydrogel alone or hydrogel with E-vitamin. Rabbit legs were immobilized for 3, 10, 20 and 30 days. Samples from tenotomized and normal tendons were embedded in parafin and in Spurr and Araldite resins by conventional methods.

With LM, a more pronounced healing process was observed in tendons treated with hydrogels if compared with controls, but this comparative study needs further investigation. During the process of repair, different stages can be identified: inflammation, revascularization and angiogenesis, cell proliferation, collagen synthesis and fibrillogenesis, and alignment/organization of collagen fibers into bundles oriented in the longitudinal axis of the tendon.
Microanatomical changes of the cerebral capillary network in aged rats: influence of treatment with a dihydropyridine-type calcium antagonist
Authors: Zaccheo D. , Amenta F.
Published: June 2020

A variety of age-dependent changes affect cerebral vasculature. Morphological changes of the brain microvasculature characterized by microvascular fibrosis , vascular convolutions , thickening or membranous changes of the capillary basal membrane and gliofibrillary proliferation have been reported with aging. These changes may be related with age-dependent impairment in behavioral performance. Increasing evidence suggests that Ca+2 antagonists of the dihydropyridine family, which are used primarily in the therapy of cardiovascular disorders may be useful for treating neurologic diseases including cerebral ischemia, age-related neurodegenerative disorders, senile dementia and epilepsy. This in view of the capability of these compounds to decrease Ca+2 overload which can result in increased cell death.

Recent studies have shown that the dihydropyridine Ca2+ antagonists delayed the expression of agerelated microvascular changes in the rat forebrain and sciatic nerve without reducing systolic blood pressure. The present study was designed to assess the influence of long term treatment with the dihydropyridine derivative darodipine [diethyl 4-2,1,3- (benzoxadiazal-4-yl)-l,4-dihydro-2,6-dimethylpyridine- 3,5-dicarboxylate, PY 108-068] on age-related microvascular changes occurring in the rat cerebral cortex and hippocampus. The brain capillary network was investigated using alkaline phosphatase histochemistry associated with image analysis.
Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy
Authors: Selinfreund R H. , Cornell-Bell A. H.
Published: June 2020

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.

The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.
SEM study of the morphological effects of kinetin and zeatin on the growth and development of the apical meristem in wheat
Authors: Hutchins G. M. , Gardner J. S.
Published: June 2020

Cytokinins are plant hormones that play a large and incompletely understood role in the life-cycle of plants. The goal of this study was to determine what roles cytokinins play in the morphological development of wheat. To achieve any real success in altering the development and growth of wheat, the cytokinins must be applied directly to the apical meristem, or spike of the plant. It is in this region that the plant cells are actively undergoing mitosis. Kinetin and Zeatin were the two cytokinins chosen for this experiment. Kinetin is an artificial hormone that was originally extracted from old or heated DNA. Kinetin is easily made from the reaction of adenine and furfuryl alcohol. Zeatin is a naturally occurring hormone found in corn, wheat, and many other plants.

Chinese Spring Wheat (Triticum aestivum L.) was used for this experiment. Prior to planting, the seeds were germinated in a moist environment for 72 hours.
Ultrastructural changes in roots of peppers resistant and susceptible to Phytophthora capsici
Authors: Ilarslan H. , Ustun A.S. , Yilmazer R.
Published: June 2020

The infection by Phytophthora capsici Leonian causes foliar blight and crown and root rot of pepper plants. The ultrastructural examination of resistant and susceptible host-pathogen interactions was conducted in the pepper cultivars Ince Sivri-35, PM217, and PM702=CM 334 following inoculation with Phytophthora capsici. Responses were characterized and compared with healthy non-inoculated controls at 2,4 and 6 days after inoculation. Numerous ultrastructural studies have been made of the interaction of various host plants Phytophthora spp. No ultrastructural studies comparing the resistant and susceptible reactions of pepper cultivars to P. capsici. have been reported. It is important to examine the ultrastructural changes in inoculated and infected tissue of resistant and susceptible pepper cultivars to P. capsici. Information reported here characterizes the processes of pathogen containment in resistant interactions and compares these with the processes occuring in susceptible interactions.

After 2 days in susceptible interactions, the pathogen grew intercellularly in roots, whereas in resistant interactions only a few intercellular hyphae were observed penetrating the host cells and forming haustoria.
Comparison of localization of metallothionein in tissues of metallothionein-deficient mice
Authors: Nishimura H. , Nishimura R , Adelson D.L. , Michaelska A.E. , Choo K.H.A. , Tohyama C.
Published: June 2020

Metallothionein (MT), a cysteine-rich heavy metal binding protein, has several isoforms designated from I to IV. Its major isoforms, I and II, can be induced by heavy metals like cadmium (Cd) and, are present in various organs of man and animals. Rodent testes are a critical organ to Cd and it is still a controversial matter whether MT exists in the testis although it is clear that MT is not induced by Cd in this tissue. MT-IV mRNA was found to localize within tongue squamous epithelium. Whether MT-III is present mainly glial cells or neurons has become a debatable topic. In the present study, we have utilized MT-I and II gene targeted mice and compared MT localization in various tissues from both MT-deficient mice and C57Black/6J mice (C57BL) which were used as an MT-positive control. For MT immunostaining, we have used rabbit antiserum against rat MT-I known to cross-react with mammalian MT-I and II and human MT-III. Immunohistochemical staining was conducted by the method described in the previous paper with a slight modification after the tissues were fixed in HistoChoice and embedded in paraffin.
Immunolocalization of lactase in the crypt and villus enterocytes of the neonatal pig intestinal epithelium
Authors: Heath Julian P. , Kömüves László G.
Published: June 2020

The crypt is the proliferative compartment of the intestinal epithelium where pluripotent stem cells continuously divide giving rise to committed daughter cells that follow a differentiation pathway as they migrate out of the crypt and along the villus axis. Certain cell antigens and markers are crypt-cell specific. Others, such as brush border enzymes, are thought to be expressed only on fully differentiated enterocytes, a theory supported by in situ hybridization analyses that reveal an absence of enzyme mRNAs in the crypt. But recent studies using immunocytochemical detection methods indicated that the brush border enzymes sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV are in fact expressed by both crypt and villus enterocytes. We are using immunogold electron microscopy to examine the effects of diet on enzyme expression in the newborn pig intestine.

Tissues were collected from the jejunum of newborn and 9 day-old piglets. Cryosections (100 nm thick) were incubated in monoclonal anti-pig lactase antibody (PBB.3.7 from Dr. A. Quaroni, Cornell Univ) followed by 17 nm gold-conjugated anti-mouse immunoglobulin G.
Ultrastructural distribution of steroid sulfatase activity and mRNA in the human kidney
Authors: Nishikawa M. , Nakazawa T. , Kanda N. , Aikawa E. , Toma H.
Published: June 2020

Steroid sulfatase (STS) is a microsomal enzyme catalyzing hydrolysis of 3β-hydroxysteroid sulfatases, including dehydroepiandrosterone sulfate, cholesterol and estrogen precursors. STS exists widely in various tissues and is involved in the metabolism of steroid hormones which play a critical role in cell proliferation and may be related to cancer induction in several organ systems. In the kidney, the proximal and distal portions of the nephrons have been suggested as possible sites of synthesis or as target tissues for steroid hormones. However, the presence and the role of STS in the human kidney have not been fully investigated. In this study, we examined the distribution of STS activity and mRNA in human renal tissue by enzyme histochemistry and in situ hybridization at the light and electron microscopic level.

Renal tissue specimens were taken from normal areas in the surgically resected kidney from patients with renal cancer. STS activity was analyzed by enzyme histochemistry using 4-methylumbelliferyl sulfate. STS-mRNA distribution was detected by in situ hybridization using an STS cDNA probe cloned from human placenta.
Improvement of lysine effect on aldehyde-ruthenium red preservation and staining of the staphylococcal glycocalyx
Authors: Fassel Theresa A. , Mozdziak Paul E. , Sanger James R. , Edmiston Charles E.
Published: June 2020

The cationic reagent ruthenium red (RR) has been used to enhance ultrastructural preservation and staining of the bacterial glycocalyx. Lysine was needed in prefixation to preserve the polysaccharide glycocalyx material for the gram-positive, coagulase-negative staphylococci species. The inclusion of lysine in the prefixative of glutaraldehyde (GA)-RR enhanced observation of elaborate and extensive glycocalyx material. However, prefixation in 75 mM lysine in GA-RR is restricted to 20 minutes because gelling or solidification frequently occurs at longer intervals resulting in loss of sample. To improve the utility of this approach, the effect of the Karnovsky ratio of 2% paraformaldehyde to 2.5% GA was investigated.

Cells of gram-positive, coagulase-negative staphylococci species, Staphylococcus epidermidis RP62, a polysaccharide producer, and Staphylococcus epidermidis M187-SN3, a polysaccharide negative mutant, were recovered from frozen storage and grown on blood agar plates. After 24 hours, cells were transferred to trypticase soy broth for 18 hours, and incubated at 35°C. Half of the cells were prefixed in 75 mM L-lysine, 0.075% RR, 2.5% GA in 0.1 M sodium cacodylate pH 7.2 for 20 minutes.
Intensity attenuation of Three-Dimensional, confocal fluorescence images of thick tissue
Authors: Kayali S. , Ancin H. , Roysam B. , Shain W. , Szarowski D.H. , Turner J.N.
Published: June 2020

The confocal scanning laser microscope (CSLM) provides three-dimensional (3-D) images from fixed tissues. These images are obtained by stacking consecutive confocal planes through the depth of a specimen. We have developed software for quantitative analysis of these data sets and have applied these methods to cell counting. A major issue in this analysis is the relative intensity of signal measured from like objects through the depth of the specimen.

The rat hippocampus was chosen to test the attenuation of the fluorescence signal allowing us to make observations in areas of higher density-pyramidal cell layer-and lower density -extra-pyramidal regions. Paraformaldehyde fixed specimens were stained with Feulgen-Schiffs acriflavine. This stain was selected for its fluorescent properties and its high DNA specificity. Specimens were mounted in media with different glycerol concentrations. Significant attenuation was observed using a 50:50 mixture of glycerol and buffer, while 100% glycerol provided significantly less attenuation. All data presented here were collected using sections mounted in 100% glycerol. 3-D images were collected from 50-, 75-, and 100-μm thick sections with a 40x oil objective, having an (x,y) resolution of 2 pixels//xm and a distance of 1 fim between optical sections. Fig. 1 is a projection of a 100-μm section of the rat hippocampus illustrating pyramidal and extra-pyramidal areas.
Treatment of the gradient of defocus in images of tilted, thin crystals
Authors: Henderson R. , Baldwin J.M.
Published: June 2020

Bacteriorhodopsin, a membrane protein from Halobacteria, forms two-dimensional crystals which often have diameters of 20 microns. Several crystal forms have been obtained with cell dimensions of about 60 Å and diffraction to beyond 3 Å resolution. The structure of one of these crystal forms has recently been determined in projection at 3.5 Å resolution using images recorded at low temperature, together with computer image analysis methods that enable the averaging of information from areas of up to 1 micron in diameter. Electron diffraction intensities have also been recorded from tilted and untilted specimens providing three-dimensional amplitude measurements to 3 Å resolution. We are now analysing images from tilted specimens that should provide phases for the Fourier components of the structure to high resolution in three dimensions.
3-D reconstruction from serial thick sections, with multiple-level profiling within stereo pairs, using the albany HVEM's “SSRECN” software
Authors: Marko M. , Banerjee R. , Leith A. , King M. , Radermacher M. , Frank J. , Parsons D.
Published: June 2020

We have designed and written a program, SSRECN, for making 3-D reconstructions from contours in parallel planes. In our work with serial thick sections, we find a need to sample the object at multiple levels in depth, and we suggest a means for doing this.

SSRECN was written with the biological electron microscopist in mind, and its capabilities cover all common requirements for such work. Additional features are added as special user needs come up. Special care was taken to make it “user friendly”; it is menu-driven, with liberal helps and explanations available on-line, and requires no prior computer experience or reading of documentation. Error traps prevent frustration due to accidental loss of data or program crashes. Its comprehensive and versatile database system provides for handling three-dimensional contour data (including depth connection instructions for reconstruction of branching structures) at all points in the program, both as single contours and as groups (i.e. individual contours of a single organelle and also those of a group of organelles of the same type).
Tantalum replicas of freeze-fractured proteoliposomes
Authors: Costello M.J. , Burgess F. , Escaig J.
Published: June 2020

Tantalum will form thin, fine-grained metal films, and under some conditions, continuous films with high resolution. We have prepared pure tantalum metal replicas of frozen fractured proteoliposomes employing a new electron gun designed for the Reichert Cryofract 190 freeze-fracture-etch device. The replicas are similar in appearance to those made with conventional platinum/carbon deposition except that tantalum replicas can be made thinner and with smaller grains, and the intramembrane particles (IMPs) which occur on fracture faces have smaller dimensions.
Membrane lipids: Electronmicroscopic correlates
Authors: Friend D. S.
Published: June 2020

The dimensions of individual membrane lipids are too small to be seen by transmission electronmicroscopy. In order to obtain even approximate information about lipid planar and transbi-layer distribution, strategies have therefore been developed using radioautography, lipase digestion, rapid freezing, freeze-fracture, and cytochemistry employing globulins and antibiotics. Each approach has its inherent limitations.
Electron microscopy of frozen, hydrated lacrosse virus
Authors: Talmon Y. , Prasad B. V. V. , Clerx J. P. M. , Chiu W. , Hewlett M. J.
Published: June 2020

LaCrosse (LAC) virus is a member of the California serogroup of the Bunyaviridae. The bunyaviruses are all negative-sense, RNA viruses whose genome consists of three segments of single stranded RNA. LAC virions are composed of multiple viral RNA strands complexed with viral nucleocapsid protein surrounded by a bilayer membrane embedded with 2 glycoproteins.

Staining and drying of LAC virus samples produce images in which the virions are collapsed and distorted (Figure 1). Fixation prior to staining, also gives disappointing results, probably due to the high sensitivity of the LAC virus to changes in the ionic strength and composition of the solution in which it is suspended.
Estrogen and progesterone receptors in carcinoma of the breast (A cytochemical, immunofluorescence and electron microscopic study)
Authors: Elliott Robert L. , Harrison Gregory O. , Coble-Landry Anne , Bullock-Ledford Lindsay , McRea Anne T. , Nutt Debbie
Published: June 2020

Tissue from 105 patients with infiltrating ductal carcinoma of the breast was assayed for the estrogen and progesterone receptor by the cytochemical dextran coated charcoal technique and by immunofluorescence with a direct dual staining procedure with Fluorocep, and also examined electron microscopically.

Tissue was fixed in Zambonis; post-fixed in 1% OsO4; embedded in a Med Cast Resin, and thin sections were cut on a ultramicrotone. They were stained with uranyl acetate and lead citrate and examined in a Hitachi H-600 electron microscope.
Ultrastructural investigation of embryonal carcinoma of the testis
Authors: Siew S. , Newton B.
Published: June 2020

Testicular germinal tumors are the most common malignant neoplasms in young men. We had the opportunity to examine a case of embryonal carcinoma of the testis by means of transmission (TEM), scanning (SEM) electron microscopy and energy dispersive x-ray analysis. The propositus was a man of 21 years of age, who complained of left testicular discomfort, which radiated to the left anterior superior spine in the inguinal area. On examination, the right testis was normal. There was slight enlargement of the left testis with a definite nodularity, medially, where there was a very firm area. The epididymis was normal on palpation. Surgical resection was performed with removal of the left testis and dissection of the retroperitoneal lymph nodes. The testis measured 5.2x3.4x3 cm. On section, it had a lobulated appearance. There was gross evidence of extension into the rete testis but the tunica albuginea was not penetrated and the epididymis was normal.