bluuebunny/crossref_metadata_2025_split huggingface dataset:
Low-temperature Scanning Electron Microscope constructed with high-excitation objective lens
Authors: Koike H. , Inoué T. 
Published: June 2020

Low temperature microscope technology can be traced back to the last century including the time of light microscope, and its history is over a hundred years. In the field of electron microscopy, low temperature techniques such as the freeze-fracture replica, freeze-sectioning, freeze-substitution, etc. were tested up to early 1960s. According to the progress of the rapid-freezing method, the freeze-substitution and freeze-etching replica methods have provided great successful results.The low temperature scanning electron microscope (LTSEM) was also tested by Echlin et al. in 1970, and thereafter, a number of LTSEM constructions were attempted. These LTSEMs are generally classified into two groups: the type with fracturing and coating facilities directly attached to the SEM column, and the group having a separated preparation chamber and a transfer device. The LTSEM so far constructed were reviewed comprehensively in greater detail. Some such instruments were designed taking account of stringent requirements of low temperature techniques. These systems, however, seemed to be too comprehensive, involving complex procedures as compared withe their resolutions. In comparison with the conspicuous results obtained by other low temperature techniques, the LTSEM can be regarded as still in the stage prior to practical application from the viewpoint of the high resolution. In consideration of these circumstances, the present paper aims at providing a new LTSEM to realize simple operation retaining the advantage of the ultrastructural preservation by the rapid-freezing and the high resolution by introducing the high-excitation objective lens.
Anatomy and cytochemistry of abscission zones by microscopical examination of nitrocellulose tissue prints
Authors: Reid Philip D. , del Campillo Elena 
Published: June 2020

Tissue printing on nitrocellulose membranes has been used to localize extensin in soybean seeds and as a matrix for histochemical localization of enzymes in bean abscission zones. Pressing hand cut sections of abscission zones into nitrocellulose membranes results in a tissue print which is seen as a three dimensional image when viewed with side illumination and shows the characteristic morphology and anatomical organization of the tissues in these segments. Changes in the cells of the abscission layer can be observed on the tissue prints as the process of abscission proceeds, and treatment of the tissue prints with antibodies to abscission specific enzymes identifies the cells in the abscission layer.Ten day old explants of Phaseolus vulgaris c.v. red kidney were induced to abscise by removing the leaf blades. Tissue prints were made on dry nitrocellulose membranes (0.45 μm pore size) at zero time, 24, 48 and 53 h and observed using either transmitted or side illumination at 10X magnification. Some tissue prints were incubated with rabbit 9.5 cellulase antibody followed by exposure to an alkaline phosphatase conjugated-goat antirabbit antibody. Color was then developed using standard alkaline phosphatase substrates. The 9.5 cellulase is an abscission specific form of the enzyme.
Changes in microtubule arrays during cotton fiber development
Authors: Seagull Robert W. 
Published: June 2020

Developing cotton fiber provides an excellent model system for the study of microtubule (MT) arrays and their involvement in cell wall deposition. The secondary wall of the cotton fiber is arranged in layers of parallel microfibrils, to produce a polylamellate configuration. Cortical MTs parallel microfibrils and undergo re-orientations which predicted new microfibril orientations. Previous studies describe the general relationship between MTs and microfibrils but provided no quantitative analysis of MT arrays. In this paper quantitative measurements of MT arrays were done during primary and secondary wall synthesis. Changes in the MT array accompany the shift from primary to secondary wall deposition.Fibers of cotton (Gossypium hirsutum, variety Acala SJ-2) were grown using ovule culture techniques. At various stages of development (days post anthesis, DPA) ovules with attached fibers were fixed in 0.1M phosphate buffered (pH 6.9) 1% glutaraldehyde for 1 h. After a 1 h wash, the fibers were post-fixed in phosphate buffered 1% OsO4. After rapid dehydration with 2,2 dimethoxypropane, cells were infiltrated with Spurr's resin. At this point fibers were removed from ovules and flat embedded between teflon coated microscope slides. Serial section reconstruction analysis of microtubule arrays was done as previously described.
Development and self-repair in hair cell epithelia
Authors: Corwin Jeffrey T. 
Published: June 2020

This presentation will attempt to explain current understanding of the cellular mechanisms of normal hair cell development and the mechanisms of hair cell regeneration. Hair cells are the mechanoreceptors that transduce sound and balance stimulation of the ear and water current stimulation of the lateral line organs into electrical activity that is transmitted to the brain. Hair cells have one true cilium, the kinocilium, and 30 to 200 or more stereocilia which are modified, actin-filled microvilli, that project from the flat apical surfaces of the cells. The stereocilia are arrayed across each cell in an “organ pipe” arrangement, with a row of short stereocilia at one end and rows of increasingly taller stereocilia proceeding from that row toward the single eccentrically positioned kinocilium. That asymmetrical surface structure defines the functional polarity of each hair cell, because stimuli that mechanically bend the stereocilia array in the direction of its tall end cause a decrease in the transmembrane potential of the hair cell and increased exocytotic release of neurotransmitter from vesicles at tonically active synaptic sites in the subnuclear region. In that way the appropriately directed bending of the stereocilia causes excitation of neurons that conduct action potentials to the brain. Bending of the stereocilia in the opposite direction causes hyperpolarization of the hair cell, reduction in the exocytosis of neurotransmitter, and a resulting decrease in the frequency of action potentials conducted to the brain.In most mature organs the orientations of the stereocilia arrays of the hair cells are aligned throughout the epithelia. However, during early development of these cells their cilia bundles are oriented in a nearly random distribution. As the cells differentiate their cilia arrays grow taller and reorient, so that neighbors come into alignment. In some epithelia, such as the auditory epithelium in the cochlea of the chicken, both the number and the maximum length of the stereocilia on hair cells vary systematically along gradients related to cell position in the epithelium. This pattern of sensory cell ultrastructure correlates with the high to low pitch tuning of the basilar membrane that supports the epithelium and with the tuning of the neurons that contact the individual cells. Cells at the distal end of the chicken cochlea have less than 50 stereocilia; cells at the proximal end have over 200. The stereocilia on proximal hair cells are short (<2 microns); those on distal hair cells are long (>5 microns). The reorientation of the stereocilia arrays and their location-specific differences in stereocilia number and length all become recognizable at approximately the time when synapses form between these cells and their neurons, but experiments have shown that these processes of hair cell differentiation can occur in the absence of neurons. Several hypotheses that attempt to explain the control of differentiation in hair cells will be covered
Cytoskeletal functions in sensory receptors
Authors: Burnside Beth 
Published: June 2020

Since the shapes of sensory receptors are so exquisitely specialized for mediating their unique functions, the cytoskeletons of sensory cells are deployed for morphogenetic and motile objectives in particularly interesting ways. Receptors erect cytoskeletal scaffolding to support two basic sorts of surface elaborations: 1) those designed to achieve the most effective presentation of specialized membrane laden with receptor proteins (photoreceptors, olfactory receptors, taste cells, and chemoreceptors), and 2) those designed to respond directly to mechanical perturbations in the cell's environment (auditory hair cells, mechanoreceptors). Each of these receptor types has specific structural requirements.Sensory receptors have built their surface elaborations upon either microtubule- or actin-based scaffoldings. Microtubule-based scaffoldings have evolved from motile cilia, and the axoneme has been modified to various ends. All ciliary-derived receptors so far described, except (curiously) those from the worm Caenorhabdites elegans, are associated with basal bodies which nucleate the assembly of surface specializations. The mechanisms for elaborating the myriad membrane specializations associated with ciliary receptors are not yet understood. Recently it has been shown that in vertebrate photoreceptors, actin is not required for the addition of membrane to the outer segment, but it is required for the proper assembly of new disks .
Mechanisms for establishing and reversing epithelial cell polarity
Authors: Nelson W. James , Wang Allan Z. 
Published: June 2020

Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs. However, the mechanisms involved in the morphogenesis of this important cell type are poorly understood. When epithelial cells, such as Madin-Darby Canine Kidney (MDCK) cells, are induced to develop polarity structurally and functionally different plasma membrane domains (apical and basal-lateral) must be established and maintained (ref. 1). This requires the sorting of constituent proteins to the correct membrane domain and the removal of incorrectly localized proteins. To understand how these domains are formed we designed an experimental situation in which cells that have established membrane polarity in one orientation are forced to reverse this orientation; by observing this reversal we should gain new insight into the mechanisms of membrane domain assembly and disassembly (Figure 1).We have now established this experimental situation in vitro. Under appropriate growth conditions in suspension culture or in a collagen gel MDCK cells are induced to form multicellular cysts. These multicellular cysts comprise a closed monolayer of cells of defined polarity (see below) that surrounds a fluid-filled lumen. We have begun to analyze morphologically the state of cell polarity of these structures using electron microscopy and immunocytochemistry with antibodies specific for marker proteins of the apical (gpl35) and basal-lateral (Na+,K+-ATPase) plasma membranes, and the tight junction (ZO-1) (Figure 2).
The organization of cell surface membrane domains
Authors: Edidin Michael 
Published: June 2020

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.
Import of proteins into chloroplasts and mitochondria
Authors: Pain D. , Murakami H. , Schnell D. J. , Blobel G. 
Published: June 2020

Although both chloroplasts and mitochondria are capable of nucleic acid and protein synthesis, their biogenesis requires the import of a large number of nuclear-encoded proteins that are synthesized on cytoplasmic ribosomes. In the case of both organelles the transfer is post-translational and energy dependent. Most of the nuclear-encoded proteins are synthesized as precursors with an amino-terminal extension known as the signal, transit or pre-sequence. Like proteins destined for the secretory pathway, targeting of nuclear-encoded chloroplast and mitochondrial proteins to the proper cellular location is mediated by these N-terminal topogenic sequences. As is the case for protein translocation across the endoplasmic reticulum or prokaryotic plasma membrane, our data suggest dual recognition of signal sequences for protein import into mitochondria or chloroplasts; first by a soluble recognition system and then by a membrane bound one. The latter has been localised to distinct regions where the inner and outer membrane of these organelles are in close contact.The in vitro import of cytoplasmically synthesized precursors into chloroplasts has been previously described. We have transcribed mRNA from a cDNA clone coding for a cytoplasmically synthesized polypeptide of the chloroplast stroma, the small subunit of ribulose 1,5,-bisphosphate carboxylase. Translation of the specific mRNA in a wheat germ cell-free system yielded a large precursor (pS), and post-translational incubation with isolated pea chloroplasts resulted in import of pS into the chloroplast stroma accompanied by conversion of pS into the mature form (S) by means of cleavage of an NH2-terminal transit sequence. Import was an energy-dependent process and required ATP hydrolysis. Nonpenetrating ATP-consuming systems had no effect on import when chloroplasts were allowed to synthesize ATP in the stroma in the dark, suggesting that the ATP exerts its effect on import within the stroma and not in the cytosol. Studies with various ionophores demonstrated that ATP hydrolysis within the stroma was not used to generate a membrane potential across membranes.
Image of PhoE Porin in projection at 3.5Å resolution
Authors: Jap B.K. , Walian P. J. , Downing K. H. 
Published: June 2020

The outer membrane of Gram-negative bacteria, such as E. coli, contains a high density of a family of pore-forming proteins, which are often called porins. One member of the family, PhoE porin, is expressed under conditions of phosphate starvation, and has been shown to favor the transport of phosphate-containing compounds and negatively charged molecules. The three-dimensional (3-D) structure of PhoE porin obtained from two-dimensional crystals, negatively stained with uranyl acetate, has been determined to a resolution of about 18Å. The projected structure of glucose-embedded PhoE porin has also been obtained to a resolution of about 6.5Å. Based on these results and previous spectroscopic studies, a 3-D model of PhoE porin has been proposed, consisting of a trimer of extended funnel-like structures. Each funnel-like structure comprises a large water filled vestibule leading into a narrower extension. The vestibule wall is formed by β-sheet which has a large fraction of the β-strands oriented perpendicular to the membrane plane. In this report, we present an extension of the resolution to 3.5Å in the projection map of PhoE porin. At this resolution we can confirm that the β-strands of the vestibule are predominantly normal to the membrane plane.We have recorded high resolution images of trehalose-embedded PhoE porin using both the conventional flood illumination technique and the spot scan illumination method. Images with high quality as judged from optical diffraction were digitized and processed to correct for crystal distortions, lens aberrations and illumination tilt. Phases obtained from several high quality images were used to determine the phases of the reflections used in reconstructing the projected map. The amplitudes of the reflections needed to generate the reconstruction were obtained from the 3-D diffraction data set of PhoE porin, which has been determined to a resolution of about 2.8Å. A typical electron diffraction pattern of PhoE porin at zero tilt angle is shown in Figure 1.
Cryo-electron microscopy of cauliflower mosaic virus
Authors: Cheng R. H. , Olson N. H. , Baker T. S. 
Published: June 2020

Cauliflower mosaic virus (CaMV), the type member of the Caulimovirus family, is one of the most extensively studied plant viruses since it was one of the first plant viruses shown to encapsidate a genome of double stranded DNA. This virus has served as a model system for studying plant gene organization, expression and replication and is potentially useful as a gene vector for the genetic manipulation of higher plants. Despite being well characterized in terms of its molecular biology, little is known about the structure, organization and assembly of mature CaMV virions.CaMV virions have a molecular mass of ˜22.8x106 daltons and a sedimentation coefficient of 208S.2 In negatively-stained samples, the diameter of the virus was determined to be 50.3±1.4 nm. The capsid consists of multiple copies of a single, phosphorylated polypeptide (37-42 kDa) which is presumed to be post-translationally modified from the 58kDa primary product of gene IV. The sizes of the virion and mass of the capsid protein indicate that there may be 420 copies of the polypeptide, arranged with T=7 icosahedral lattice symmetry, although no direct evidence exists to support this model. Projected images of CaMV particles, examined by conventional electron microscopy procedures, are difficult to interpret since the details are relatively smooth and featureless. The organization of the circular doubled-stranded DNA molecule (˜4.9xl06 daltons) is unknown: neutron diffraction experiments indicate that it is mainly confined to the region between radii 15.0-21.5 nm. The current study is aimed at obtaining a reliable three-dimensional structural determination of CaMV by examining purified virus samples using recently developed cryo-electron microscopy techniques and image analysis procedures.
The hyaline layer in early development of the sea urchin: Cell-extracellular matrix interactions
Authors: McClay David R. , Alliegro Mark C. , Black Steven D 
Published: June 2020

The hyaline layer is released by the sea urchin zygote starting about 30 seconds after fertilization. This layer is elevated above the surface of the zygote where it serves as a protective layer, a barrier to polyspermy, and as an adhesive substrate for the early stages of development. We have been interested in learning how the hyaline layer is stored in the egg, how it is released after fertilization, and how it functions as an extracellular matrix during early development. The layer contains about fifteen abundant proteins plus a number of others that may be present or may contaminate preparations of the layer during isolation procedures. Our goal was to identify the major components of the hyaline membrane, to follow assembly of the structure after fertilization, and to identify molecules that are active as adhesive substrates for cells.Using double-label immunofluorescence and ultrastructural immunogold localization of polyclonal and monoclonal antibodies, the components of the hyaline layer have been localized to four vesicle classes in the unfertilized egg. Centrifugation studies on unfertilized eggs and double-label immunofluorescent studies have shown that the four classes each contain a distinct group of proteins. The four vesicles were followed through oogenesis to learn when proteins of each class were synthesized by the oocyte. Each of the four classes (cortical granules, basal lamina vesicles, apical vesicles and echinonectin vesicles) were synthesized, localized, and relocalized in the oocyte by unique spatial and temporal pathways.
Ultrastructure of colonic biopsies in gardner's syndrome
Authors: Siew S. , Lev R. , Lebenthal E. , Rossi T. 
Published: June 2020

Gardner's Syndrome is a familial condition which is inherited as an autosomal Mendelian dominant with a high degree of penetrance. It is characterized by the development of multiple adenomatous polyps in the gastrointestinal tract with a particular predilection for the colon. This multiple polyposis is associated with desmoid tumors. The condition carries a high malignant potential and carcinoma of the colon may develop by the age of forty. The gross and histologic features of the polyps have been defined. We undertook a blind study of 19 colonic endoscopic biopsies by means of transmission electron microscopy (TEM) in order to elucidate the applications of this technique in the investigation and diagnosis of Gardner's Syndrome.Endoscopy of the colon was performed on 8 patients; 6 males and 2 females. Their ages ranged from 2-17 years with a mean of 9.1. Two of the cases were siblings from a family with Gardner's Syndrome, who showed evidence of the disease, another two were siblings from a family with Gardner's Syndrome, who did not manifest the disease. The remaining 4 patients were normal controls. Biopsies obtained by means of endoscopy were fixed immediately either for histologic examination or TEM. The tissue was processed in the conventional manner.
TEM studies of primitive neuroectodermal tumors with neuronal and glial differentiation
Authors: Harb Joseph M. 
Published: June 2020

The term primitive neuroectodermal tumor (PNET) was originally applied in 1973 by Hart and Earle to refer to a group of undifferentiated cerebral neoplasms in children. Since that report, few studies have utilized the transmission electron microscope (TEM) to establish the identity of the tumor cells and to clarify the propensity of the PNETs for pluripotential differentiation. Of particular interest are reports which suggest the possibility of simultaneous neuronal and glial differentiation within the same cell. It would be of significant interest to know if the propensity to divergent differentiation can be explained by a single population of neoplastic cells that have the potential to differentiate in multiple directions. We will present a small series of cases with ultrastructural evidence that some of the cells in the tumors express both neuronal and glial features.The first case is a primary cerebral neuroblastoma which was associated with a population of neoplastic glial cells. At the ultrastructural level neuroblast and glial cell populations were identified. We also recognized a population of cells which displayed a continuous basal lamina and rare, poorly developed junctional complexes. Of particular interest was yet another, smaller, population of cells which displayed both dense-core granules and microtubules typical of neuroblasts and abundant cytoplasmic filaments typical of glial cells (Figure 1). In two other cases, the astrocytic nature of the cells was confirmed at the ultrastructural level on the basis of prominent glial filaments in the cytoplasm. However, some of the astrocytes in one of the cases were invested partially by a distinct basal lamina (Figure 2), and there were dense-core granules in the cytoplasm (Figure 2, inset).