Science.gov

Sample records for cells identified ultrastructurally

  1. Ultrastructural characteristics of novel epithelial cell types identified in human pathologic liver specimens with chronic ductular reaction.

    PubMed

    De Vos, R; Desmet, V

    1992-06-01

    Previous immunohistochemical studies on human liver biopsies with chronic ductular reaction revealed the presence of "small cells" with bile-duct type cytokeratin profile in the periportal area. This study identified similar cells by electron microscopy. The authors studied 13 human liver specimens with various liver diseases, but all characterized by chronic ductular reaction. In all specimens, variable numbers of "small cells" with common epithelial characteristics were identified in the periportal area. They could be classified into three types. Type I cells showed an oval cell shape and oval nucleus, early or established formation of junctional complexes with adjacent cells, a full assortment of cytoplasmic organelles, and bundles of tonofilaments. Type II cells showed features of bile-duct cell differentiation, including lateral interdigitations, apical microvilli, basal pinocytotic vacuoles, and basement membrane formation. In contrast, type III cells displayed additional features indicating hepatocellular differentiation, such as a more prominent nucleus, formation of a hemicanaliculus, and glycogen rosettes. It is concluded that these small cells of epithelial nature display variable differentiation characteristics of either bile-duct type cells or hepatocytes. These findings support the existence of bipotential progenitor epithelial cells in human liver. They may have implications for liver regeneration and carcinogenesis.

  2. [Ultrastructure of peritoneal mesothelial cells].

    PubMed

    Obradovic, M M; Stojimirovic, B B; Trpinac, D P; Milutinovic, D D; Obradovic, D I; Nesic, V B

    2001-01-01

    The introduction of peritoneal dialysis (PD) as a respectable modality of renal replacement therapy some three decades ago, suddenly drew attention of many authors to peritoneal membrane as insufficiently investigated structure. In order to explain the pathological changes in peritoneum due to renal diseases, it became necessary to explore the normal peritoneal structure. The aim of this study was to examine the morphology of peritoneal lining cells in healthy persons. Biopsies of the peritoneum were performed on 20 volunteer kidney donors. Tissue samples were taken during renal transplantation. Special care was taken in getting appropriate samples without artificial damage because of the extreme fragility of the peritoneal tissue. The preparing procedure was standard for routine HE staining and for plastic embedded semifine and fine sections studies. Semifine sections were made on ultramicrotome, stained with Toluidin blue and studied by light microscope, while fine sections were made by ultramicrotome and studied by transmission electron microscope. One layer of cuboidal or flattened lining cells present over the lamina propria connective tissue presented mesothelium. The cells were overlapped like tiles on the roof. Lateral parts of their interdigitated membranes were interconnected with different types of cell junctions: unpermeable, adhesion and communication junctions; inhibiting intercellular transport. Cell surface was often covered with great number of microvilli and lamellar bodies. A single kinocilia was also often present on apical cell surface. Nuclei were euchromatic with well developed nucleoli. Cytoplasm was filled with a great number of ribosomes, mitochondria, cisterns of rough endoplasmatic reticulum and Golgi apparatus, lamellar bodies and lipid inclusions. Numerous pinocytic vesicles on all parts of the membrane as well as in the cytoplasm indicating active endocytosis, egsocytosis and transcytosys in the process of secretion and reabsorption

  3. Effects of ultrasound upon endothelial cell ultrastructure

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    A number of new brain applications for therapeutic ultrasound are emerging including drug delivery through BBB opening, enhancement of angiogenesis, sonothrombolysis and neuromodulation. Safety remains important as alterations in the cytoskeleton and tight junctions of endothelial cells have been described. In this study we characterize the in vitro effects of ultrasound on cell morphology using a new human brain cell line (hCMEC/D3). Changes in ultrastructure were analyzed with antibodies against tubulin, actin and catenin. Transport was analyzed by measuring transferrin uptake. No significant changes were seen after continuous wave ultrasound treatment of hCMEC/D3 cells grown in Opticell{trade mark, serif} chambers. We could not observe disassembled actin stress fibers or variations in the microtubule network. However, severe damage occurred in cells cultured in petri dishes.

  4. Ultrastructure study of apple meristem cells during cryopreservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ultrastructure of apple (Malus x domestica Borkh.) meristem cells was studied before and after cold acclimation (CA) and during the steps of PVS2 vitrification. We compared cells of in vitro grown shoots of two cultivars, Grushovka Vernenskaya and Voskhod. Cells of the two cultivars were simila...

  5. Ultrastructure Study of Apple Meristem Cells During Cryopreservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ultrastructure of apple (Malus x domestica Borkh.) meristem cells was studied before and after cold acclimation (CA) and during the steps of PVS2 vitrification. We compared cells of in vitro grown shoots of two cultivars, Grushovka Vernenskaya and Voskhod. Cells of the two cultivars were simila...

  6. Combined fluorescence and ultrastructural mapping of living cells

    NASA Astrophysics Data System (ADS)

    Kohen, Elli; Hirschberg, Joseph G.; Kohen, Cahide; Prince, Jeffrey; Suckewer, Szymon; Santus, Rene C.; Morliere, Patrice; Dubertret, Louis

    1990-05-01

    The topographic analysis of fluorescence distribution has been carried out pixel-by-pixel by one dimensional, two-dimensional microspectrofluorometry and three-dimensional confocal fluorescence microscopy. Fluorescence emission spectra of NAD(P)H and benzo(a)pyrene (or metabolites) were recorded at different excitation wavelengths. Cell bioenergetics are monitored in normal and malignant cells as well as cells with genetic defects by coenzyme responses to microinjections of substrates and modifiers from key metabolic pathways in presence and absence of inhibitors and drugs active on mitochondrial structure and function. Cooperative interactions between organelles involved in detoxification mechanisms are observed in cells treated with fluorescent cytotoxic agents. Such interactions can be directly mapped by the fluorescence of cytotoxic agents, their reaction products or vital probes such as NBD ceramide for the Golgi apparatus. To identify the organelles involved parallel electron microscopic studies are carried out in cells first treated with the cytotoxic agent and then incubated with an electron opaque material. A recently developed combined X-ray laser microscope (COXRALM) holds the promise of carrying out combined phase-fluorescence-and X-ray microscopic observations of fluorescence and ultrastructural correlations in live cell probing. As further versatility is gained in such methods it may become possible to obtain a very detailed structure and function mapping of living cells within the context of cytomatrix analysis, metabolic compartmentation and organelle interactions.

  7. Ultrastructural and Cytochemical Properties of Peripheral Blood Cells of Piebald Naked Carp (Gymnocypris eckloni).

    PubMed

    Zheng, Z X; Tang, Y; Fang, J; Peng, X; Fan, J D; Cui, H M; Yang, L Z

    2017-02-01

    The ultrastructural and cytochemical properties of peripheral blood cells of Gymnocypris eckloni were investigated by transmission electron microscopy and a range of cytochemical techniques to provide clear insight into the structure and function of blood cells from this fish. Ultrastructurally, erythrocytes, leucocytes (neutrophils, eosinophils, lymphocytes, monocytes), thrombocytes and plasma cells were identified in the peripheral blood of G. eckloni. The most special ultrastructural characteristics of blood cells in this fish were that neutrophils exhibited only one type of cytoplasmic granules containing an eccentric, spherical or oval electron-dense core, and eosinophils presented two types of granules with non-uniform electronic density and without crystalloids in their cytoplasm. Neutrophils, eosinophils, lymphocytes, monocytes and thrombocytes were positive for periodic acid-Schiff and α-naphthyl acetate esterase staining. Intense peroxidase positive staining was observed in neutrophils and monocytes, but not in eosinophils, lymphocytes and thrombocytes. Neutrophils, eosinophils and monocytes were stained positively for acid phosphatase, whereas lymphocytes and thrombocytes did not stain. Leucocytes and thrombocytes were negative for alkaline phosphatase and Sudan black B staining. Erythrocytes were negative for all cytochemical staining. The cytochemical and ultrastructural features of peripheral blood cells of G. eckloni were similar to those of other fish species. However, some important differences were identified in G. eckloni.

  8. Immunohistochemical and ultrastructural study of anterior pituitary cells in the female Afghan pika, Ochotona rufescens rufescens.

    PubMed

    Nakamura, F; Suzuki, Y; Yoshimura, F

    1986-01-01

    An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's "corticotrophs" of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus.

  9. [Ultrastructure of human umbilical cord mesenchymal stem cells].

    PubMed

    Qiao, Shu-Min; Chen, Guang-Hua; Wang, Yi; Wu, De-Pei

    2012-04-01

    The purpose of this study was to observe the ultrastructure of human umbilical cord mesenchymal stem cells (hUCMSC). hUCMSC from full-term newborn umbilical cord were isolated and cultured by collagenase digestion, and then subcultured, amplification, and cell morphology was observed by microscopy. The immunophenotype and trilineage differentiation potential of hUCMSCs at passage 3 were analyzed. Transmission electron microscopy and scanning electron microscopy were used to observe the ultrastructure of hUCMSC. The results indicated that appearance of hUCMSC was spindle-shaped and polygonal, and nuclei were observed. hUCMSC expressed immunophenotype CD44, CD73, CD105, did not express CD34, CD45, CD31 and human leukocyte antigen HLA-DR. hUCMSC were capable of adipogenic, osteogenic, and cartilage differentiation; the short and thick microvilli processes were seen at the surface of hUCMSC by scanning electron microscope. Two different cell morphologies of hUCMSC were seen under transmission electron microscope, the one was a quiescent period in which a large and round or oval nucleus only one nucleolus were seen, cytoplasmic organelles were less; the other was in a relatively active period in which one or two nuclei in the same one cell were observed, the organelles were rich, structure was clear, expansion of the mitochondria was visible. It is concluded that the cells successfully isolated and cultured from umbilical cord, which possess biological characteristics of MSC and display two different states of ultrastructure.

  10. Ulcerative colitis: ultrastructure of interstitial cells in myenteric plexus.

    PubMed

    Rumessen, J J; Vanderwinden, J-M; Horn, T

    2010-10-01

    Interstitial cells of Cajal (ICC) are key regulatory cells in the gut. In the colon of patients with severe ulcerative colitis (UC), myenteric ICC had myoid ultrastructural features and were in close contact with nerve terminals. In all patients as opposed to controls, some ICC profiles showed degenerative changes, such as lipid droplets and irregular vacuoles. Nerve terminals often appeared swollen and empty. Glial cells, muscle cells, and fibroblast-like cells (FLC) showed no alterations. FLC enclosed macrophages (MLC), which were in close contact with naked axon terminals. The organization and cytological changes may be of pathophysiological significance in patients with UC.

  11. Ultrastructure of human Leydig cells at early gonadal embryogenesis.

    PubMed

    Makabe, S; Naguro, T; Heyn, R; Motta, P M

    1995-01-01

    The ultrastructure of human Leydig cells at different stages of the testicular prenatal development is described by means of transmission and scanning electron microscopy. Between 5 and 7 weeks of gestation (w.g.) the interstitial tissue of the gonad is filled with small undifferentiated mesenchymal cells, migrating primordial germ cells and blood vessels. When the embryo is 7 to 8 weeks-old Leydig cells (LC) appear in basically two morphological patterns, light and dark cells. Their most significative feature is the development of the smooth endoplasmic reticulum (SER) as a dense tubulo-vesicular network and the presence of numerous pleomorphic mitochondria with mainly lamellar cristae. At 14 and 16 w.g. the testicular interstitium reaches the maximum development; the cytoplasm of the LC shows a widespread network of anastomosing tubules of the SER and mitochondria with tubular cristae. Fetal LC show a partial cell coat, lack the crystals of Reinke, have few lipid droplets and show no signs of massive cell degeneration, at least until 16 w.g. These ultrastructural modifications in fetal LC are in accordance with the changes in both steroidogenic activity and hCG levels reported by the literature to occur at this stage of development. Junctional complexes were often observed among LC from 7 to 8 w.g. onwards.

  12. Ultrastructure of autophagy in plant cells: a review.

    PubMed

    van Doorn, Wouter G; Papini, Alessio

    2013-12-01

    Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.

  13. Ultrastructural observations reveal the presence of channels between cork cells.

    PubMed

    Teixeira, Rita Teresa; Pereira, Helena

    2009-12-01

    The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

  14. Odor Sensing Cell Ultrastructure by Electron Microscopy.

    DTIC Science & Technology

    conditions in which it could readily oxidize to dehydroascorbic acid. Glucose oxidase and urease were immobilized in acrylamide gel which shows good...the initial studies on immobilized enzymes, the completion of the observations on the regeneration of olfactory cells and centriole migration during

  15. The Ultrastructural Signature of Human Embryonic Stem Cells.

    PubMed

    Underwood, Jean M; Becker, Klaus A; Stein, Gary S; Nickerson, Jeffrey A

    2017-04-01

    The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc.

  16. How effectively does a clinostat mimic the ultrastructural effects of microgravity on plant cells?

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1990-01-01

    Columella cells of seedlings of Zea mays L. cv. Bear Hybrid grown in the microgravity of orbital flight allocate significantly larger relative-volumes to hyaloplasm and lipid bodies, and significantly smaller relative-volumes to dictyosomes, plastids, and starch than do columella cells of seedlings grown at 1 g. The ultrastructure of columella cells of seedlings grown at 1 g and on a rotating clinostat is not significantly different. However, the ultrastructure of cells exposed to these treatments differs significantly from that of seedlings grown in microgravity. These results indicate that the actions of a rotating clinostat do not mimic the ultrastructural effects of microgravity in columella cells of Z. mays.

  17. Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

    PubMed Central

    Rodal, Avital A.; Kozubowski, Lukasz; Goode, Bruce L.; Drubin, David G.; Hartwig, John H.

    2005-01-01

    Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. PMID:15525671

  18. Ultrastructural identification of Ricinus communis agglutinin-1 positive cells in primary dissociated cell cultures of human embryonic brain.

    PubMed

    Bobryshev, Y; Ashwell, K

    1994-12-01

    While Ricinus communis agglutinin 1 (RCA-1) can be used as a specific marker to study the development and differentiation of microglial cells in human embryogenesis, little is known about the structural heterogeneity and nature of RCA-1+ cells. To analyse the structural peculiarities of RCA-1+ cells, we have used primary dissociated cultures of human embryonic brain. These have been used as models for investigating many of the aspects of central nervous system (CNS) HIV infection. We have shown that primary dissociated cultures from human embryos as young as 10 weeks gestation contain RCA-1+ cells. The RCA-1+ cells exist in two forms, those without (type I) and those with (type II) processes. The former have a poorly developed ultrastructure, while the latter have well developed ultrastructural features, such as rough endoplasmic reticulum with short cisternae, abundant ribosomes, mitochondria, lysosomes and vacuoles. Furthermore, some of these cells with processes have well developed cytoskeletal features. In this paper, the classification of RCA-1+ cells of embryonic human brain is considered and their morphology compared to microglia identified in rodent CNS.

  19. The effects of ultraviolet C radiation on the ultrastructure of the liver cells of mole rats.

    PubMed

    Tekın, Saban; Türker, Hüseyin; Güven, Turan; Yel, Mustafa

    2016-01-01

    The aim of this study was to elucidate the ultrastructural changes in the liver cells of mole rats (Spalax leucodon) exposed to ultraviolet radiation (UVR). Thirteen mole rats used in this study were caught from nature. They were divided into four groups. The first group was separated as a control and was not given any radiation. The rest were exposed to ultraviolet C (UVC) radiation for 7, 14, and 21 days. The electron microscopic examinations revealed that significant ultrastructural changes occurred in the liver tissue. These changes were the reduction in cytoplasmic organelles, dilatation in rough endoplasmic reticulum, impairment of nucleus membrane, and broadened and vacuolated mitochondria in the cytoplasm. Also, UVC radiation caused significant changes in liver enzymes of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gama-glutamiltransferase values. After long-term exposure to radiation, some excessive ultrastructural changes occurred. These results indicated that longer exposure to UVR would cause more ultrastructural effects on the liver cells and liver enzymes.

  20. Cadmium-induced ultrastructural changes in Euglena cells

    SciTech Connect

    Duret, S.; Bonaly, J.; Bariaud, A.; Vannereau, A.; Mestre, J.C.

    1986-02-01

    The ultrastructure of Euglena gracilis grown in the presence of Cd showed only numerous myelin-like structures in mitochondria, chloroplasts altered in shape, and thylakoid arrangement and increase of osmiophilic plastoglobuli. These alterations indicate that respiratory processes are the initial target of Cd toxicity.

  1. Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development.

    PubMed

    Segado, Patricia; Domínguez, Eva; Heredia, Antonio

    2016-02-01

    The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall.

  2. Ultrastructural characteristics of 5BrdU labeling retention cells including stem cells of regenerating feathers in chicken.

    PubMed

    Alibardi, Lorenzo; Wu, Ping; Chuong, Cheng-Ming

    2014-07-01

    Feathers regenerate from stem cells localized in a region of the follicle indicated as the bulge of the collar. Stem cells are slow cycling cells and some of these cells can be identified after labeling experiments using 5-bromo-deoxyuridine to detect label retaining cells (5BrdU LRCs). The present electron microscopic analysis of 5BrdU LRCs has described the ultrastructural characteristics of small cells present in the bulge region of the follicle in regenerating feathers of chickens that include stem cells. Labeled feather stem cells are smaller than 10 lm in average diameter, possess large nuclei with high nuclear/cytoplasmic ratio, and contain evenly distributed ribosomes, sparse bundles of intermediate filaments, scarce or no endoplasmic reticulum, and few mitochondria. The nuclei are mainly euchromatic with a variable amount of heterochromatin clumps and the nucleoli show developed granular and fibrillar components. These features indicate that feather stem cells are transcriptionally active cells for ribosomal and proteins synthesis. The cell surface of feather stem cells often shows small and irregular folds resembling microvilli in contact with the surrounding cells. The latter characteristics may be related to the exchange of molecules and/or with the migration of stem cells among other epithelial cells of the collar epithelium.

  3. Ultrastructural analysis of cell component distribution in the apical cell of Ceratodon protonemata

    NASA Technical Reports Server (NTRS)

    Walker, L. M.; Sack, F. D.

    1995-01-01

    A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longtitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the moss Ceratodon. The first 35 micrometers of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tip-most 5 micrometers and evenly distributed throughout the remaining 30 micrometers. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.

  4. Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc.

    PubMed

    Gruber, H E; Hanley, E N

    2009-06-01

    The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.

  5. Pollen and microsporangium development in Hovenia dulcis (Rhamnaceae): a different type of tapetal cell ultrastructure.

    PubMed

    Gotelli, Marina M; Galati, Beatriz G; Zarlavsky, Gabriela; Medan, Diego

    2016-07-01

    Despite that there is some literature on pollen morphology of Rhamnaceae, studies addressing general aspects of the microsporogenesis, microgametogenesis, and anther development are rare. The aim of this paper is to describe the ultrastructure of pollen grain ontogeny with special attention to tapetum cytology in Hovenia dulcis. Anthers at different stages of development were processed for transmission and scanning electron microscopy, bright-field microscopy, and fluorescence microscopy. Different histochemical reactions were carried out. The ultrastructural changes observed during the development of the tapetal cells and pollen grains are described. Large vesicles containing carbohydrates occur in the tapetal cell cytoplasm during the early stages of pollen development. Its origin and composition are described and discussed. This is the first report on the ontogeny and ultrastructure of the pollen grain and related sporophytic structures of H. dulcis.

  6. Ultrastructural Changes in Murine Peritoneal Cells Following Cyclophosphamide Administration

    PubMed Central

    Chin, K. N.; Hudson, G.

    1974-01-01

    Peritoneal cells were studied at intervals of between 6 h and 30 days following a single intravenous injection of a sublethal dose of cyclophosphamide. With the electron microscope, evidence of cell damage and death could be seen at 6 h, and by 12 h large numbers of dead cells were noted, either lying free or within the cytoplasm of macrophages. Most of the damaged cells were lymphocytes but degenerating blast cells, eosinophils, neutrophils and mast cells were also identified. Nuclei were seen in which margination of chromatin had occurred but nuclei of uniform density were also prominent and showed irregular shape and lobulation. Macrophages exhibited all stages of phagocytosis and digestion and a few phagocytes of atypical appearance were noted. By 24 h most of the dead cells lay within the cytoplasm of macrophages which showed many phagocytic inclusions as well as lipid droplets. By 6 days, the peritoneal cells had regained normal appearances although the proportion of lymphoid cells was still reduced. By the 18th day, all features were indistinguishable from normal. The changes observed showed a general similarity to those noted previously in the lymphoreticular cells of the Peyer's patch; they provide no evidence that the environment of the peritoneal cavity protects cells against the action of cyclophosphamide. ImagesFigs. 4-6Figs. 7-9Figs. 10-12Figs. 1-3 PMID:4447790

  7. Cutaneous epithelioid cell histiocytoma: immunohistochemical and ultrastructural findings suggesting endothelial origin.

    PubMed

    Manente, L; Schmitt, I; Onetti, A M; Peris, K; Caracciolo, E; Chimenti, S

    1997-10-01

    A 13-year-old boy presented with a polypoid nodule localized in the groin. Although the clinical and histopathological features corresponded to previously described diagnostic criteria of epithelioid cell histiocytoma, immunohistochemical and ultrastructural findings suggested vascular differentiation of the epithelioid cells. In particular, the immunohistochemical negativity of the epithelioid cell elements for Factor XIIIa failed to substantiate the previously forwarded hypothesis that this lesion represents a dendrocytoma. Instead, the presence of histiocytoid, vacuolated cells occasionally containing sparse red blood cells, positive staining for Factor VIII-related antigen, and ultrastructural evidence of endothelial characteristics in epithelioid neoplastic cells favor the hypothesis that "epithelioid cell histiocytoma" is of endothelial origin. We suggest the descriptive term cutaneous histiocytoid hemangioendothelioma for this lesion.

  8. Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures

    PubMed Central

    1989-01-01

    The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under- surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA- 3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion

  9. Ultrastructure of Zika virus particles in cell cultures

    PubMed Central

    Barreto-Vieira, Debora Ferreira; Barth, Ortrud Monika; da Silva, Marcos Alexandre Nunes; Santos, Carolina Cardoso; Santos, Aline da Silva; F, Joaquim Batista; de Filippis, Ana Maria Bispo

    2016-01-01

    Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible. PMID:27581122

  10. An ultrastructural study of sporidium formation during infection of a rhabditid nematode by large gun cells of Haptoglossa heteromorpha.

    PubMed

    Glockling, S L; Beakes, G W

    2000-10-01

    Recently fired gun cells of Haptoglossa heteromorpha, an aplanosporic nematode parasite, were examined ultrastructurally. The everted tubes of the fired cells had penetrated the cuticle of a nematode, and infective sporidia were developing inside the host body. The nematode cuticle was penetrated by the narrow, walled part of the tube below the needle chamber. The lower unwalled part of the tube tail formed the sporidium. The developing sporidium had a multilayered fibrous outer coating and the plasma membrane was separated from the wall in places. Sporidia contained biphasic membrane-bound vesicles that had been generated by the Golgi dictyosome during gun cell development. Immediately following gun cell firing, the nuclear envelope of the sporidium nucleus was not apparent, and the sporidium nucleus contained clusters of electron-dense particles concentrated in the nucleolar region. We compare the structures and organelles found in the mature gun cell with those in the fired cell and attempt to identify the membranous layers around the sporidium.

  11. Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

    PubMed Central

    Scholz, Matthew J.; Weiss, Taylor L.; Jinkerson, Robert E.; Jing, Jia; Roth, Robyn; Goodenough, Ursula; Posewitz, Matthew C.

    2014-01-01

    Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes. PMID:25239976

  12. Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells

    SciTech Connect

    Bhargava, U.; Bar-Lev, M.; Bellows, C.G.; Aubin, J.E.

    1988-01-01

    When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na beta-glycerophosphate, they form discrete three-dimensional nodular structures with the histological and immunohistochemical appearance of woven bone. The present investigation was undertaken to verify that bone-like features were identifiable at the ultrastructural level. The nodules formed on top of a fibroblast-like multilayer of cells. The upper surface of the nodules was lined by a continuous layer of cuboidal osteoblastic cells often seen to be joined by adherens junctions. Numerous microvilli, membrane protrusions, and coated pits could be seen on the upper surface of these cells, their cytoplasm contained prominent RER and Golgi membranes, and processes extended from their lower surfaces into a dense, highly organized collagenous matrix. Some osteocyte-like cells were completely embedded within this matrix; they also displayed RER and prominent processes which extended through the matrix and often made both adherens and gap junctional contacts with the processes of other cells. The fibroblastic cells not participating in nodule formation were surrounded by a less dense collagenous matrix and, in contrast to the matrix of the nodules, it did not mineralize. An unmineralized osteoid-like layer was seen directly below the cuboidal top layer of cells. A mineralization front was detectable below this in which small, discrete structures resembling matrix vesicles and feathery mineral crystals were evident and frequently associated with the collagen fibrils. More heavily mineralized areas were seen further into the nodule. Electron microprobe and electron and X-ray diffraction analysis confirmed the mineral to be hydroxyapatite.

  13. Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

    PubMed

    Manea, Claudiu Marius; Rusu, Mugurel Constantin; Constantin, Daniel; Mănoiu, Valentina Mariana; Moldovan, Lucia; Jianu, Adelina Maria

    2014-01-01

    Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

  14. Potential ultrastructural changes in rat epididymal cell types induced by Boswellia papyrifera and Boswellia carterii incense.

    PubMed

    Ahmed, Mukhtar; Al-Daghri, Nasser; Harrath, Abdul Halim; Alokail, Majed S; Aladakatti, Ravindranath H; Ghodesawar, Mukhtar Ahmed G; Alwasel, Saleh

    2013-08-01

    Boswellia papyrifera and Boswellia carterii, known as Arabian incense, diffuses smoke, contaminating the air, which adversely affects human health. Therefore, this study was designed to ascertain the effect of these plants on histopathological and ultrastructure changes in cauda epididymis of Albino rats. Animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Our study indicates a significant reduction in epithelial heights. Cells showed signs of degeneration. The ultrastructural study revealed that the cauda epididymis was affected, including its cell types. Furthermore, a decrease in the size of mitochondria, Golgi complex, and both ERs was observed. In all treated groups, plasma fructose decreased considerably, indicating the sign of reduced energy, vital for motility and other sperm functions. The results of this study suggest that these plants systematically affect cauda epididymal cell types and its lumen through its potential toxicity.

  15. [Effects of allelochemical EMA isolated from Phragmites communis on algal cell membrane lipid and ultrastructure].

    PubMed

    Li, Feng-min; Hu, Hong-ying; Chong, Yun-xiao; Men, Yu-jie; Guo, Mei-ting

    2007-07-01

    In order to reveal the antialgal mechanisms of allelochemicals, effects of the allelochemical eathyl-2-methyl acetoacetate (EMA) on cell membrane lipid and ultrastructure of Chlorella pyrenoidosa, Microcystis aeruginosa and Chlorella vulagaris were studied in this paper. The lipid fatty acids of the algal membrane were isolated following the Bligh and Dye method and quantified by gas chromatograph/mass spectrometry. The ultrastructure of algal cells was observed with TEM. The results showed that EMA increased the contents of linolenic acid and linolic acid with increment of 14%, while decreased the content of myristic acid and cetylic acid in C. pyrenoidosa, membrane. The content of unsaturated fatty acids C18:1 and C18:2 increased 12% and 10% in M. aeruginosa with the addition of EMA, while the content of saturated fatty acids C18:0 and C16:0 decreased. EMA showed no significant change in the fatty acid composition in C. vulagaris under the experiment condition. EMA broke off cell wall of C. pyrenoidosa and M. aeruginosa. EMA damaged the cell membrane and the inclusion of algal cell leaked out. Nuclear and mitochondrial structure was damaged with the addition of EMA. EMA showed no significant change in the ultrastructure of C. vulgaris.

  16. [Ultrastructural study of TNT effect on the callus cells and the cells of intact plants of Yucca gloriosa L].

    PubMed

    Gogoberidze, M; Zaalishvili, G; Ramishvili, M; Gogava, M; Chelidze, N

    2009-01-01

    Intracellular distribution of assimilated 2,4,6-trinitrotoluene (TNT) in callus cells, flower buds and leaves of intact Yucca gloriosa L. plants using electron microscope radioautography. The radiotracer was detected in vacuoles, plastids, mitochondrion, endoplasmic reticulum and cytoplasm. It was found that in dedifferentiated callus cells TNT was incorporated in the vacuoles in greater quantities in comparison with the cells of intact plant. Correspondingly the ultrastructural integrity of the dedifferentiated cells is less damaged.

  17. Ultrastructural characterization of normal and abnormal chondrogenesis in micromass rat embryo limb bud cell cultures.

    PubMed

    Renault, J Y; Caillaud, J M; Chevalier, J

    1995-02-01

    Inhibition of chondrogenesis in limb bud cell micromass cultures has been proposed as a short-term teratogen detection test. Validation studies were performed by testing large series of reference compounds and comparing their teratogenic potential with their ability to inhibit chondrogenesis; however, there are few reports describing the histological and ultrastructural changes associated with inhibition of chondrogenesis in vitro. The objective of this study was to provide a qualitative description of the histological and ultrastructural alterations induced by three chondrogenesis inhibitors: retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and doxylamine succinate (DS), a nonteratogen compound. In addition, in order to have a basis for the interpretation of the morphological alterations induced by the test compounds, the histological and ultrastructural changes which occur during the time course of chondrogenesis in control cultures were described and compared with those in rat embryo limb buds. We found that RA at 0.5 micrograms/ml led to a marked decrease in the number and size of cartilaginous foci; most cells lacked morphological signs of differentiation but their ability to proliferate was unaffected. At concentrations of 2 micrograms/ml and more, 6AN delayed cell proliferation, reduced staining of the extracellular matrix, and induced the formation of endoplasmic cisternae. DS at 50 micrograms/ml affected both differentiation and proliferation; pigment deposits were observed in chondrocytes, suggesting phospholipid metabolism disorders. In conclusion, this study showed that inhibition of chondrogenesis in this simple cell culture system can be associated with different types of histological and ultrastructural alterations. Examination of these alterations can provide useful information about the teratogenic potential of tested compounds and their mechanism of action.

  18. Avian minor salivary glands: an ultrastructural study of the secretory granules in mucous and seromucous cells.

    PubMed

    Olmedo, L A; Samar, M E; Avila, R E; de Crosa, M G; Dettin, L

    2000-01-01

    Ultrastructural descriptions in birds are scarce thus, in this study we have characterized the secretory granules of mucous and seromucous cells from the palatine and lingual salivary glands of birds with different diets. The samples were taken from the tongue and palatine mucosa of chicken (Gallus gallus), quail (Coturnix coturnix), chimango (Milvago chimango) and white heron (Egretta thula). The samples were processed for observation by transmission electron microscopy (TEM) employing 4% Karnovsky solution for fixation. The most noteworthy finding was the heterogeneous ultrastructural appearance of the secretory granules. Differences in substructure were found between the four species, between the palatine and lingual glands in the same species and even within the same acinus and the same cell. At variance with other authors, these differences cannot be attributed to the type of fixative solution used taking into account that all the samples were processed in the same way. Previous histochemical studies have shown the presence of sulfated and non sulfated glycoconjugates in these glands which can be associated to the maturation of the granules. These granules are probably representative of peculiar storage of the secretory products that would give rise to a heterogeneous and complex ultrastructural pattern of granules in the mucosa and seromucosa cells of these avian species.

  19. The Ultrastructural Changes of the Sertoli and Leydig Cells Following Streptozotocin Induced Diabetes

    PubMed Central

    Kianifard, Davoud; Sadrkhanlou, Rajab Ali; Hasanzadeh, Shapour

    2012-01-01

    Objective(s) This investigation was conducted to evaluate the effects of diabetes on the structure and function of testicular tissue. Materials and Methods Diabetes was induced in male adult rats by a single intraperitoneal injection of streptozotocin. Body and testicular weight, hormonal analyses, histological and ultrastructural analyses were measured. Results The body and testicular weights were dropped significantly (P< 0.05) in diabetic rats in comparison with control rats. On the other hand, in diabetic rats, the blood glucose level increased significantly (P< 0.05). The blood plasma levels of testosterone, 17-β estradiol, progesterone, FSH and LH were reduced in diabetic rats. Histomorphological studies were revealed reduction in diameter of seminiferous tubules and germinal epithelium height, edema in interstitial tissue, germ cell depletion, decrease in cellular population and activity with disruption of spermatogenesis in diabetic rats. Ultrastructural study showed the mitochondrial change and reduction of smooth endoplasmic reticulum in Sertoli and presence of lipid droplets in Leydig cells of diabetic rat’s testes. Conclusion The results of the present study confirmed that, the ultrastructural changes of Sertoli and Leydig cells, brought about by streptozotocin induced diabetes, because of the alterations in pituitary gonadotropins, and these changes influence the normal spermatogenesis in rats. PMID:23493249

  20. Cell death and phagocytosis of haematopoietic elements at the onset of haematopoiesis in the mouse spleen: an ultrastructural study.

    PubMed Central

    Sasaki, K; Iwatsuki, H; Suda, M; Itano, C

    1993-01-01

    The spleen in fetal and early postnatal mice contains a variety of proliferating haematopoietic cells as well as 2 kinds of phagocytes, scavenger macrophages and mast cells, laden with large heterogeneous inclusions. Their phagocytotic activity is directed towards extruded erythrocyte nuclei, erythrocytes and dying haematopoietic cells. The splenic cords after 18 d of gestation become filled with proliferating haematopoietic cells, and the cords contain a small number of free haematopoietic cells undergoing degeneration. The early signs of cell death can be observed in the nuclear structures: hyperchromasia of the nuclear membrane or nuclear dissolution. Erythroblast nuclei are amongst the most frequent elements engulfed. Phagocytes also take up and digest degenerating blood cells, i.e. erythrocytes, erythroblasts and neutrophil granulocytes. Since the digestion processes are ultrastructurally different for the various haematopoietic elements, the origins of heterolysosomes enclosed by phagocytes can be identified by electron microscopy. Mast cells, originally classified as secretory cells, phagocytose erythroid line cells in the spleen. Cell death in several haematopoietic cell lines is discussed in relation to programmed cell death in the developing spleen. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8270466

  1. Distribution and ultrastructural characteristics of dark cells in squamous metaplasias of the respiratory tract epithelium. [Rats

    SciTech Connect

    Klein-Szanto, A.J.P.; Nettesheim, P.; Pine, A.; Martin, D.

    1981-05-01

    Dark epithelial basal cells were found in both carcinogen-induced and non-carcinogen-induced squamous metaplasias of the tracheal epithelium. Formaldehyde-induced squamous metaplasias exhibited 4% dark cells in the basal layer. Metaplasias induced by vitamin A deficiency and those induced by dimethylbenz(a)anthracene (DMBA) without atypia showed 18-20% basal dark cells. DMBA-induced metaplasias with moderate to severe atypia exhibited 50% basal dark cells. The labeling index of basal cells in metaplastic epithelia, regardless of the inducing agent, was 16-18%, ie, the same as that of the normal esophageal stratified squamous epithelium. The percentage of labeled dark basal cells per total dark cell population was approximately 19% in the non-carcinogen-induced metaplasias and in the DMBA-induced metaplasias without atypia. In the atypical metaplasias induced by DMA this percentage increased to 26. On the basis of ultrastructural observations, five types of dark epithelial cells could be distinguished in the metaplastic epithelia. Each type of squamous metaplasia could thus be recognized by a determined numerical distribution of dark cells in the basal layer and a specific pattern of distribution of the ultrastructurally defined dark cell categories.

  2. Biochemistry and cell ultrastructure changes during senescence of Beta vulgaris L. leaf.

    PubMed

    Romanova, Alla K; Semenova, Galina A; Ignat'ev, Alexander R; Novichkova, Natalia S; Fomina, Irina R

    2016-05-01

    The comparative study of biochemical and ultrastructure features in senescing sugar beet (Beta vulgaris L.) leaves was carried out. One group of plants was grown under normal conditions in washed river sand and poured in turn with nitrate-containing mineral solution or water (N plants). Another group of plants, after 1 month of normal growth, was further grown with nitrate omitted in the nutritive solution (defN plants). The starting point of normal leaf senescence in N plants was identified by the maximal content of soluble protein. Soluble carbohydrate pools were statistically constant in senescing N plants, whereas glucose pools varied noticeably. A decrease in the contents of soluble protein and chlorophyll (a + b) in the course of senescing was typical for N plant leaves. The cell membrane in N plant leaves remained mostly intact; the central vacuoles in the leaf cells were large, and their membranes remained intact. The chloroplasts and mitochondria in senescing N plant leaves became swollen. The vesicles that were present in the cytoplasm of N plant leaves were especially large in the oldest leaves. It was concluded that senescing of sugar beet leaves at sufficient nitrate nutrition occurs according to a "vacuolar" scenario. In the case of nitrate deficiency, the content of soluble carbohydrates in defN leaves first reached maximum and then decreased in older leaves; the protein and chlorophyll (a + b) contents were totally lower than those in normal leaves and continuously decreased during the experiments. Chloroplasts in mesophyll cells of defN plant leaves became more rounded; starch grains in chloroplasts degraded and the number and size of lipid globules increased. The multitude of membrane impairments and lots of large vesicles-"crystals" appeared during the experiment. The results showed the controlling action of nitrogen nutrition in the senescing of sugar beet leaves.

  3. Ultrastructural changes in muscle cells of patients with collagen VI-related myopathies

    PubMed Central

    Tagliavini, Francesca; Sardone, Francesca; Squarzoni, Stefano; Maraldi, Nadir Mario; Merlini, Luciano; Faldini, Cesare; Sabatelli, Patrizia

    2013-01-01

    Summary Collagen VI is an extracellular matrix protein expressed in several tissues including skeletal muscle. Mutations in COL6A genes cause Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD) and Myosclerosis Myopathy (MM). Collagen VI deficiency causes increased opening of the mitochondrial permeability transition pore (mPTP), leading to ultrastructural and functional alterations of mitochondria, amplified by impairment of autophagy. Here we report for the first time ultrastructural studies on muscle biopsies from BM and UCMD patients, showing swollen mitochondria with hypodense matrix, disorganized cristae and paracrystalline inclusions, associated with dilated sarcoplasmic reticulum and apoptotic changes. These data were supported by scanning electron microscopy analysis on BM and UCMD cultured cells, showing alterations of the mitochondrial network. Morphometric analysis also revealed a reduced short axis and depicted swelling in about 3% of mitochondria. These data demonstrate that mitochondrial defects underlie the pathogenetic mechanism in muscle tissue of patients affected by collagen VI myopathies. PMID:24596691

  4. GABAergic and glycinergic pathways to goldfish retinal ganglion cells: an ultrastructural double label study

    SciTech Connect

    Muller, J.F.

    1987-01-01

    An ultrastructural double label has been employed to compare GABAergic and glycinergic systems in the inner plexiform layer (IPL) of the goldfish retina. Electron microscope autoradiography of /sup 3/H-GABA and /sup 3/H-glycine uptake was combined with retrograde HRP-labeling of ganglion cells. When surveyed for distribution, GABAergic and glycinergic synapses were found onto labeled ganglion cells throughout the IPL. This reinforces previous physiological work that described GABAergic and glycinergic influences on a variety of ganglion cells in goldfish and carp; These physiological effects often reflect direct inputs.

  5. Ultrastructural study of Mayaro virus replication in BHK-21 cells.

    PubMed

    Mezencio, J M; de Souza, W; Fonseca, M E; Rebello, M A

    1990-01-01

    The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed in Aedes albopictus cells.

  6. Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

    PubMed Central

    Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O.; Aubrey, Doug P.

    2016-01-01

    The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that

  7. Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

    DOE PAGES

    Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; ...

    2016-06-24

    The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by

  8. Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

    SciTech Connect

    Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O.; Aubrey, Doug P.

    2016-06-24

    The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic

  9. Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability.

    PubMed

    Pattathil, Sivakumar; Ingwers, Miles W; Victoriano, Olivia L; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O; Aubrey, Doug P

    2016-01-01

    The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that

  10. Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

    PubMed

    de Almeida, Sinara Mônica Vitalino; da Silva, Lúcia Patrícia Bezerra Gomes; de Lima, Luiza Rayanna Amorim; Longato, Giovanna Barbarini; Padilha, Rafael José Ribeiro; Alves, Luiz Carlos; Brayner, Fábio André; Ruiz, Ana Lucia Tasca Gois; de Carvalho, João Ernesto; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2016-11-01

    The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60μM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

  11. Effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of summer maize.

    PubMed

    Ren, Baizhao; Cui, Haiyan; Camberato, James J; Dong, Shuting; Liu, Peng; Zhao, Bin; Zhang, Jiwang

    2016-08-01

    A field experiment was conducted to study the effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of two summer maize hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The ambient sunlight treatment was used as control (CK) and shading treatments (40 % of ambient sunlight) were applied at different growth stages from silking (R1) to physiological maturity (R6) (S1), from the sixth leaf stage (V6) to R1 (S2), and from seeding to R6 (S3), respectively. The net photosynthetic rate (P n) was significantly decreased after shading. The greatest reduction of P n was found at S3 treatment, followed by S1 and S2 treatments. P n of S3 was decreased by 59 and 48 % for DH605, and 39 and 43 % for ZD958 at tasseling and milk-ripe stages, respectively, compared to that of CK. Additionally, leaf area index (LAI) and chlorophyll content decreased after shading. In terms of mesophyll cell ultrastructure, chloroplast configuration of mesophyll cells dispersed, and part of chloroplast swelled and became circular. Meanwhile, the major characteristics of chloroplasts showed poorly developed thylakoid structure at the early growth stage, blurry lamellar structure, loose grana, and a large gap between slices and warping granum. Then, plasmolysis occurred in mesophyll cells and the endomembrane system was destroyed, which resulted in the dissolution of cell membrane, karyotheca, mitochondria, and some membrane structures. The damaged mesophyll cell ultrastructure led to the decrease of photosynthetic capacity, and thus resulted in significant yield reduction by 45, 11, and 84 % in S1, S2, and S3 treatments, respectively, compared to that of CK.

  12. Ultrastructure of Gentiana tibetica proembryogenic cells before and after cooling treatments.

    PubMed

    Mikula, A; Tykarska, T; Kuras, M

    2005-01-01

    The influence of increased concentrations of sucrose, 0.4 M sorbitol, DMSO and vitrification solution (PVS2) on the ultrastructure of non-frozen and frozen suspensions of Gentiana tibetica King ex Hook. F.tissue cells was investigated. Embryogenic aggregates were composed of three groups of cells of different size with various types of plastids. The ultrastructural changes resulting from increasing the sucrose concentration in the medium from 3 to 6 percent for 4 weeks and from treatment with 0.4 M sorbitol for 48 h were similar. Observations showed replacement of large vacuoles by numerous small ones, condensation of cytoplasm, accumulation of starch, and fragmentation of endoplasmic reticulum. Treatment with PVS2 led to degradation of starch, coalescence of amyloplasts and to shrinking of nucleoli from the third group of cells when originating from 6 percent sucrose medium. The mitochondria initially had various shapes, but after PVS2 treatment showed only spherical shapes with sparse cristae. After programmed freezing of tissue protected by sorbitol and DMSO, lethal damage was observed: membrane and nuclei degradation, and cell destruction. Reversible changes after freezing were observed in tissue pretreated with vitrification solution: dilation of cell membranes, mitochondria with electron-lucent vessels, aggregation of numerous vesicles, and degradation of starch in amyloplasts. In cells cooled by a vitrification method, cell organelles appeared normal as early as 5 h after thawing, and anomalies were not observed after 48 h of post-thawing culture.

  13. An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus.

    PubMed

    Pretorius, Etheresia; du Plooy, Jenny; Soma, Prashilla; Gasparyan, Armen Yuri

    2014-07-01

    The study suggests that patients with systemic lupus erythematosus (SLE) present with distinct inflammatory ultrastructural changes such as platelets blebbing, generation of platelet-derived microparticles, spontaneous formation of massive fibrin network and fusion of the erythrocytes membranes. Lupoid platelets actively interact with other inflammatory cells, particularly with white blood cells (WBCs), and the massive fibrin network facilitates such an interaction. It is possible that the concerted actions of platelets, erythrocytes and WBC, caught in the inflammatory fibrin network, predispose to pro-thrombotic states in patients with SLE.

  14. Ultrastructural organization of contractile proteins in rat glomerular mesangial cells.

    PubMed Central

    Drenckhahn, D.; Schnittler, H.; Nobiling, R.; Kriz, W.

    1990-01-01

    Glomerular mesangial cells of the rat kidney contain actin, nonmuscle myosin, tropomyosin, and the muscular Z-line protein, alpha-actinin. This was shown for actin, myosin, and alpha-actinin by immunoblotting as well as by immunoelectron microscopy. Tropomyosin was localized in mesangial cells by immunofluorescence. In cultured mesangial cells, actin, myosin, and alpha-actinin constitute a considerable amount of the total cellular protein contents. In mesangial cells in situ actin, myosin and alpha-actinin were found to be colocalized within conspicuous microfilament bundles that traverse the cell body or major processes in various directions and project into either the tonguelike pericapillary processes, which run toward mesangial angles, or into the microvilluslike lateral extensions that abut on the perimesangial portion of the glomerular basement membrane (GBM). Thereby, the GBM of opposing mesangial angles as well as of opposing portions of the perimesangial GBM are regularly interconnected by filament bundles within mesangial cells that contain actin, myosin, and alpha-actinin. The authors suggest that the major function of actin-, myosin-, and alpha-actinin-containing filament bundles in mesangial cells is to create an isometric tension (or minute isotonic contractions) to counteract the distending forces of the rather high intracapillary hydraulic pressure and its resulting pressure gradients across the capillary wall and across the perimesangial GBM. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:2260624

  15. Platinum-Based Drugs Differentially Affect the Ultrastructure of Breast Cancer Cell Types

    PubMed Central

    Al-Adawi, Kawther; Al-Nabhani, Abdurahman; Al-Kindi, Mohamed

    2017-01-01

    Breast cancer (BC) is the most common cause of cancer-related death worldwide. Although platinum-based drugs (PBDs) are effective anticancer agents, responsive patients eventually become resistant. While resistance of some cancers to PBDs has been explored, the cellular responses of BC cells are not studied yet. Therefore, we aim to assess the differential effects of PBDs on BC ultrastructure. Three representative cells were treated with different concentrations and timing of Cisplatin, Carboplatin, and Oxaliplatin. Changes on cell surface and ultrastructure were detected by scanning (SEM) and transmission electron microscope (TEM). In SEM, control cells were semiflattened containing microvilli with extending lamellipodia while treated ones were round with irregular surface and several pores, indicating drug entry. Prolonged treatment resembled distinct apoptotic features such as shrinkage, membrane blebs, and narrowing of lamellipodia with blunt microvilli. TEM detected PBDs' deposits that scattered among cellular organelles inducing structural distortion, lumen swelling, chromatin condensation, and nuclear fragmentation. Deposits were attracted to fat droplets, explained by drug hydrophobic properties, while later they were located close to cell membrane, suggesting drug efflux. Phagosomes with destructed organelles and deposits were detected as defending mechanism. Understanding BC cells response to PBDs might provide new insight for an effective treatment. PMID:28377926

  16. Ultrastructure of Fibre and Parenchyma Cell Walls During Early Stages of Culm Development in Dendrocalamus asper

    PubMed Central

    GRITSCH, CRISTINA SANCHIS; MURPHY, RICHARD J.

    2005-01-01

    • Background and Aims The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. • Methods The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol–HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. • Key Results The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. • Conclusions The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation. PMID:15665037

  17. [The effect of apoptosis inductor dexamethasone on the energy exchange and ultrastructure of plant cells].

    PubMed

    Rakhmatullina, D F; Gordon, L Kh; Ponomareva, A A; Ogorodnikova, T I

    2009-01-01

    Changes in respiration and cell ultrastructure induced by long-term incubation with dexamethasone (DM) in excised roots of 5-day old wheat (Triticum aestivum L.) seedlings were investigated. During 5 h incubation of roots with DM, oxygen consumption was inhibited by 20-30%, while respiratory coefficient did not change and its value was about 1. DM prevented from glucose-induced activation of respiration, which indicated blockade of glycolysis and decrease in oxygen uptake by this apoptotic inductor. It has been suggested that the respiratory inhibition by DM might be also connected with the influence of DM on the 1st segment ofmitochondrial electron transport chains. This suggestion is supported by the fact that succinate prevented DM-induced inhibition of respiration. Furthermore, stabilization of intracellular pH by dipeptide carnosine abolished inhibitory effect of DM on respiration. Probably depression of oxygen consumption by DM is also due to acidification of cytoplasm. Strong vacuolization of cytoplasm, one of the characteristics of cell death, occurred in 5 h after treatment of roots with DM. Vacuolization was to a great extent prevented by carnosine. The ultrastructure of root cells after long-term (23 h) treatment with DM was disturbed, and oxygen consumption was also dramatically decreased. These effects of DM were in part prevented by carnosine. The data obtained suggest that DM causes acidification of cytoplasm, disturbance of energy exchange and cytoplasm vacuolization in root cells, and induces death of these cells.

  18. Ultrastructural changes in tracheal epithelial cells exposed to oxygen

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Harrison, G. A.; Turnbill, C.; Black, S.

    1977-01-01

    White albino rats were sacrificed after 24, 36, 48, 72, and 96 h of exposure to 100% O2 at 1 atm. Tissue was prepared for the scanning electron microscope (SEM) by Critical Point Drying and for the transmission electron microscope (TEM) by plastic embedding. Scanning microscopy showed a loss of microvilli after 48 h of exposure. Cilia appeared relatively normal with SEM, but TEM revealed changes in the outer membrane. In TEM, nonciliated cells appeared swollen and often encroached on the ciliated cells. A heavy mucous blanket remained even after processing. All the changes observed that are induced by oxygen exposure contribute to mucostasis, reducing and/or halting mucociliary clearance.

  19. An ultrastructural study, effects of Proteus vulgaris OX19 on the rabbit spleen cells.

    PubMed

    Gul, Nursel; Ozkorkmaz, Ebru Gokalp; Kelesoglu, Ilknur; Ozluk, Aydin

    2013-01-01

    Effects of Proteus vulgaris OX19 on the spleen cells of rabbits were investigated. Control group (n=5) and Proteus treated group (n=5) of New Zealand male rabbits were used in this study. Bacteria were injected to the rabbits in five days periods with increasing dosages for one month. Thin sections were examined by transmission electron microscope (Jeol 100CXII). Ultrastructural changes were defined in spleen tissue cells due to the antigenic stimulation of bacteria. Spleen cells observed in control group were in normal structure and cells were in close contact with each other. However, spleen cells of Proteus treated group displayed structural changes with regard to the control group in electron microscopic examinations. Chemotaxis of macrophages, forming of pseudopodia and presence of phagocytic vacuoles were observed. Lymphocytes, the major cells of spleen revealed mitotic activity. In addition, chromatin condensation in nucleus and dilatations in perinuclear space were significant. Interactions of lymphocytes and macrophages were noteworthy.

  20. Influence of parathyroid hormone on bone cell ultrastructure

    SciTech Connect

    Matthews, J.L.; Talmage, R.V.

    1981-05-01

    A study in rats demonstrated that morphologic changes in the bone osteocytes and osteoblasts are produced following parathyroid hormone (PTH) injection into thyroparathyroidectomized animals. It further showed that similar changes occur in normal rats as the result of extended fasting. The most significant morphologic alterations involved surface microvilli and blebs as determined by scanning electron microscopy. Transmission electron microscopy studies showed alterations in the cisternae of the rough endoplasmic reticulum. Additionally, cell shape varied markedly from the control cuboidal morphology. These morphologic changes occurred during peak periods of plasma calcium change and returned to control morphology as plasma calcium levels normalized. The study supports the concept that osteocytes and lining cells on the surface of bone play a role in maintenance of plasma calcium concentrations. (JMT)

  1. Ultrastructure of the suberized styloid crystal cells in Agave leaves.

    PubMed

    Wattendorff, J

    1976-01-01

    Styloid calcium oxalate crystal idioblasts of Agave americana L. are suberized. Where the crystals do not touch the cell wall directly they are enclosed in a suberinic sheath which is connected with the suberinic wall layer. No polysaccharides are laid down as a tertiary wall layer, nor could any polysaccharides be found in the crystal sheath. These results contradict those of Arnott (1973) but agree fully with those of Rothert and Zalenski (1899).

  2. Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development1[OPEN

    PubMed Central

    Segado, Patricia; Domínguez, Eva

    2016-01-01

    The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall. PMID:26668335

  3. Ultrastructure and immunocytochemical characteristics of cells in the octopus cell area of the rat cochlear nucleus: comparison with multipolar cells.

    PubMed

    Alibardi, Lorenzo

    2003-01-01

    Cells in the octopus cell area of the rat ventral cochlear nucleus have been connected to the monaural interpretation of spectral patterns of sound such as those derived from speech. This is possible by their fast onset of firing after each octopus cell and its dendrites have been contacted by many auditory fibres carrying different frequencies. The cytological characteristics that make these large cells able to perform such a function have been studied with ultrastructural immunocytochemistry for glycine, GABA and glutamate, and compared to that of other multipolar neurons of other regions of the ventral cochlear nucleus. Cells in the octopus cell area have an ultrastructure similar to large-giant D-multipolar neurons present in other areas of the cochlear nucleus, from which they differ by the presence of a larger excitatory axo-somatic synaptic input and larger mitochondria. Octopus cells are glycine and GABA negative, and glutamate positive with different degree. Large octopus cells receive more axo-somatic boutons than smaller octopus cells. Fusiform octopus cells are found sparsely within the intermediate acoustic striae. These cells are large to giant excitatory neurons (23-35 microm) with 62-85% of their irregular perimeter covered with large axo-somatic synaptic boutons. Most boutons contain round vesicles and are glycine and GABA negative but glutamate positive. The latter excitatory boutons represent about 70% of the input to octopus cells. Glycine positive boutons with flat and pleomorphic vesicles account for 9-10% of the input while GABA-ergic boutons with pleomorphic vesicles represent about 20% of the synaptic input. Other few, multipolar cells within the rat octopus cell area are surrounded by more inhibitory than excitatory terminals which contain flat and pleomorphic vesicles, a feature distinctive from that of true octopus cells. The latter resemble multipolar cells seen outside the octopus cell area that project to the contralateral inferior

  4. Ultrastructural study of the blood cells of the coelacanth Latimeria chalumnae (Rhipidistia: Coelacanthini).

    PubMed

    Jarial, M S

    2005-04-01

    The blood cells in the renal capillaries of the coelacanth Latimeria chalumnae Smith were studied by transmission electron microscopic methods. On the basis of ultrastructural similarities of cytoplasmic granules of the leukocytes and by comparison with those of the fish and mammalian cells, erythrocytes and three types of granular leukocytes, namely neutrophils, eosinophils and basophils, and three types of agranular leukocytes, i.e., lymphocytes, monocytes and thrombocytes are characterized. The presence of granular and agranular leukocytes in the blood of Latimeria suggests that these cells appeared early in vertebrate evolution. The display of nuclear blebs on the cytoplasmic phase of the nuclear membrane and the presence of nuclear fragments in the cytoplasm of some erythrocytes suggest that these cells undergo apoptosis in order to delete older erythrocytes from the blood stream. The relatively small size of its nucleated erythrocytes and the striking resemblance of the ultrastructural features of its leukocytes to those of higher vertebrate leukocytes support the view that Latimeria is a close living relative of tetrapods.

  5. Ultrastructural aspects of Cystoisospora belli (syn. Isospora belli) in continuous cell lines.

    PubMed

    Resende, Deisy V; Assis, Dnieber C; Ribeiro, Múcio F Barbosa; Cabrine-Santos, Marlene; Frenkel, Jacob K; Correia, Dalmo; Oliveira-Silva, Márcia B

    2014-06-01

    Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli, and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT-8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp.

  6. Comparative effectiveness of a clinostat and a slow-turning lateral vessel at mimicking the ultrastructural effects of microgravity in plant cells

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1990-01-01

    The object of this research was to determine how effectively the actions of a clinostat and a fluid-filled, slow-turning lateral vessel (STLV) mimic the ultrastructural effects of microgravity in plant cells. We accomplished this by qualitatively and quantitatively comparing the ultrastructures of cells grown on clinostats and in an STLV with those of cells grown at 1 g and in microgravity aboard the Space Shuttle Columbia. Columella cells of Brassica perviridis seedlings grown in microgravity and in an STLV have similar structures. Both contain significantly more lipid bodies, less starch, and fewer dictyosomes than columella cells of seedlings grown at 1 g. Cells of seedlings grown on clinostats have significantly different ultrastructures from those grown in microgravity or in an STLV, indicating that clinostats do not mimic microgravity at the ultrastructural level. The similar structures of columella cells of seedlings grown in an STLV and in microgravity suggest that an STLV effectively mimics microgravity at the ultrastructural level.

  7. [The effect of microgravity on osteogenic cells in culture. Ultrastructural research].

    PubMed

    Berezovskaia, O P; Rodionova, N V

    1998-01-01

    In order to estimate the influence of microgravity on osteogenic cells a comparative ultrastructural study of preosteoblastic cells of line MN7, cultivated in space and in ground conditions has been carried out. Morphometric analysis has revealed that in cells exposed during 9 days on board the biosatellite Cosmos 2229 endoplasmic reticulum was not so expensive and the number of myeline-like structures increased as compared to the ground control. Percentage of cells with nucleoli is lower in the flight samples as well. The increase in specific volume of lipid droplets and changed structure of mitochondria testifies to deviation in energy metabolism. The reaction of osteogenic cells to microgravity resulting in suppression of specific functions may contribute to alterations in the bone metabolism under the space flight conditions.

  8. Ultrastructural interaction between spermatozoon and human oviductal cells in vitro.

    PubMed

    Vigil, Pilar; Salgado, Ana María; Cortés, Manuel E

    2012-04-01

    The oviduct is an important organ for successful mammalian reproduction. In this work, human oviducts were inseminated and their explants analyzed using scanning electron microscopy in order to study, at a finer ultrastructual level, the interaction between spermatozoon and oviduct in vitro. Results show unequivocally a spermatozoon tightly attached through the acrosomal region of its head to several cilia of the human tubal epithelial cells. This finding proves that spermatozoa do indeed adhere to the endosalpinx, a fact of utmost relevance for the physiology of the reproductive process, since it supports the idea of a spermatozoa reservoir being formed in the oviduct, which is also briefly discussed.

  9. Comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities.

    PubMed

    Plopper, C G; Mariassy, A T; Wilson, D W; Alley, J L; Nishio, S J; Nettesheim, P

    1983-12-01

    Three types of nonciliated epithelial cells in mammalian conducting respiratory airways are thought to be secretory: mucous (goblet) cells, serous epithelial cells, and Clara cells. Mucous and serous cells are considered to be the secretory cells of the trachea. Clara cells are considered to be the secretory cells of the most distal conducting airways or bronchioles. To ascertain if mucous and serous epithelial cells are common to the tracheal epithelium of mammalian species, we characterized the ultrastructure and population densities of tracheal epithelial cells in six species: hamster (H), rat (Rt), rabbit (Rb), cat (C), Bonnet monkey (M. radiata) (B), and sheep (S). Following fixation by airway infusion with glutaraldehyde/paraformaldehyde, tracheal tissue was processed for light and electron microscopy (EM) by a selective embedding technique. Tracheal epithelium over cartilage was quantitated by light microscopy and characterized by transmission EM. Mucous cells were defined by abundant large nonhomogeneous granules, numerous Golgi complexes, basally located nuclei and granular endoplasmic reticulum (GER). The percentage of mucous cells in the tracheal epithelium was: H (0%), Rt (0.5%), Rb (1.3%), C (20.2%), B (8%), S (5.1%). Serous cells had homogeneous, electron-dense granules and extensive GER. Serous cells were present only in rats (39.2%). Clara cells had homogeneous electron-dense granules, abundant agranular endoplasmic reticulum (AER) and basal GER. Clara cells were found in hamsters (41.4%) and rabbits (17.6%). In sheep trachea, 35.9% of the epithelial cells had small electron-lucent granules, abundant AER and numerous Golgi complexes. In Bonnet monkey trachea, 16% of the epithelial cells had small electron-lucent granules, numerous polyribosomes, perinuclear Golgi apparatus and moderate GER. In cat trachea, 5.4% of the epithelial cells lacked granules, and had moderate numbers of mitochondria, moderate amounts of polyribosomes, a central nucleus, and

  10. The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses

    SciTech Connect

    Terrier, Olivier; Moules, Vincent; Carron, Coralie; Cartet, Gaeelle; Frobert, Emilie; Yver, Matthieu; Traversier, Aurelien; Wolff, Thorsten; Naffakh, Nadia; and others

    2012-10-10

    Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

  11. The ultrastructure of camel blood platelets: a comparative study with human, bovine, and equine cells.

    PubMed

    Gader, Abdel Galil M Abdel; Ghumlas, Abeer K Al; Hussain, Mansour F; Haidari, Ahmed Al; White, James G

    2008-02-01

    Previous studies indicated that the camel has a very active haemostatic mechanism with a short bleeding time and thrombocytosis. However, platelet function, when tested by agonist-induced aggregation and PFA 100 closure time, showed marked inhibition compared to humans. Since camels are also far more resistant to long exposure to excessive heat and high body temperature than humans, it seemed worthwhile to explore fundamental morphological differences between human and camel platelets and those from other species. The present study has examined the ultrastructure of camel platelets and compared them with the fine structures of human, bovine and equine thrombocytes. Camel platelets, like bovine and equine cells, are discoid in shape and about two-thirds the size of human platelets. A circumferential coil of microtubular supports the disk-like form of camel platelets. Their cytoplasm, like bovine and equine platelets, is filled with alpha granule twice as large as those in human platelets, but lacking the organized matrix of equine alpha granules. Dense bodies are present in camel platelets with whip-like extensions not present on bovine or equine thrombocytes, but found on occasional human platelet dense bodies. Camel platelets, like bovine and equine thrombocytes, lack an open canalicular system (OCS) and must secrete granule products by fusion with the cell wall rather than an OCS. Future studies will determine if the differences in ultrastructural anatomy protect camel platelets from heat more than human thrombocytes.

  12. Ultrastructural characteristics of nurse cell-larva complex of four species of Trichinella in several hosts.

    PubMed

    Sacchi, L; Corona, S; Gajadhar, A A; Pozio, E

    2001-06-01

    The nurse cell-larva complex of nematodes of the genus Trichinella plays an important role in the survival of the larva in decaying muscles, frequently favouring the transmission of the parasite in extreme environmental conditions. The ultrastructure of the nurse cell-larva complex in muscles from different hosts infected with T. nativa (a walrus and a polar bear), T. spiralis (horses and humans), T. pseudospiralis (a laboratory mouse) and T. papuae (a laboratory mouse) were examined. Analysis with transmission electron microscope showed that the typical nurse cell structure was present in all examined samples, irrespective of the species of larva, of the presence of a collagen capsule, of the age of infection and of the host species, suggesting that there exists a molecular mechanism that in the first stage of larva invasion is similar for encapsulated and non-encapsulated species.

  13. Cytogenetical and ultrastructural effects of copper on root meristem cells of Allium sativum L.

    PubMed

    Liu, Donghua; Jiang, Wusheng; Meng, Qingmin; Zou, Jin; Gu, Jiegang; Zeng, Muai

    2009-04-01

    Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.

  14. Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout, and first description of erythrophore ultrastructure in salmonids.

    PubMed

    Djurdjevič, Ida; Kreft, Mateja Erdani; Sušnik Bajec, Simona

    2015-11-01

    Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble-like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short-range interactions, an important role of the cellular environment and long-range interactions between chromatophores in promoting adult pigment pattern

  15. Ultrastructure of single cells, callus-like and monospore-like cells in Porphyra yezoensis ueda on semisolid culture medium

    NASA Astrophysics Data System (ADS)

    Mei, Junxue; Shen, Songdong; Jiang, Ming; Fei, Xiugeng

    2003-06-01

    It had been demonstrated that individual cells or protoplasts isolated from Porphyra thallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi-solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi-solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi-solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus-like cells formed on semi-solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2-layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.

  16. Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins

    SciTech Connect

    Lea, P.J.; Pawlowski, A.; Persad, S.D.; Menon, I.A.; Haberman, H.F.

    1988-01-01

    Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.

  17. Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells.

    PubMed Central

    Ryser, U; Keller, B

    1992-01-01

    A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels. PMID:12297662

  18. Ultrastructural evaluation of parathyroid glands and thyroid C cells of cattle fed Solanum malacoxylon.

    PubMed Central

    Collins, W. T.; Capen, C. C.; Döbereiner, J.; Tokarnia, C. H.

    1977-01-01

    Fine structural alterations of thyroid C cells and parathyroid chief cells were evaluated after feeding dried leaves of the calcinogenic plant, Solanum malacoxylon, to cattle for 1, 6 and 32 days. Thyroid C cells initially were degranulated in response to the hypercalcemia, and parathyroid chief cells accumulated secretory granules. There was hypertrophy of thyroid C cells with well-developed secretory organelles but few secretory granules in the cytoplasm after 6 days of feeding S. malacoxylon. Inactive chief cells with dispersed profiles of endoplasmic reticulum and increased lysosomal bodies predominated in the parathyroid glands. Multiple foci of soft tissue mineralization were present in the heart, lung, and kidney. Thyroid C cells underwent hypertrophy and hyperplasia after 32 days of S. malacoxylon, and parathyroid chief cells were inactive or atrophic in response to the long-term hypercalcemia. Severe soft tissue mineralization was present throughout the cardiovascular system, lung, kidney, and spleen. These ultrastructural changes in thyroid C cells and parathyroid chief cells plus the widespread soft tissue mineralization observed after feeding cattle small amounts of S. malacoxylon are consistent with the recent evidence that leaves of this plant are a potent source of the active metabolite, 1,25-dihydroxycholecalciferol, of vitamin D. Images Figure 7 Figure 8 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:869016

  19. Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron.

    PubMed

    Bassett, Mark L; Dahlstrom, Jane E; Taylor, Matthew C; Koina, Mark E; Maxwell, Lesley; Francis, Douglas; Jain, Sanjiv; McLean, Allan J

    2003-07-01

    Hepatic sinusoidal endothelial cells form an important interface between the vascular system, represented by the sinusoids, and the space of Disse that surrounds the hepatocyte microvilli. This study aimed to assess the light microscopic and ultrastructural effects of acute exposure of hepatic sinusoidal endothelial cells to colloidal iron by injection of rats with iron polymaltose. Eight minutes after a single intravenous injection of iron polymaltose sinusoidal endothelial cells showed defenestration, and thickening and layering as assessed by transmission electron microscopy. Kupffer cells and stellate cells appeared activated. These changes were not observed in control animals, experiments using equivalent doses of maltose, or experiments using colloidal carbon except for Kupffer cell activation due to colloidal carbon. No significant light microscopic changes were seen in study or control animals. The findings indicate that acute exposure to colloidal iron causes changes in hepatic sinusoidal endothelial cells, stellate cells and Kupffer cells. This may be the result of a direct toxic effect of iron or increased production of reactive oxygen species. These observations suggest a possible mechanism for defenestration of sinusoidal endothelial cells in ageing and in disease states.

  20. Air pollution effects on the ultrastructure of Phlomis fruticosa mesophyll cells

    SciTech Connect

    Psaras, G.K.; Christodoulakis, N.S.

    1987-04-01

    Plant physiologists and environmental scientists suggest that a basic effect of air pollution on plants leads towards the minimization of their productivity. On the other hand the action of individual pollutants on intact plants has been studied from biochemical as well as structural viewpoint. Thus the study of plant responses to SO/sub 2/ exposure revealed that this agent causes acute and chronic injury. Chronic injury results in chlorosis and subsequent necrosis due to destruction of chlorophylls and final chloroplast lysis. It has been documented that ultrastructural characteristics of leaves are affected prior to any visible injury. Electron microscope examination of SO/sub 2/ fumigated plant-attached leaves of Vicia faba revealed chloroplast thylakoids starting to swell whilst photosynthesis rate was drastically reduced. The first light microscope-detected effects of air pollution on the leaf structure of plants common in natural ecosystems of Athens metropolitan area, have been reported. A chlorosis phenomenon in Urginea maritima leaves as well as an indication of detrimental effects of Phlomis fruticosa mesophyll chloroplasts were documented. In this work further investigation has been undertaken in order to elucidate the precise effects of air pollution on the ultrastructure of the photosynthesizing mesophyll cells.

  1. Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action.

    PubMed

    Cushnie, T P Tim; O'Driscoll, Noëlle H; Lamb, Andrew J

    2016-12-01

    Efforts to reduce the global burden of bacterial disease and contend with escalating bacterial resistance are spurring innovation in antibacterial drug and biocide development and related technologies such as photodynamic therapy and photochemical disinfection. Elucidation of the mechanism of action of these new agents and processes can greatly facilitate their development, but it is a complex endeavour. One strategy that has been popular for many years, and which is garnering increasing interest due to recent technological advances in microscopy and a deeper understanding of the molecular events involved, is the examination of treated bacteria for changes to their morphology and ultrastructure. In this review, we take a critical look at this approach. Variables affecting antibacterial-induced alterations are discussed first. These include characteristics of the test organism (e.g. cell wall structure) and incubation conditions (e.g. growth medium osmolarity). The main body of the review then describes the different alterations that can occur. Micrographs depicting these alterations are presented, together with information on agents that induce the change, and the sequence of molecular events that lead to the change. We close by highlighting those morphological and ultrastructural changes which are consistently induced by agents sharing the same mechanism (e.g. spheroplast formation by peptidoglycan synthesis inhibitors) and explaining how changes that are induced by multiple antibacterial classes (e.g. filamentation by DNA synthesis inhibitors, FtsZ disruptors, and other types of agent) can still yield useful mechanistic information. Lastly, recommendations are made regarding future study design and execution.

  2. Ultrastructural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of eicosanoid metabolism

    SciTech Connect

    Wilson, D.E.; Anderson, K.M. ); Seed, T.M. )

    1990-01-01

    Human glioblastoma cells incubated in the presence of inhibitors of eicosanoid biosynthesis show decreased cellular proliferation without cytotoxicity. The authors studied the ultrastructural morphology of a human glioblastoma cell line cultured with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, or 5,8,11,14-eicosatetraynoic acid, a cyclooxygenase and lipoxygenase inhibitor. When glioblastoma cells were treated for 3 days with antiproliferative concentrations of either agent, they shared many morphological characteristics, including evidence for increased astrocytic differentiation with only limited signs of toxicity. The inhibited glioma cells demonstrated an increase in the number and length of astrocytic processes containing greater numbers of glial filaments, and the NDGA-treated cells also demonstrated extensive lateral pseudopod formation along the processes. The glioblastoma cell shape also become more elongated, losing the usual nuclear lobularity and nuclear inclusions, especially in NDGA-treated cells. Many cytoplasmic organelles packed the cytosol of the inhibited glioma cells, including prominent Golgi apparatus, dilated smooth endoplasmic reticulum evolving into dilated vesicles, cytoplasmic vacuoles, and numerous concentric laminations. There was limited evidence for toxicity, however, as the mitochondria were more pleomorphic with some mitochondrial distension and disruption of the cristae along with an increase in cytoplasmic vacuolization. The authors conclude that the inhibitors of eicosanoid biosynthesis. NDGA and 5,8,11,14-eicosatetraynoic acid, not only suppress glioblastoma cell proliferation, but also include increased astrocytic differentiation.

  3. Ultrastructural features of degenerated cardiac muscle cells in patients with cardiac hypertrophy.

    PubMed Central

    Maron, B. J.; Ferrans, V. J.; Roberts, W. C.

    1975-01-01

    Degenerated cardiac muscle cells were present in hypertrophied ventricular muscle obtained at operation from 12 (38%) of 32 patients with asymmetric septal hypertrophy (hypertrophic cardiomyopathy) or aortic valvular disease. Degenerated cells demonstrated a wide variety of ultrastructural alterations. Mildly altered cells were normal-sized or hypertrophied and showed focal changes, including preferential loss of thick (myosin) filaments, streaming and clumping of Z band material, and proliferation of the tubules of sarcoplasmic reticulum. Moderately and severely degenerated cells were normal-sized or atrophic and showed additional changes, including extensive myofibrillar lysis and loss of T tubules. The appearance of the most severely degenerated cells usually reflected the cytoplasmic organelle (sarcoplasmic reticulum, glycogen, or mitochondria) which underwent proliferation and filled the myofibril-free areas of these cells. Moderately and severely degenerated cells were present in areas of fibrosis, had thickened basement membranes, and had lost their intercellular connections. These observations suggest that degenerated cardiac muscle cells have poor contractile function and may be responsible for impaired cardiac performance in some patients with chronic ventricular hypertrophy. Images Fig 1 Fig 2 Fig 3 Figs 4-6 Figs 7-8 Fig 9 Fig 10 Fig 11 Figs 12-15 Fig 16 Fig 17 Figs 18-21 Figs 22-23 Fig 24 Fig 25 Fig 26 Fig 27 Figs 28-29 Fig 30 Figs 31-32 Fig 33 PMID:124533

  4. Ultrastructural Characterization of Mammary Analogue Secretory Carcinoma of the Salivary Glands: A Distinct Entity from Acinic Cell Carcinoma?

    PubMed

    Guilmette, Julie; Nielsen, Gunnlaugur P; Faquin, William C; Selig, Martin; Nosé, Vânia; Chi, Anthony W S; Sadow, Peter M

    2017-02-13

    Mammary analogue secretory carcinoma (MASC) of the salivary glands is a recently described neoplasm of the salivary glands with a characteristic morphology complemented by a specific cytogenetic translocation and gene rearrangements. Although immunophenotypic and cytogenetic differences allow for a more reliable distinction, ultrastructural features can also provide important information about the relationship between MASC, classic acinic cell carcinoma (AciCC), and AciCC intercalated duct cell-predominant variant. Following approval from the hospital's institutional review board, 7 cases of MASC, 8 cases of classic AciCC, and 4 cases of AciCC intercalated duct cell-predominant variant were retrieved from the pathology files of Massachusetts General Hospital from 2012 to 2015. Electron microscopy was performed using formalin-fixed, paraffin-embedded tissue. Ultrastructural features of all 19 neoplasms of the salivary glands were recorded. The predominant cell-types observed in MASC are those with intercalated/striated duct cell differentiation. These features include prominent invaginations of the cell surface studded with microvilli, and some intra- and intercellular lumina also with a microvillous surface. Classic AciCC dominant cell-type recapitulates acinar cell differentiation. These cells contain large intracytoplasmic zymogen-like granules. AciCC intercalated duct cell-predominant variant showed both cell populations in various proportions with the intercalated/striated duct cell type usually being the dominant one. MASC presents with distinctive ultrastructural features that allows its proper differentiation from classic AciCC. However, significant ultrastructural features overlaps between both AciCC intercalated duct cells-predominant and classic AciCC and MASC. These findings indicate a very close proximity between these tumors.

  5. Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters.

    PubMed

    Díaz, Seila; Renault, Tristan; Villalba, Antonio; Carballal, María Jesús

    2011-09-09

    Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.

  6. Macro- and microvascular endothelial cells in vitro: maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

    SciTech Connect

    Stolz, D.B.; Jacobson, B.S. )

    1991-02-01

    Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of {sup 35}S-methionine-labeled whole cells. Two-dimensional gel analysis of {sup 35}S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture.

  7. Hygroscopic swelling and shrinkage of latewood cell wall micropillars reveal ultrastructural anisotropy.

    PubMed

    Rafsanjani, Ahmad; Stiefel, Michael; Jefimovs, Konstantins; Mokso, Rajmund; Derome, Dominique; Carmeliet, Jan

    2014-06-06

    We document the hygroscopic swelling and shrinkage of the central and the thickest secondary cell wall layer of wood (named S2) in response to changes in environmental humidity using synchrotron radiation-based phase contrast X-ray tomographic nanoscopy. The S2 layer is a natural fibre-reinforced nano-composite polymer and is strongly reactive to water. Using focused ion beam, micropillars with a cross section of few micrometres are fabricated from the S2 layer of the latewood cell walls of Norway spruce softwood. The thin neighbouring cell wall layers are removed to prevent hindering or restraining of moisture-induced deformation during swelling or shrinkage. The proposed experiment intended to get further insights into the microscopic origin of the anisotropic hygro-expansion of wood. It is found that the swelling/shrinkage strains are highly anisotropic in the transverse plane of the cell wall, larger in the normal than in the direction parallel to the cell wall's thickness. This ultrastructural anisotropy may be due to the concentric lamellation of the cellulose microfibrils as the role of the cellulose microfibril angle in the transverse swelling anisotropy is negligible. The volumetric swelling of the cell wall material is found to be substantially larger than the one of wood tissues within the growth ring and wood samples made of several growth rings. The hierarchical configuration in wood optimally increases its dimensional stability in response to a humid environment with higher scales of complexity.

  8. Hygroscopic swelling and shrinkage of latewood cell wall micropillars reveal ultrastructural anisotropy

    PubMed Central

    Rafsanjani, Ahmad; Stiefel, Michael; Jefimovs, Konstantins; Mokso, Rajmund; Derome, Dominique; Carmeliet, Jan

    2014-01-01

    We document the hygroscopic swelling and shrinkage of the central and the thickest secondary cell wall layer of wood (named S2) in response to changes in environmental humidity using synchrotron radiation-based phase contrast X-ray tomographic nanoscopy. The S2 layer is a natural fibre-reinforced nano-composite polymer and is strongly reactive to water. Using focused ion beam, micropillars with a cross section of few micrometres are fabricated from the S2 layer of the latewood cell walls of Norway spruce softwood. The thin neighbouring cell wall layers are removed to prevent hindering or restraining of moisture-induced deformation during swelling or shrinkage. The proposed experiment intended to get further insights into the microscopic origin of the anisotropic hygro-expansion of wood. It is found that the swelling/shrinkage strains are highly anisotropic in the transverse plane of the cell wall, larger in the normal than in the direction parallel to the cell wall's thickness. This ultrastructural anisotropy may be due to the concentric lamellation of the cellulose microfibrils as the role of the cellulose microfibril angle in the transverse swelling anisotropy is negligible. The volumetric swelling of the cell wall material is found to be substantially larger than the one of wood tissues within the growth ring and wood samples made of several growth rings. The hierarchical configuration in wood optimally increases its dimensional stability in response to a humid environment with higher scales of complexity. PMID:24671938

  9. Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum.

    PubMed

    Tanaka, Atsuko; De Martino, Alessandra; Amato, Alberto; Montsant, Anton; Mathieu, Benjamin; Rostaing, Philippe; Tirichine, Leila; Bowler, Chris

    2015-11-01

    The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

  10. Ultrastructure of ECL cells in Mastomys after long-term treatment with H2 receptor antagonist loxtidine.

    PubMed

    Vigen, Reidar Alexander; Kidd, Mark; Modlin, Irvin M; Chen, Duan; Zhao, Chun-Mei

    2012-06-01

    Gastric ECL-cell hyperplasia and carcinoids (ECLoma) develop after 1 year in rats treated with omeprazole or 2 months in Mastomys treated with loxtidine. The aim of this study was to examine the ultrastructure of ECL cells in Mastomys after loxtidine treatment with an attempt to evaluate whether an impairment of autophagy was involved in the tumorigenesis. Mastomys were given loxtidine for 8 or 27 weeks. Morphological analysis of ECL cells showed that (1) cell size was not increased after 8 or 27 weeks; (2) secretory vesicles, a hallmark feature of welldifferentiated ECL cells, were unchanged after 8 weeks but reduced after 27 weeks; (3) granules were reduced after 8 or 27 weeks; (4) microvesicles were unchanged after the treatment; and (5) vacuoles and lipofuscin bodies were found occasionally after 8 weeks but not at 27 weeks. In addition, the appearance of ECL-cell ultrastructure differed between loxtidine-treated Mastomys and rats treated with omeprazole or subjected to antrectomy, but was similar between Mastomys treated with loxtidine for 27 weeks and mice deficient in CCK(2) receptor. We suggest that the ultrastructure of ECL cells in Mastomys after long-term treatment with loxtidine displayed an impaired formation of vacuoles and lipofuscin bodies, markers of the autophagic pathway.

  11. Effects of Waterlogging on Leaf Mesophyll Cell Ultrastructure and Photosynthetic Characteristics of Summer Maize

    PubMed Central

    Ren, Baizhao; Zhang, Jiwang; Dong, Shuting; Liu, Peng; Zhao, Bin

    2016-01-01

    A field experiment was performed to study the effects of waterlogging on the leaf mesophyll cell ultrastructure, chlorophyll content, gas exchange parameters, chlorophyll fluorescence, and malondialdehyde (MDA) content of summer maize (Zea mays L.) hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The waterlogging treatments were implemented for different durations (3 and 6 days) at the third leaf stage (V3), the sixth leaf stage (V6), and the 10th day after the tasseling stage (10VT). Leaf area index (LAI), chlorophyll content, photosynthetic rate (Pn), and actual photochemical efficiency (ΦPSII) were reduced after waterlogging, indicating that waterlogging significantly decreased photosynthetic capacity. The chloroplast shapes changed from long and oval to elliptical or circular after waterlogging. In addition, the internal structures of chloroplasts were degenerated after waterlogging. After waterlogging for 6 d at V3, the number of grana and grana lamellae of the third expanded leaf in DH605 were decreased by 26.83% and 55.95%, respectively, compared to the control (CK). Those in ZD958 were reduced by 30.08% and 31.94%, respectively. Waterlogging increased MDA content in both hybrids, suggesting an impact of waterlogging on membrane integrity and thus membrane deterioration. Waterlogging also damaged the biological membrane structure and mitochondria. Our results indicated that the physiological reactions to waterlogging were closely related to lower LAI, chlorophyll content, and Pn and to the destruction of chloroplast ultrastructure. These negative effects resulted in the decrease of grain yield in response to waterlogging. Summer maize was the most susceptible to damage when waterlogging occurred at V3, followed by V6 and 10VT, with damage increasing in the wake of waterlogging duration increasing. PMID:27583803

  12. Immunocytochemical and ultrastructural identification of pituitary cell types in the protogynous Thalassoma duperrey during adult sexual ontogeny

    USGS Publications Warehouse

    Parhar, I.S.; Nagahama, Y.; Grau, E.G.; Ross, R.M.

    1998-01-01

    Protogynous wrasses (Thalassoma duperrey): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypophyseal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin I?? subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin II?? subunit containing cells were seen in the centroventral parts of the PPD and along the periphery of the pars intermedia (PI). The pars intermedia was composed of melanotropic cells and somatolactin cells that lined the neurohypohysis. Distinct ultrastructural differences in corticotropic and somatotropic cells were not observed between the four groups. In all groups, prolactin cells in the ventral-most RPD could be immature cells or actively secreting prolactin. Gonadotropic II cells of PM and F had relatively higher incidence of "nuclear budding" and cell organelles compared to TM and SM. Besides gonadotropic, the active melanotropic and somatolactin cells might be associated with some aspect(s) of reproduction.

  13. [Effects of silicon on the ultrastructures of wheat radical cells under copper stress].

    PubMed

    Zhang, Dai-Jing; Ma, Jian-Hui; Yang, Shu-Fang; Chen, Hui-Ting; Liu, Pei; Wang, Wen-Fei; Li, Chun-Xi

    2014-08-01

    To explore the alleviation effect of silicon on wheat growth under copper stress, cultivar Aikang 58 was chosen as the experimental material. The growth, root activities and root tip ultrastructures of wheat seedlings, which were cultured in Hoagland nutrient solution with five different treatments (control, 15 mg x L(-1) Cu2+, 30 mg x L(-1) Cu2+, 15 mg x L(-1) Cu2+ and 50 mg x L(-1) silicon, 30 mg x L(-1) Cu2+ and 50 mg x L(-1) silicon), were fully analyzed. The results showed that root length, plant height and root activities of wheat seedlings were significantly restrained under the copper treatments compared with the control (P < 0.01), while these restraining effects were alleviated after adding silicon to copper-stress Hoagland nutrient solution. Under copper stress, the cell wall and cell membrane of wheat seedling root tips suffered to varying degrees of destruction, which caused the increase of intercellular space and the disappearance of some organelles. After adding silicon, the cell structure was maintained intact, although some cells and organelles were still slightly deformed compared with the control. In conclusion, exogenous silicon could alleviate the copper stress damages on wheat seedlings and cellular components to some extent.

  14. Ultrastructure of the epithelial cells associated with tooth biomineralization in the chiton Acanthopleura hirtosa.

    PubMed

    Shaw, Jeremy A; Macey, David J; Brooker, Lesley R; Stockdale, Edward J; Saunders, Martin; Clode, Peta L

    2009-04-01

    The cusp epithelium is a specialized branch of the superior epithelium that surrounds the developing teeth of chitons and is responsible for delivering the elements required for the formation of biominerals within the major lateral teeth. These biominerals are deposited within specific regions of the tooth in sequence, making it possible to conduct a row by row examination of cell development in the cusp epithelium as the teeth progress from the unmineralized to the mineralized state. Cusp epithelium from the chiton Acanthopleura hirtosa was prepared using conventional chemical and microwave assisted tissue processing, for observation by light microscopy, conventional transmission electron microscopy (TEM) and energy filtered TEM. The onset of iron mineralization within the teeth, initiated at row 13, is associated with a number of dramatic changes in the ultrastructure of the apical cusp cell epithelium. Specifically, the presence of ferritin containing siderosomes, the position and number of mitochondria, and the structure of the cell microvilli are each linked to aspects of the mineralization process. These changes in tissue development are discussed in context with their influence over the physiological conditions within both the cells and extracellular compartment of the tooth at the onset of iron mineralization.

  15. Structure of xanthan gum and cell ultrastructure at different times of alkali stress

    PubMed Central

    de Mello Luvielmo, Márcia; Borges, Caroline Dellinghausen; de Oliveira Toyama, Daniela; Vendruscolo, Claire Tondo; Scamparini, Adilma Regina Pippa

    2016-01-01

    The effect of alkali stress on the yield, viscosity, gum structure, and cell ultrastructure of xanthan gum was evaluated at the end of fermentation process of xanthan production by Xanthomonas campestris pv. manihotis 280-95. Although greater xanthan production was observed after a 24 h-alkali stress process, a lower viscosity was observed when compared to the alkali stress-free gum, regardless of the alkali stress time. However, this outcome is not conclusive as further studies on gum purification are required to remove excess sodium, verify the efficiency loss and the consequent increase in the polymer viscosity. Alkali stress altered the structure of xanthan gum from a polygon-like shape to a star-like form. At the end of the fermentation, early structural changes in the bacterium were observed. After alkali stress, marked structural differences were observed in the cells. A more vacuolated cytoplasm and discontinuities in the membrane cells evidenced the cell lysis. Xanthan was observed in the form of concentric circles instead of agglomerates as observed prior to the alkali stress. PMID:26887232

  16. Structure of xanthan gum and cell ultrastructure at different times of alkali stress.

    PubMed

    Luvielmo, Márcia de Mello; Borges, Caroline Dellinghausen; Toyama, Daniela de Oliveira; Vendruscolo, Claire Tondo; Scamparini, Adilma Regina Pippa

    2016-01-01

    The effect of alkali stress on the yield, viscosity, gum structure, and cell ultrastructure of xanthan gum was evaluated at the end of fermentation process of xanthan production by Xanthomonas campestris pv. manihotis 280-95. Although greater xanthan production was observed after a 24h-alkali stress process, a lower viscosity was observed when compared to the alkali stress-free gum, regardless of the alkali stress time. However, this outcome is not conclusive as further studies on gum purification are required to remove excess sodium, verify the efficiency loss and the consequent increase in the polymer viscosity. Alkali stress altered the structure of xanthan gum from a polygon-like shape to a star-like form. At the end of the fermentation, early structural changes in the bacterium were observed. After alkali stress, marked structural differences were observed in the cells. A more vacuolated cytoplasm and discontinuities in the membrane cells evidenced the cell lysis. Xanthan was observed in the form of concentric circles instead of agglomerates as observed prior to the alkali stress.

  17. Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

    PubMed

    Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

    2014-04-01

    The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

  18. Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their differentiated Three-Dimensional Derivatives: A Comparative Study

    PubMed Central

    Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah

    2014-01-01

    Abstract The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential. PMID:24606239

  19. Physiological and ultrastructural features of human induced pluripotent and embryonic stem cell-derived skeletal myocytes in vitro.

    PubMed

    Skoglund, Gunnar; Lainé, Jeanne; Darabi, Radbod; Fournier, Emmanuel; Perlingeiro, Rita; Tabti, Nacira

    2014-06-03

    Progress has recently been made toward the production of human skeletal muscle cells from induced pluripotent stem (iPS) cells. However, the functional and ultrastructural characterization, which is crucial for disease modeling and drug discovery, remains to be documented. We show, for the first time to our knowledge, that the electrophysiological properties of human iPS-derived skeletal myocytes are strictly similar to those of their embryonic stem (ES) cell counterparts, and both are typical of aneural mammalian skeletal muscle. In both cell types, intracellular calcium signaling that links membrane depolarization to contraction occurs in the absence of extracellular Ca(2+), a unique feature of skeletal muscle. Detailed analysis of the Ca(2+) signal revealed diverse kinetics of the rising phase, and hence various rates in the release of Ca(2+) from the sarcoplasmic reticulum. This was mirrored by ultrastructural evidence of Ca(2+) release units, which varied in location, shape, and size. Thus, the excitation-contraction coupling machinery of both iPS- and ES-derived skeletal myocytes was functional and specific, but did not reach full maturity in culture. This is in contrast with the myofibrillar network, which displayed the same organization as in adult skeletal muscle. Overall, the present study validates the human iPS-based skeletal myocyte model in comparison with the embryonic system, and provides the functional and ultrastructural basis for its application to human skeletal muscle diseases.

  20. DYNAMICS OF ACRIDINE ORANGE-CELL INTERACTION. II. DYE-INDUCED ULTRASTRUCTURAL CHANGES IN MULTIVESICULAR BODIES (ACRIDINE ORANGE PARTICLES).

    PubMed

    ROBBINS, E; MARCUS, P I; GONATAS, N K

    1964-04-01

    The brilliantly fluorescent cytoplasmic particles that accumulate in HeLa cells treated with acridine orange, previously referred to as acridine orange particles, are shown to represent acid phosphatase positive multivesicular bodies (MVB). Dynamic changes in the ultrastructure of these organelles may be induced by varying the concentration of extracellular dye and the length of exposure to the dye. Low concentrations of dye for long intervals of time lead to marked hypertrophy of the MVB and accumulation of myelin figures within them, the acid phosphatase activity being retained. High concentrations of dye for short time intervals lead initially to a diffuse distribution of dye through out the cytoplasm (cytoplasmic reddening) as viewed in the fluorescence microscope. When cells are stained in this way and incubated in a dye-free medium, the diffusely distributed dye is segregated into MVB within 1 hour. Ultrastructurally, these MVB show dilatation but no myelin figures. The process of dye segregation is energy dependent and will not occur in starved cells. This energy dependence and the occurrence of segregation via dilatation of the MVB rather than ultrastructural transformation, i.e. formation of new binding sites, suggests that the process involves an active transport mechanism. Of the various energy sources supplied to starved cells, only glucose, mannose, and pyruvate are fully effective in supporting dye segregation. Blockage of the tricarboxylic acid cycle with malonate inhibits the effects of pyruvate but not of glucose, demonstrating the efficacy of both the tricarboxylic acid and glycolytic cycles in supplying energy for the process.

  1. Ultrastructural studies of allergic contact dermatitis in man. Infiltrating cells at the earliest phase of spongiotic bulla formation.

    PubMed

    Komura, J; Oguchi, M; Aoshima, T; Ofuji, S

    1980-01-01

    The kind and fine structure of mononuclear cells appearing in the epidermis at about 6 h of allergic contact dermatitis were examined by electron microscopy. They were monocytes and lymphocytes, the number being about equal. The ultrastructure of monocytes was that described for normal ones in blood, and apparently actively moving, streching the intercellular connections of the keratinocytes. Lymphocytes displayed a round or oval nucleus with some electron-dense cytoplasm which contained ribosomes and polysomes but only occasional mitochondria and Golgi complexes.

  2. Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions.

    PubMed

    Zaganescu, Raluca; Barbu Tudoran, Lucian; Pall, Emoke; Florea, Adrian; Roman, Alexandra; Soanca, Andrada; Mihaela Mihu, Carmen

    2015-09-01

    This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three-dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro- and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast-like or star-like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells.

  3. Populus trichocarpa cell wall chemistry and ultrastructure trait variation, genetic control and genetic correlations.

    PubMed

    Porth, Ilga; Klápště, Jaroslav; Skyba, Oleksandr; Lai, Ben S K; Geraldes, Armando; Muchero, Wellington; Tuskan, Gerald A; Douglas, Carl J; El-Kassaby, Yousry A; Mansfield, Shawn D

    2013-02-01

    The increasing ecological and economical importance of Populus species and hybrids has stimulated research into the investigation of the natural variation of the species and the estimation of the extent of genetic control over its wood quality traits for traditional forestry activities as well as the emerging bioenergy sector. A realized kinship matrix based on informative, high-density, biallelic single nucleotide polymorphism (SNP) genetic markers was constructed to estimate trait variance components, heritabilities, and genetic and phenotypic correlations. Seventeen traits related to wood chemistry and ultrastructure were examined in 334 9-yr-old Populus trichocarpa grown in a common-garden plot representing populations spanning the latitudinal range 44° to 58.6°. In these individuals, 9342 SNPs that conformed to Hardy-Weinberg expectations were employed to assess the genomic pair-wise kinship to estimate narrow-sense heritabilities and genetic correlations among traits. The range-wide phenotypic variation in all traits was substantial and several trait heritabilities were > 0.6. In total, 61 significant genetic and phenotypic correlations and a network of highly interrelated traits were identified. The high trait variation, the evidence for moderate to high heritabilities and the identification of advantageous trait combinations of industrially important characteristics should aid in providing the foundation for the enhancement of poplar tree breeding strategies for modern industrial use.

  4. RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

    PubMed Central

    1975-01-01

    A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs. PMID:1171872

  5. The effects of diethylcarbamazine on the ultrastructure of lung cells in vivo.

    PubMed

    Florêncio, M S; Saraiva, K L A; Peixoto, C A

    2005-06-01

    The pulmonary surfactant synthesis is disturbed in experimentally induced asthma, as are the intracellular storage capacity and its physical activity. These alterations may also be present in chronic asthmatic patients, and therefore the dysfunction of the pulmonary surfactant system may play an important role in the pathophysiology of asthma. Some clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in patients with bronchial asthma showing that DEC is effective in terminating acute attacks of bronchial asthma. The present study aimed to analyze the ultrastructural alterations of lung cells after treatment in vivo with diethylcarbamazine. After 12 days of treatment with DEC, when compared with control samples, type II pneumocytes showed active nuclei with abundant euchromatin and evident nucleoli, and a substantially greater number of mature secretion vesicle. On the other hand, type I pneumocytes showed no morphological alterations. After DEC treatment, lung macrophages also presented several characteristics of cellular activation such as nuclei with a prominence of euchromatin and central nucleoli as well as an abundance of early and late endossomes distributed throughout the cytoplasm. These results confirm that DEC exerts a role in the activation of important pulmonary cellular pathways, which are probably related to the clinical improvement of asthma symptoms after DEC treatment.

  6. Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales, Streptophyta) with special reference to cell division and thallus formation

    PubMed Central

    Mikhailyuk, Tatiana; Holzinger, Andreas; Massalski, Andrzej; Karsten, Ulf

    2014-01-01

    Representatives of the closely related genera, Interfilum and Klebsormidium, are characterized by unicells, dyads or packets in Interfilum and contrasting uniseriate filaments in Klebsormidium. According to the literature, these distinct thallus forms originate by different types of cell division, sporulation (cytogony) versus vegetative cell division (cytotomy), but investigations of their morphology and ultrastructure show a high degree of similarity. Cell walls of both genera are characterized by triangular spaces between cell walls of neighbouring cells and the parental wall or central space among the walls of a cell packet, exfoliations and projections of the parental wall and cap-like and H-like fragments of the cell wall. In both genera, each cell has its individual cell wall and it also has part of the common parental wall or its remnants. Therefore, vegetative cells of Interfilum and Klebsormidium probably divide by the same type of cell division (sporulation-like). Various strains representing different species of the two genera are characterized by differences in cell wall ultrastructure, particularly the level of preservation, rupture or gelatinization of the parental wall surrounding the daughter cells. The differing morphologies of representatives of various lineages result from features of the parental wall during cell separation and detachment. Cell division in three planes (usual in Interfilum and a rare event in Klebsormidium) takes place in spherical or short cylindrical cells, with the chloroplast positioned perpendicularly or obliquely to the filament (dyad) axis. The morphological differences are mainly a consequence of differing fates of the parental wall after cell division and detachment. The development of different morphologies within the two genera mostly depends on characters such as the shape of cells, texture of cell walls, mechanical interactions between cells and the influence of environmental conditions. PMID:26504365

  7. Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae): response to fixatives, anatomical distribution, histochemical contents and ultrastructural features.

    PubMed

    Rocha, Juliana Silva; Chiarini-Garcia, Hélio

    2007-03-01

    Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughout the body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gastrointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between the two species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content. In H. malabaricus, the tissues fixed with Helly's solution contained mast cells that were basophilic, metachromatic and had heparin in their cytoplasmic granules, while the tissues fixed with Karnovsky's solution contained eosinophilic and orthochromatic mast cells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic, orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionally seen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky's solution revealed that the cytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two species belonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneous characteristics, possibly due to their functional properties or to their sensitivity to fixatives.

  8. [Quantitative changes in the ultrastructure of myocardial cells in Japanese quail during hypergravity, hypodynamia and space flight].

    PubMed

    Bózner, A; Boda, K; Dostál, J; Matĕjková, Z; Devecka, V

    1993-03-01

    The experimental work aimed at the quantitative ultrastructure of the myocardial cells of the Japanese quail Coturnix coturnix japonica during hypergravitation, hypodynamism and space flight in a Soviet satellite. For the determination of quantitative changes of the myocardial ultrastructure a morphometrical method was used with parameters like the number of mitochondria, average mitochondrial size, relative mitochondrial volume, deficiency of cristae and relative volume of myofibrils. The quails were observed in 3 groups. The absolute control consisted of quails living in normal Earth conditions, in the laboratory group the quails were exposed to conditions of hypergravitation and hypodynamism in a specially constructed centrifuge, and in the flying group the quails were exposed to space flight in a Soviet orbital station MIR. In the group of absolute controls no pathological changes of the myocardial ultrastructure were found. In the flying group there were no significant changes, with the exception of decreased relative volume of myofibrils, which however agrees with the findings on symptoms corresponding to human and animal heart weakness during space flights. In the laboratory group, pathological changes were observed in each of the fractions. The most significant pathological findings were found in the group controls in the center and in hypergravitation combined with hypodynamism. It can be concluded that the laboratories can simulate conditions induced by the start and flight of space ships. (Fig. 2, Ref, 8.)

  9. Ultrastructural Characterization of Hyperactive Endothelial Cells, Pericytes and Fibroblasts in Hypertrophic and Nodular Port Wine Stain Lesions.

    PubMed

    Gao, L; Yin, R; Wang, H; Guo, W; Song, W; Nelson, J S; Tan, W; Wang, G

    2017-02-09

    Port wine stain (PWS) is a congenital vascular malformation of human skin involving the superficial vascular plexus,(1-4) but the molecular pathogenesis of these lesions remains incompletely understood.(5-8) We herein performed a transmission electron microscopy (TEM) study to determine the main pathological characteristics and ultrastructure of various cell types, including endothelial cells (ECs), pericytes, fibroblasts and keratinocytes, in hypertrophic and nodular PWS. The study was approved by the Investigational Review Board at the Xijing Hospital, Xi'an, China. This article is protected by copyright. All rights reserved.

  10. Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells

    PubMed Central

    Costello, M. Joseph; Brennan, Lisa A.; Gilliland, Kurt O.; Johnsen, Sönke; Kantorow, Marc

    2016-01-01

    An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12–15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial

  11. Identifying ECM Mediators of Tumor Cell Dormancy

    DTIC Science & Technology

    2008-05-01

    proliferating cells. f) At time of euthanasia, stage of estrous will be determined by vaginal smear so that inter- animal variation due to estrous...ratio of the number of distinct peptides identified for each protein divided by the total number of peptides identified in the two hour run are...displayed as the peptide count ratio. Using this peptide counting label-free quantification approach, surprisingly, suggested that the three fibrillar

  12. Assessing light-independent effects of hypericin on cell viability, ultrastructure and metabolism in human glioma and endothelial cells.

    PubMed

    Huntosova, Veronika; Novotova, Marta; Nichtova, Zuzana; Balogova, Lucia; Maslanakova, Maria; Petrovajova, Dana; Stroffekova, Katarina

    2017-04-01

    Cell exposure to light-independent effects of photosensitizers (PS) used in PDT is clinically relevant when PS affect the pro-apoptotic cascade. In many malignant cells, Hypericin (Hyp) has PS displayed light-dependent anti-proliferative and cytotoxic effects with no cytotoxicity in the dark. Recent studies have shown that Hyp also exhibited light-independent cytotoxic effects in a wide range of concentrations. The molecular mechanisms underlying Hyp light-independent (dark) toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and these mechanisms are not understood in detail. Here, we demonstrate that in human glioma and endothelial cells, Hyp displayed light-independent effects at several sub-cellular levels (ultrastructure, mitochondria function and metabolism, and protein synthesis). Taking together previously published and our present results, the findings strongly suggest that Hyp light independent effects: (i) depend on the cell type and metabolism; (ii) underlying molecular mechanisms are due to Hyp interaction with the multiple target molecules including Bcl2 family of proteins. In addition, the findings suggest that Hyp without illumination can be explored as an adjuvant therapeutic drug in combination with chemo- or radiation cancer therapy.

  13. ABCB5 identifies immunoregulatory dermal cells

    PubMed Central

    Schatton, Tobias; Yang, Jun; Kleffel, Sonja; Uehara, Mayuko; Barthel, Steven R.; Schlapbach, Christoph; Zhan, Qian; Dudeney, Stephen; Mueller, Hansgeorg; Lee, Nayoung; de Vries, Juliane C.; Meier, Barbara; Vander Beken, Seppe; Kluth, Mark A.; Ganss, Christoph; Sharpe, Arlene H.; Waaga-Gasser, Ana Maria; Sayegh, Mohamed H.; Abdi, Reza; Scharffetter-Kochanek, Karin; Murphy, George F.; Kupper, Thomas S.; Frank, Natasha Y.; Frank, Markus H.

    2015-01-01

    Summary Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly-defined immunomodulatory cell populations poses a barrier to this field. Here we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5+ DIRCs suppressed T-cell proliferation, evaded immune rejection, homed to recipient immune tissues and induced Tregs in vivo. In fully MHC-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T-cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5+ DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy. PMID:26321644

  14. [Ultrastructure of primordial germ cells of Lacerta muralis and Lacerta viridis. Comparison with Lacerta vivipara (author's transl)].

    PubMed

    Hubert, J

    1974-01-01

    The ultrastructure of primordial germ cells migrating through the embryo and situated in genital crests is described in Lacerta viridis and Lacerta muralis. It is compared with the germ cells of Lacerta vivipara previously studied. The cytoplasmic ultrastructure is the same in the three Lacertilians, but several nuclear differences are observed. The outline of the nucleus is regular and his shape is spheric in Lacerta vivipara, but in the two others Lacertilians the nucleus shows a very sinuous outline with deep cytoplasmic invaginations. There are fibrous nuclear bodies in the three Lacertilians but the germ cells of Lacerta muralis have in addition granular bodies. The nucleolus in Lacerta viridis and Lacerta muralis is very different from the annular nucleolus of the germ cells of Lacerta vivipara. It is possible to distinguish three types of nucleoli: a compact nucleolus in Lacerta viridis and Lacerta muralis, a granular cords nucleolus in Lacerta viridis, a vacuolar nucleolus in Lacerta muralis. Each type can be seen during the migration of the gonocytes or during the colonisation of the genital region. Probably there is a relationship between these different architectures and the activity of nucleolus in synthesis and liberation of ribosomal RNA. However the nuclear fibrillar area which we have previosly called "masse para nucléolaire" in Lacerta vivipara, is present in the germ cells of Lacerta viridis and Lacerta muralis.

  15. Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

    PubMed Central

    Junquera, Concepción; Martínez-Ciriano, Carmen; Castiella, Tomás; Serrano, Pedro; Azanza, María Jesús; Ramón y Cajal Junquera, Santiago

    2007-01-01

    Abstract Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duo-denum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce per-inuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular con-tacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium show-ing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single

  16. High resolution imaging of the ultrastructure of living algal cells using soft x-ray contact microscopy

    SciTech Connect

    Ford, T.W.; Cotton, R.A.; Page, A.M.; Tomie, T.; Majima, T.; Stead, A.D.

    1995-12-31

    Soft x-ray contact microscopy provides the biologist with a technique for examining the ultrastructure of living cells at a much higher resolution than that possible by various forms of light microscopy. Readout of the developed photoresist using atomic force microscopy (AFM) produces a detailed map of the carbon densities generated in the resist following exposure of the specimen to water-window soft x-rays (2--4nm) produced by impact of a high energy laser onto a suitable target. The established high resolution imaging method of transmission electron microscopy (TEM) has inherent problems in the chemical pre-treatment required for producing the ultrathin sections necessary for this technique. Using the unicellular green alga Chlamydomonas the ultrastructural appearance of the cells following SXCM and TEM has been compared. While SXCM confirms the basic structural organization of the cell as seen by TEM (e.g., the organization of the thylakoid membranes within the chloroplast; flagellar insertion into the cytoplasm), there are important differences. These are in the appearance of the cell covering and the presence of carbon-dense spherical cellular inclusions.

  17. Silicon alleviates cadmium toxicity by enhanced photosynthetic rate and modified bundle sheath's cell chloroplasts ultrastructure in maize.

    PubMed

    Vaculík, Marek; Pavlovič, Andrej; Lux, Alexander

    2015-10-01

    Silicon was shown to alleviate the negative effects of various biotic and abiotic stresses on plant growth. Although the positive role of Si on toxic and heavy metal Cd has been already described, the mechanisms have been explained only partially and still remain unclear. In the present study we investigated the effect of Si on photosynthetic-related processes in maize exposed to two different levels of Cd via measurements of net photosynthetic rate (AN), chlorophyll a fluorescence and pigment analysis, as well as studies of leaf tissue anatomy and cell ultrastructure using bright-field and transmission electron microscopy. We found that Si actively alleviated the toxic syndromes of Cd by increasing AN, effective photochemical quantum yield of photosystem II (ϕPSII) and content of assimilation pigments, although did not decrease the concentration of Cd in leaf tissues. Cadmium did not affect the leaf anatomy and ultrastructure of leaf mesophyll's cell chloroplasts; however, Cd negatively affected thylakoid formation in chloroplasts of bundle sheath cells, and this was alleviated by Si. Improved thylakoid formation in bundle sheath's cell chloroplasts may contribute to Si-induced enhancement of photosynthesis and related increase in biomass production in C4 plant maize.

  18. Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells

    PubMed Central

    1983-01-01

    We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined. PMID:6220105

  19. Phenotypic, ultra-structural and functional characterization of bovine peripheral blood dendritic cell subsets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dendritic cells (DC) are multifunctional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets...

  20. Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

    PubMed

    Fridman, Sophie; Rana, Krishen J; Bron, James E

    2013-10-01

    Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level.

  1. Changes in ultrastructure and Fourier transform infrared spectrum of Salmonella enterica serovar typhimurium cells after exposure to stress conditions.

    PubMed

    Alvarez-Ordóñez, A; Prieto, M

    2010-11-01

    The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H₂O₂) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.

  2. [Changes of ultrastructure of the capillary endotheliocytes of ischemized and nonaffected muscular tissue after transplantation of human hemopoietic stem cells of fetal liver in experiment in vivo].

    PubMed

    Saliutin, R V; Zadorozhna, T D; Medvets'kyĭ, E B; Driuk, M F; Petrenko, A Iu

    2010-04-01

    In experiment was investigated ultrastructure of the capillaries endothelial cells and histological peculiarities of muscular tissue on various stages after transplantation of hemopoietic stem cells of fetal liver (HSCFL). There was proved, that in ischemic environment HSCFL stimulate processes of angiogenesis, and in the case of transplantation into intact muscular tissue they are differentiating into the tissue macrophages, not interfering with muscular tissue structure.

  3. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in Rhesus monkeys exposed to ozone

    SciTech Connect

    Castleman, W.L.; Dungworth, D.L.; Schwartz, L.W.; Tyler, W.S.

    1980-03-01

    The pathogenesis of acute respiratory bronchiolitis was examined in Rhesus monkeys exposed to 0.8 ppM ozone for 4 to 50 hours. Epithelial injury and renewal were qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4 to 12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18%) was measured after 50 hours of exposure. Most (67 to 80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20 to 33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell types most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal.

  4. Ultrastructural changes in the parenchymal liver cells of rats treated with high doses of rifampicin.

    PubMed Central

    Piriou, A.; Maissiat, R.; Jacqueson, A.; Warnet, J. M.; Claude, J. R.

    1987-01-01

    Ultrastructural study of hepatic parenchyma was carried out in female Wistar rats after they had received high doses (400 mg X kg-1) of rifampicin for 1, 2, 4, 6 and 8 days. Morphological changes in the endoplasmic reticulum, Golgi apparatus and mitochondria were observed as early as day 1 of intoxication. These changes corroborate the biochemical data available regarding RFP-induced fatty liver. Images Fig. 1 Fig. 2 Fig. 3 & 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:3580280

  5. Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves.

    PubMed

    Espelie, K E; Wattendorff, J; Kolattukudy, P E

    1982-07-01

    Styloid-calcium-oxalate-crystal-containing idioblasts possess an interior cell-wall layer which has a lamellar ultrastructure. Idioblasts were isolated by centrifugation of an Agave americana leaf homogenate through 2M sucrose. The aliphatic monomers of the polymeric material from an idioblast fraction were primarily ω-hydroxy acids (32%) and dicarboxylic acids (35%), with C18:1 dicarboxylic acid being the most dominant monomer (25%). Nitrobenzene oxidation of the idioblasts yielded syringaldehyde and vanillin in a ratio of 0.46:1. The major class of wax associated with the idioblasts was free fatty acids (34%). A major homologue of both the fatty acid and fatty alcohol fractions of this wax was C22. The hydrocarbon fraction of the wax had a broad chainlength distribution with a large amount of even-numbered (47%) and shorter-chain homologues. The ultrastructure, the composition of the aliphatic and aromatic components of the polymeric material as well as the composition of the wax show that the idioblast cell wall is suberized. The wax and cutin polymer of the epidermis of A. americana leaves were chemically characterized for comparative purposes.

  6. Ultrastructural study of sperm cells in Acanthocolpidae: the case of Stephanostomum murielae and Stephanostomoides tenuis (Digenea)

    PubMed Central

    Bakhoum, Abdoulaye J.S.; Justine, Jean-Lou; Bray, Rodney A.; Bâ, Cheikh T.; Marchand, Bernard

    2015-01-01

    The mature spermatozoa of Stephanostomum murielae and Stephanostomoides tenuis are described by transmission electron microscopy. They present several ultrastructural features previously reported in other digeneans. Their spermatozoa possess two axonemes of different length showing the 9 + ‘1’ trepaxonematan pattern, four attachment zones, two mitochondria (with an anterior moniliform one in S. murielae), a nucleus, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. The main differences between the mature spermatozoon of S. murielae and S. tenuis are the maximum number of cortical microtubules, the morphology of the anterior spermatozoon extremity and the anterior mitochondrion. This study is the first concerning members of the family Acanthocolpidae. The main ultrastructural characteristics discussed are the morphology of the anterior and posterior spermatozoon extremities, antero-lateral electron dense material, external ornamentations, spine-like bodies and number and morphology of mitochondria. In addition, the phylogenetic significance of all these ultrastructural features is discussed and compared to molecular results in order to highlight the complex relationships in the Digenea. PMID:25699200

  7. Interrelationship among testicular cells in wall lizard Hemidactylus flaviviridis (Rüppell): an ultrastructural seasonal and experimental study.

    PubMed

    Khan, U W; Rai, Umesh

    2004-04-01

    The present study was aimed at investigating ultrastructure of different testicular cells and their interactions through various junctional specializations during different phases of reproductive cycle in wall lizard H. flaviviridis to develop an integrated approach of cell-cell interaction in control of testicular functions. Specialized steroid synthesizing cell organelles such as smooth endoplasmic reticulum (SER) and long slender mitochondria with tubulo-vesicular cristae were predominantly seen in Leydig as well as Sertoli cells during spermatogenically active phase, suggesting their active involvement in steroid biosynthesis. Peritubular cells also exhibited marked seasonal variations. Multi-layered fibroblast-like peritubular cells during regressed phase became single layered myoid-like during spermatogenically active phase. The presence of various types of junctions, including gap and tight junctions (occluding junctions) and adhering junctions such as desmosomes, septate-like junction, ectoplasmic specializations and tubulo-bulbar complexes, were demonstrated among testicular cells in wall lizard H. flaviviridis. However, the nature and degree of junctional (environmental) interaction varied with the reproductive state of the wall lizard. Further, administration of dihydrotestosterone in wall lizards during regressed phase resulted in increase of lipid droplets in Leydig cells and accumulation of germ cell debris in seminiferous tubules. Some of the Sertoli cells were seen darker in response to testosterone treatment probably due to its inhibitory effect on lipid metabolism. These results suggest that testosterone either directly or via inhibiting pituitary basal gonadotropin secretion has suppressive effect on testicular cells.

  8. Basal epithelial cells of human prostate gland are not myoepithelial cells. A comparative immunohistochemical and ultrastructural study with the human salivary gland.

    PubMed Central

    Srigley, J. R.; Dardick, I.; Hartwick, R. W.; Klotz, L.

    1990-01-01

    The hypothesis that basal epithelial cells of the human prostate are of myoepithelial origin was investigated using immunohistochemical and ultrastructural methodologies. The immunohistologic analyses show significant phenotypic differences between prostatic basal cells and myoepithelial cells of the salivary gland. Although both cell types stain intensely with the 312C8-1 monoclonal antibody, only true myoepithelial cells demonstrated significant amounts of muscle-specific actin as decorated by the HHF35 monoclonal antibody. Furthermore, using double-labeling experiments, the prostatic basal cells were strongly decorated with a fluorescein-tagged basal cell-specific keratin but were negative with the rhodamine-tagged phalloidin, a chemical that binds specifically to actin microfilaments. Ultrastructural studies also showed an absence of thin microfilament bundles, dense bodies, and micropinocytotic vesicles in the prostatic basal cells. The current investigations show that the prostatic acini do not have a basal myoepithelium. Although some authors have suggested a stem cell role for prostatic basal cells, the weight of experimental work argues against this hypothesis. The exact role of the basal epithelial cells of the prostate is not known, although they may serve endocrine, paracrine, or other regulatory functions and may be involved in modulating signals between prostatic stroma and epithelium. Images Figure 3 Figure 1 Figure 2 Figure 4 PMID:1691595

  9. Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.

    PubMed

    Barile, Lucio; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Moccetti, Tiziano; Vassalli, Giuseppe

    2012-01-01

    The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34⁺ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  10. Sperm-cell ultrastructure of North American sturgeons. IV. The pallid sturgeon (Scaphirhynchus albus Forbes and Richardson, 1905)

    USGS Publications Warehouse

    DiLauro, M.N.; Walsh, R.A.; Peiffer, M.; Bennett, R.M.

    2001-01-01

    Sperm-cell morphology and ultrastructure in the pallid sturgeon (Scaphirhynchus albus) were examined using transmission and scanning electron microscopy. Metrics and structure were compared with similar metrics obtained from other published descriptions of sturgeon sperm cells. General morphology was found to be similar to that of sperm cells of the white (Acipenser transmontanus), lake (A. fulvescens), stellate (A. stellatus), Chinese (A. sinensis), Russian (A. gueldenstaedti colchicus), and shortnose (A. brevirostrum) sturgeons, which all shared a gradual tapering of the nuclear diameter from posterior to anterior, unlike that of the Atlantic sturgeon (A. oxyrhynchus). The sperm cell of the pallid sturgeon was similar in size to that of the Atlantic sturgeon, being only slightly larger. The sperm cell of the pallid sturgeon differed from those of other sturgeons chiefly in the acrosomal region, where the posterolateral projections (PLP) have the shape of an acute triangle and are arranged in a spiral about the longitudinal axis of the cell. The PLP were longer than those of other sturgeons, being twice the length of those of the Atlantic sturgeon and 58% longer than those of the lake sturgeon. Also, in cross section the acrosome had the shape of a hollow cone rather than the cap of an oak tree acorn, as was found in ultrastructural studies of other sturgeons. In addition, we were able to confirm that the structural arrangement of the distal centriole of the midpiece is identical with that of the proximal centriole: nine sets of microtubular triplets around the periphery of the centriole. This information is of potential use to fishery biologists, forensic biologists, zoologists, reproductive physiologists, taxonomists, evolutionary biologists, and aquaculturists.

  11. Studies on the effects of microgravity on the ultrastructure and functions of cultured mammalian cells (L-6)

    NASA Technical Reports Server (NTRS)

    Sato, Atsushige

    1993-01-01

    The human body consists of 10(exp 13) cells. Understanding the mechanisms by which the cells sense and respond to microgravity is very important as the basis for space biology. The cells were originally isolated aseptically from mammalian bodies and cultured in vitro. A set of cell culture vessels was developed to be applied to three kinds of space flight experiments. Experiment 1 is to practice the cell culture technique in a space laboratory and obtain favorable growth of the cells. Aseptic handling in tryspin treatment and medium renewal will be tested. The cells, following space flight, will be returned to the ground and cultured continuously to investigate the effects of space flight on the cellular characteristics. Experiment 2 is to examine the cytoskeletal structure of the cells under microgravity conditions. The cytoskeletal structure plays essential roles in the morphological construction, movements, axonal transport, and differentiation of the cells. The cells fixed during space flight will be returned and the cytoskeleton and ultrastructure observed using electron microscopy and fluorescence microscopy. Experiment 3 is to study the cellular productivity of valuable substances. The waste medium harvested during space flight are returned and quantitated for the cellular products. The effects of microgravity on mammalian cells will be clarified from the various aspects.

  12. Ultrastructural Studies of Germ Cell Development and the Functions of Leydig Cells and Sertoli Cells associated with Spermatogenesis in Kareius bicoloratus (Teleostei, Pleuronectiformes, Pleuronectidae)

    PubMed Central

    Kang, Hee-Woong; Kim, Sung Hwan; Chung, Jae Seung

    2016-01-01

    The ultrastructures of germ cells and the functions of Leydig cells and Sertoli cells during spermatogenesis inmale Kareius bicoloratus (Pleuronectidae) were investigated by electron microscope observation. Each of the well-developed Leydig cells during active maturation division and before spermiation contained an ovoid vesicular nucleus, a number of smooth endoplasmic reticula, well-developed tubular or vesicular mitochondrial cristae, and several lipid droplets in the cytoplasm. It is assumed that Leydig cells are typical steroidogenic cells showing cytological characteristics associated with male steroidogenesis. No cyclic structural changes in the Leydig cells were observed through the year. However, although no clear evidence of steroidogenesis or of any transfer of nutrients from the Sertoli cells to spermatogenic cells was observed, cyclic structural changes in the Sertoli cells were observed over the year. During the period of undischarged germ cell degeneration after spermiation, the Sertoli cells evidenced a lysosomal system associated with phagocytic function in the seminiferous lobules. In this study, the Sertoli cells function in phagocytosis and the resorption of products originating from degenerating spermatids and spermatozoa after spermiation. The spermatozoon lacks an acrosome, as have been shown in all teleost fish spermatozoa. The flagellum or sperm tail of this species evidences the typical 9+2 array of microtubules. PMID:27294207

  13. Mast Cell Interaction with Neutrophils in Human Gastric Carcinomas: Ultrastructural Observations

    PubMed Central

    Branca, Giovanni; Alberto Caruso, Rosario

    2016-01-01

    Aim. The role of mast cells in cell-cell immune interactions has received increasing attention, although their functional interaction with neutrophils still remains to be clarified in tumors. The aim of the present study was to investigate the association between mast cells and neutrophils in a series of gastric carcinomas (GC). Patients and Methods. 52 surgically resected GC specimens were routinely processed for both light and electron microscopy. Only cases showing both mast cells and neutrophils in the tumor stroma were considered in the analysis. Results. Only 9 GC (M : F = 5 : 4; age range: 50–82 years) showed both mast cells and neutrophils in the tumor stroma. At ultrathin sections, we identified heterotypic aggregation and intermingling of mast cells and neutrophils. Mast cells had mature phenotype and showed full complement of granules with homogeneous, scroll, particle, and mixed pattern. In addition, we found normal-appearing or early apoptosis showing neutrophils. Conclusion. Our histological findings showed the likely interaction between mast cells and neutrophils in GC. We hypothesize that the granular content of mast cells may be released in small quantity through a mechanism called “kiss-and-run fusion,” which is alternative to well-known massive anaphylactic or piecemeal degranulation. PMID:27882290

  14. Tendon's ultrastructure.

    PubMed

    Tresoldi, Ilaria; Oliva, Francesco; Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; Bei, Roberto; Modesti, Andrea

    2013-01-01

    The structure of a tendon is an important example of complexity of ECM three-dimensional organization. The extracellular matrix (ECM) is a macromolecular network with both structural and regulatory functions. ECM components belong to four major types of macromolecules: the collagens, elastin, proteoglycans, and noncollagenous glycoproteins. Tendons are made by a fibrous, compact connective tissue that connect muscle to bone designed to transmit forces and withstand tension during muscle contraction. Here we show the ultrastructural features of tendon's components.

  15. Ultrastructural localization of F-actin using phalloidin and quantum dots in HL-60 promyelocytic leukemia cell line after cell death induction by arsenic trioxide.

    PubMed

    Izdebska, Magdalena; Gagat, Maciej; Grzanka, Dariusz; Grzanka, Alina

    2013-06-01

    Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD-streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes.

  16. Pathological and ultrastructural changes in cultured cells induced by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

    PubMed

    Rivers, David B; Uçkan, Fevzi; Ergin, Ekrem; Keefer, Donald A

    2010-12-01

    The ectoparasitic wasp Nasonia vitripennis produces a proteinaceous venom that induces death in fly hosts by non-paralytic mechanisms. Previous in vitro assays have suggested that the primary cause of cell and tissue death is oncosis, a non-programmed cell death (PCD) pathway characterized by cellular swelling and lysis. However, ultrastructural analyses of BTI-TN-5B1 cells exposed to LC(99) doses of wasp venom revealed cellular changes more consistent with apoptosis and/or non-apoptotic PCD than oncosis or necrosis: By 3h after incubation with venom, susceptible cells displayed indentations in the nuclear membranes, large nucleoli, and extensive vacuolization throughout the cytoplasm. In the vast majority of venom treated cells, annexin V bound to the plasma membrane surface within 15 min after treatment, a characteristic consistent with translocation of phosphatidylserine to the cell surface during the early stages of apoptosis. Likewise, mitochondrial transmembrane potential was depressed in cells within 15 min in venom-treated cells, an event that occurred in the absence of mitochondrial swelling or rupturing of cristae. Active caspase 3 was detected by fluorescent labeling in nearly all venom treated cells 3h after exposure to venom, and in turn, the potent caspase 3 inhibitor Z-VAD-FMK attenuated the morphological changes elicited by wasp venom and afforded protection to BTI-TN-5B1-4 cells.

  17. Membrane associated qualitative differences in cell ultrastructure of chemically and high pressure cryofixed plant cells.

    PubMed

    Zechmann, Bernd; Müller, Maria; Zellnig, Günther

    2007-06-01

    Membrane contrast can sometimes be poor in biological samples after high pressure freezing (HPF) and freeze substitution (FS). The addition of water to the FS-medium has been shown to improve membrane contrast in animal tissue and yeast. In the present study we tested the effects of 1% and 5% water added to the FS-medium (2% osmium with 0.2% uranyl acetate in anhydrous acetone) on the quality and visibility of membranes in high pressure frozen leaf samples of Cucurbita pepo L. plants and compared them to chemically fixed cells (3% glutaraldehyde post-fixed with 1% osmium tetroxide). The addition of water to the FS-medium drastically decreased the amounts of well preserved cells and did not significantly improve the quality nor visibility of membranes. In samples that were freeze substituted in FS-media containing 1% and 5% water the width of thylakoid membranes was found to be significantly increased of about 20% and the perinuclear space was up to 76% wider in comparison to what was found in samples which were freeze substituted without water. No differences were found in the thickness of membranes between chemically and cryofixed cells that were freeze substituted in the FS-medium without water. Nevertheless, in chemically fixed cells the intrathylakoidal space was about 120% wider than in cryofixed cells that were freeze substituted with or without water. The present results demonstrate that the addition of water to the FS-medium does not improve membrane contrast but changes the width of thylakoid membranes and the perinuclear space in the present plant material. The addition of water to the FS-medium is therefore not as essential for improved membrane contrast in the investigated plant samples as it was observed in cells of animal tissues and yeast cells.

  18. Ultrastructure and cytochemical localization of laccase in two strains of Leptosphaerulina briosiana (Pollaci) Graham and Luttrell.

    PubMed Central

    Simon, L T; Bishop, D S; Hooper, G R

    1979-01-01

    Substrate specificity tests were used to identify the presence of laccase in two strains of Leptosphaerulina briosiana (Poll.) Graham and Luttrell, an ascomycete which causes leaf spot in alfalfa. Cytochemical localization of monophenol monooxygenase (laccase) as well as the ultrastructures of the two strains were investigated. Laccase was observed in the outer layers of the cell walls of both strains. The ultrastructures of vegetative hyphae of both strains were typical of those found in most ascomycetes. Images PMID:104971

  19. Immunohistochemical and Ultrastructural Study of the Lamellae of Oocytes in Atretic Follicles in Relation to Different Processes of Cell Death

    PubMed Central

    Escobar, M.L.; Echeverría, O.M.; García, G.; Ortiz, R.; Vázquez-Nin, G.H.

    2015-01-01

    Atresia is the process through which non-selectable oocytes are eliminated; it involves apoptosis and/or autophagy. This study used immunohistochemical and ultrastructural techniques to characterize the lamellae present in the cytoplasm of oocytes in follicles in the process of atresia in prepubertal and adult Wistar rats. The results indicate that the lamellae are positive to tubulin and myosin immunodetection under light and electron microscopy. Labeling is greater with anti-tubulin and lesser with anti-myosin. Our observations indicate that lamellae are present in oocytes at the initial antral stage in prepubertal rats; that is, from day 14 post-birth to adult age. We were able to determine that the increase in altered lamellae principally occurs in the apoptotic cells rather than in the autophagic cells. PMID:26428888

  20. Histogenesis and ultrastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial cells of host origin.

    PubMed Central

    Vajkoczy, P.; Olofsson, A. M.; Lehr, H. A.; Leiderer, R.; Hammersen, F.; Arfors, K. E.; Menger, M. D.

    1995-01-01

    In previous studies we have demonstrated that syngeneic and xenogeneic pancreatic islet grafts are revascularized within a 10 to 14-day period after transplantation. With the combined use of intravital and electron microscopy, as well as immunohistochemistry using a set of species-specific or -crossreacting antibodies to endothelial cell antigens, we investigated 1) the origin of the endothelium of the newly formed capillaries in free pancreatic islet isografts (hamster-->hamster) and xenografts (rat-->hamster), and 2) the ultrastructural characteristics of these microvessels. Intravital microscopy demonstrated that newly formed microvessels grow from the vascular bed of the host muscle tissue into the islet grafts. Immunohistochemical analysis of host tissue and transplanted islets with antibodies against factor VIII (recognizing both hamster and rat factor VIII), bovine PECAM-1 (CD31; endoCAM, crossreacting with hamster but not rat PECAM-1), and rat ICAM-1 (CD54, non-crossreacting with hamster ICAM-1) showed that the transplanted rat islets were revascularized by endothelium of hamster (host) origin. At an ultrastructural level, the endothelial lining of the newly formed microvessels showed diaphragmatic fenestration, a characteristic feature of endothelial cells of pancreatic islets in situ. On the basis of these findings we suggest that pancreatic islet transplantation may take a unique position in the field of organ transplantation, since the generally proposed mechanisms of endothelial cell-dependent antigen recognition as a trigger of graft rejection may not be transferred to islet grafts, containing microvessels lined by endothelial cells of host origin. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7539980

  1. Ultrastructural changes and programmed cell death of trophocytes in the gonad of Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada, Eutardigrada, Isohypsibiidae).

    PubMed

    Poprawa, Izabela; Hyra, Marta; Kszuk-Jendrysik, Michalina; Rost-Roszkowska, Magdalena Maria

    2015-03-01

    The studies on the fates of the trophocytes, the apoptosis and autophagy in the gonad of Isohypsibius granulifer granulifer have been described using transmission electron microscope, light and fluorescent microscopes. The results presented here are the first that are connected with the cell death of nurse cells in the gonad of tardigrades. However, here we complete the results presented by Węglarska (1987). The reproductive system of I. g. granulifer contains a single sack-like hermaphroditic gonad and a single gonoduct. The gonad is composed of three parts: a germarium filled with proliferating germ cells (oogonia); a vitellarium that has clusters of female germ cells (the region of oocytes development); and a male part filled with male germ cells in which the sperm cells develop. The trophocytes (nurse cells) show distinct alterations during all of the stages of oogenesis: previtello-, vitello- and choriogenesis. During previtellogenesis the female germ cells situated in the vitellarium are connected by cytoplasmic bridges, and form clusters of cells. No ultrastructural differences appear among the germ cells in a cluster during this stage of oogenesis. In early vitellogenesis, the cells in each cluster start to grow and numerous organelles gradually accumulate in their cytoplasm. However, at the beginning of the middle of vitellogenesis, one cell in each cluster starts to grow in order to differentiate into oocyte, while the remaining cells are trophocytes. Eventually, the cytoplasmic bridges between the oocyte and trophocytes disappear. Autophagosomes also appear in the cytoplasm of nurse cells together with many degenerating organelles. The cytoplasm starts to shrink, which causes the degeneration of the cytoplasmic bridges between trophocytes. Apoptosis begins when the cytoplasm of these cells is full of autophagosomes/autolysosomes and causes their death.

  2. Response of the common cutworm Spodoptera litura to lead stress: changes in sex ratio, Pb accumulations, midgut cell ultrastructure.

    PubMed

    Shu, Yinghua; Zhou, Jialiang; Lu, Kai; Li, Keqing; Zhou, Qiang

    2015-11-01

    When cutworm Spodoptera litura larvae were fed on the diets with different lead (Pb) concentrations for one or five generations, changes in growth and food utilization were recorded; Pb accumulations were detected by Atomic Absorption Spectrophotometer; changes in midgut cell ultrastructure were observed by Transmission Electron Microscopy (TEM). The effects of Pb stress on S. litura growth and food utilization differed significantly between insects of the 1st and 5th generation. The male-female rate of 200mgkg(-1) Pb treatment from the 1st generation and 50mgkg(-1) Pb treatment from the 5th generation was significantly higher than control. No significant difference of Pb accumulations was found in larvae, pupae and adults between the 1st and 5th generation. No significant difference of Pb accumulations in corresponding tissues of larvae was found between male and female. Compared to fat body, hemolymph, head, foregut and hindgut, the highest Pb accumulation was found in migut of larvae exposed to 200mgkg(-1) Pb. TEM showed that expanded intercellular spaces were observed in Pb-treated midgut cells. The nuclei were strongly destroyed by Pb stress, evidenced by chromatin condensation and destroyed nuclear envelope. Mitochondria became swollen with some broken cristae after exposure to Pb. Therefore, neither gender nor progeny difference was present in Pb accumulations of S. litura, although effects of Pb stress on S. litura growth and food utilization differed from different generations and genders. Pb accumulations in midgut caused pathological changes in cells ultrastructure, possibly reflected the growth and food utilization of S. litura.

  3. Human lung-derived mature mast cells cultured alone or with mouse 3T3 fibroblasts maintain an ultrastructural phenotype different from that of human mast cells that develop from human cord blood cells cultured with 3T3 fibroblasts.

    PubMed Central

    Dvorak, A. M.; Furitsu, T.; Estrella, P.; Ishizaka, T.

    1991-01-01

    Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1750506

  4. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in rhesus monkeys exposed to ozone.

    PubMed Central

    Castleman, W. L.; Dungworth, D. L.; Schwartz, L. W.; Tyler, W. S.

    1980-01-01

    The pathogenesis of acute respiratory bronchiolitis was examined in rhesus monkeys exposed to 0.8 ppm ozone fpr 4--50 hours. Epithelial injury and renewal was qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4--12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18% was measured after 50 hours of exposure. Most (67--80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20--33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell type most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal. Images Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 23 Figure 24 Figure 25 Figure 9 Figure 10 Figure 26 Figure 27 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:6767409

  5. Quantitative ultrastructural analysis of the human parietal cell during acid inhibition and increase of gastric potential difference by glucagon.

    PubMed Central

    Ivey, K J; Tarnawski, A; Sherman, D; Krause, W J; Ackman, K; Burks, M; Hewett, J

    1980-01-01

    Glucagon inhibits gastric acid secretion and increases the negativity of gastric mucosal potential difference (PD) in man. To test the hypothesis that the increased negativity of PD after glucagon in man could be due to decreased parietal cell canalicular membrane area, a quantitative ultrastructural analysis was carried out. Four healthy volunteers with normal gastric mucosa were submitted to biopsy before and 20 minutes after intravenous injection of 2 mg glucagon (G). This time corresponded with the maximal change in PD and a decrease in gastric acid secretion. Canalicular and tubulovesicular membrane area of 80 parietal cells (40 cells before glucagon and 40 cells after glucagon) were quantified by the Loud morphometric method. After glucagon, the oxyntic cell canalicular membrane area was reduced by one-fourth (P less than 0.05), while tubulovesicular membrane area showed an increase (P less than 0.05) at the same time. The decrease in the area of parietal cell canalicular membrane caused by glucagon may in part be responsible for increased negativity of the gastric PD caused by this hormone. Images Fig. 2 PMID:7364316

  6. Three-dimensional ultrastructural analysis of cells in the periodontal ligament using focused ion beam/scanning electron microscope tomography

    PubMed Central

    Hirashima, Shingo; Ohta, Keisuke; Kanazawa, Tomonoshin; Okayama, Satoko; Togo, Akinobu; Uchimura, Naohisa; Kusukawa, Jingo; Nakamura, Kei-ichiro

    2016-01-01

    The accurate comprehension of normal tissue provides essential data to analyse abnormalities such as disease and regenerative processes. In addition, understanding the proper structure of the target tissue and its microenvironment may facilitate successful novel treatment strategies. Many studies have examined the nature and structure of periodontal ligaments (PDLs); however, the three-dimensional (3D) structure of cells in normal PDLs remains poorly understood. In this study, we used focused ion beam/scanning electron microscope tomography to investigate the whole 3D ultrastructure of PDL cells along with quantitatively analysing their structural properties and ascertaining their orientation to the direction of the collagen fibre. PDL cells were shown to be in contact with each other, forming a widespread mesh-like network between the cementum and the alveolar bone. The volume of the cells in the horizontal fibre area was significantly larger than in other areas, whereas the anisotropy of these cells was lower than in other areas. Furthermore, the orientation of cells to the PDL fibres was not parallel to the PDL fibres in each area. As similar evaluations are recognized as being challenging using conventional two-dimensional methods, these novel 3D findings may contribute necessary knowledge for the comprehensive understanding and analysis of PDLs. PMID:27995978

  7. A formalin fixative for immunochemical and ultrastructural studies on gastrointestinal endocrine cells.

    PubMed Central

    Robinson, G; Dawson, I

    1979-01-01

    Using indirect immunofluorescence, indirect immunoperoxidase, and unlabelled antibody enzyme techniques, gastrin, pancreatic glucagon, insulin, and somatostatin were localised in sections of both wax- and resin-embedded tissues that had been fixed in a buffered formalin solution. Ultrastructural preservation of the resin-embedded samples was also adequate for combined electron microscopy and light microscope immunochemistry. As the fixative concerned is stable it can be permanently available in surgical units. It is suggested, therefore, that this fixative should prove useful as an alternative to buffered formaldehyde, which must be freshly prepared from paraformaldehyde powder, in institutions where specimen collection is difficult or which have to refer cases with an endocrine involvement to other laboratories for immunochemical and fine structural examination. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:372249

  8. Ultrastructural Studies on Oocyte Development and Vitellogenesis associated with Follicle Cells in Female Scapharca subcrenata (Pelecypoda: Arcidae) in Western Korea

    PubMed Central

    Kim, Sung Han

    2016-01-01

    Ultrastructural studies on oocyte development and vitellogenesis in oocytes, and the functions of follicle cells during oogenesis and oocyte degeneration were investigated to clarifyb the reproductive mechanism on vitellogenesis of Scapharca subcrenata using electron microscope observations. In this study, vitellogenesis during oogenesis in the oocytes occured by way of autosynthesis and heterosynthesis. Of two processes of vitellogenesis during oogenesis, the process of endogenous autosynthesis involved the combined activity of the Golgi complex, mitochondria and rough endoplasmic reticulum. However, the process of exogenous heterosynthesis involved endocytotic incorporation of extraovarian precursors at the basal region of the oolema of the early vitellogenic oocytes before the formation of the vitelline coat. In this study, follicle cells, which attached to the previtellogenic and vitellogenic oocytes, were easily found. In particular, the follicle cells were involved in the development of previtellogenic oocytes by the supply of nutrients, and vitellogenesis in the early and late vitellogenic oocytes by endocytosis of yolk precursors. Based on observations of follicle cells attached to degenerating oocytes after spawning, follicles of this species are involved in lysosomal induction of oocyte degeneration for the resorption phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves. In this study, the functions of follicle cells can accumulate reserves of lipid granules and glycogen particles for vitellogenesis from degenerating oocytes after spawning. PMID:27796004

  9. Ultrastructural analysis of oral exfoliated epithelial cells of tobacco smokers and betel nut chewers: A scanning electron microscopy study

    PubMed Central

    Khan, Sameera Shamim; Shreedhar, Balasundari; Kamboj, Mala

    2016-01-01

    Background: The study was undertaken to correlate epithelial surface pattern changes of oral exfoliated cells of tobacco smokers and betel nut chewers and also to compare them with patients of oral squamous cell carcinoma (OSCC) and healthy individuals. Materials and Methods: In this cross-sectional study, a total of fifty persons were included in the study, out of which thirty formed the study group (15 each tobacco smokers and betel nut chewers) and twenty formed the control group (ten each of OSCC patients – positive control and ten normal buccal mucosa – negative control). Their oral exfoliated cells were scraped, fixed, and studied under scanning electron microscope (SEM). The statistical analysis was determined using ANOVA, Tukey honestly significant difference, Chi-square test, and statistical SPASS software, P < 0.05. Results: OSCC, Individual cell modifications, intercellular relationships and surface characteristics observed by scanning electron microscopy between OSCC, tobacco smokers, betel nut chewers compared to normal oral mucosa have been tabulated. Conclusion: In normal oral mucosa, cell surface morphology depends on the state of keratinization of the tissue. Thus, it could prove helpful in detecting any carcinomatous change at its incipient stage and also give an insight into the ultra-structural details of cellular differentiations in epithelial tissues. PMID:28182055

  10. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage.

    PubMed

    Hillel, Alexander T; Taube, Janis M; Cornish, Toby C; Sharma, Blanka; Halushka, Marc; McCarthy, Edward F; Hutchins, Grover M; Elisseeff, Jennifer H

    2010-01-01

    The production of engineered cartilage from mesenchymal stem cells is a rapidly developing field. Potential applications include the treatment of degenerative joint disease as well as the treatment of traumatic and surgical bone injury. Prior to clinical application, however, further characterization of the morphology, ultrastructure, biocompatibility, and performance of the engineered tissue is warranted. To achieve this, human mesenchymal stem cells (hMSCs) were grown in vitro in pellet culture for 3 weeks in chondrogenic medium conditions. The resultant engineered cartilage was compared to native adult and fetal tissue. Routine histology, special stains, and ultrastructural and quantitative histomorphometric analyses were performed. The engineered tissue demonstrated a similar chondrocyte phenotype, collagen fibril appearance, and matrix distribution when compared to native cartilage. By histomorphometric analysis, the cell density of the engineered cartilage was between that of native fetal and adult cartilage. The cell-to-matrix ratio and cellular area fraction of engineered cartilage samples was significantly greater than in adult samples, but indistinguishable from fetal cartilage samples, supporting the hypothesis that hMSC-engineered cartilage regeneration may mimic fetal cartilage development.

  11. Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells.

    PubMed

    Whelan, Donna R; Bell, Toby D M

    2015-12-18

    Single molecule localization microscopy (SMLM) and synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffraction limit, whereas IR spectroscopy objectively measures the holistic biochemistry of an entire sample, thereby revealing any variations in overall composition. Both tools are currently applied extensively to detect cellular response to disease, chemical treatment, and environmental change. Here, these two techniques have been applied correlatively at the single cell level to probe the biochemistry of common fixation methods and have detected various fixation-induced losses of biomolecular composition and cellular ultrastructure. Furthermore, by extensive honing and optimizing of fixation protocols, many fixation artifacts previously considered pervasive and regularly identified using IR spectroscopy and fluorescence techniques have been avoided. Both paraformaldehyde and two-step glutaraldehyde fixation were identified as best preserving biochemistry for both SMLM and IR studies while other glutaraldehyde and methanol fixation protocols were demonstrated to cause significant biochemical changes and higher variability between samples. Moreover, the potential complementarity of the two techniques was strikingly demonstrated in the correlated detection of biochemical changes as well as in the detection of fixation-induced damage that was only revealed by one of the two techniques.

  12. Ultrastructural analysis of olfactory ensheathing cells derived from olfactory bulb and nerve of neonatal and juvenile rats.

    PubMed

    Gómez, Rosa M; Ghotme, Kemel; Botero, Lucía; Bernal, Jaime E; Pérez, Rosalía; Barreto, George E; Bustos, Rosa Helena

    2016-02-01

    Olfactory nerve derived and olfactory bulb derived olfactory ensheathing cells (OECs) have the ability to promote axonal regeneration and remyelination, both of which are essential in a successful cell transplant. Thus, morphological identification of OECs is a key aspect to develop an applicable cell therapy for injuries to the nervous system. However, there is no clear definition regarding which developmental stage or anatomical origin of OECs is more adequate for neural repair. In the present study, an ultrastructural comparison was made between OECs recovered from primary cultures of olfactory nerve and bulb in two developmental stages. The most notorious difference between cells obtained from olfactory nerve and bulb was the presence of indented nuclei in bulb derived OECs, suggesting a greater ability for possible chemotaxis. In neonatal OECs abundant mitochondria, lipid vacuoles, and smooth endoplasmic reticulum were detected, suggesting an active lipid metabolism, probably involved in synthesis of myelin. Our results suggest that neonatal OECs obtained from olfactory bulb have microscopic properties that could make them more suitable for neural repair.

  13. Ultrastructure of the attack of eosinophils stimulated by blood mononuclear cell products on schistosomula of Schistosoma mansoni.

    PubMed Central

    Caulfield, J. P.; Lenzi, H. L.; Elsas, P.; Dessein, A. J.

    1985-01-01

    Purified human eosinophils were treated with peripheral blood mononuclear cell supernatants containing eosinophil cytotoxic enhancing activity (ECEA). Schistosomula of Schistosoma mansoni which had been coated either with antibody (Ab) from the sera of infected patients or with the lectin concanavalin A (Con A) were incubated with ECEA-treated and untreated cells for 2 minutes to 12 hours and examined ultrastructurally. Killing was assayed at 18 hours. ECEA caused an increase in the killing of Ab-coated worms, but Con-A-coated worms were not killed by either ECEA-treated or untreated cells. Eosinophils began to degranulate on Ab-coated worms within 2 minutes and continued to degranulate, so that by 12 hours about half of the parasites had greater than 50% of their surface covered by discharge material. The ECEA-treated cells degranulated more than the untreated cells. There was much less discharge material on Con-A-coated worms than on Ab-coated worms. Eosinophils adhered to discharge material on the surface of both Ab- and Con-A-coated parasites. At 3 and 12 hours, lysed cells and cell fragments were also seen adhering to discharge material. In the absence of discharge material the cells adhered to residual glycocalyx or to the tegumental outer membrane. These studies suggest that eosinophils kill schistosomula by progressively degranulating onto their surface over many hours and that the increased toxicity caused by ECEA is due to an increase in discharge. Images Figure 3 Figure 4 Figure 5 Figure 1 Figure 2 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:4037065

  14. A new nidovirus (NamDinh virus NDiV): Its ultrastructural characterization in the C6/36 mosquito cell line

    SciTech Connect

    Thuy, Nguyen Thanh; Huy, Tran Quang; Nga, Phan Thi; Morita, Kouichi; Dunia, Irene; Benedetti, Lucio

    2013-09-15

    We describe the ultrastructure of the NamDinh virus (NDiV), a new member of the order Nidovirales grown in the C6/36 mosquito cell line. Uninfected and NDiV-infected cells were investigated by electron microscopy 24–48 h after infection. The results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. Mature virions of NDiV were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. Free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. The size of the round-shaped particles with a complete internal structure was 80 nm in diameter. This is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus NDiV, a missing evolutionary link in the emergence of the viruses with the largest RNA genomes. - Highlights: • NamDinh virus (NDiV), a new member of the order Nidovirales was tested in cultured cell line. • The morphogenesis and ultrastructure of NDiV were investigated by electron microscopy. • The viral nucleocapsid-like particles clustered and scattered in the cytoplasm. • NDiVs were released as budding particles on the cell surface. • The size of the viral particles with a complete internal structure was 80 nm in diameter.

  15. Stem cell banking: between traceability and identifiability.

    PubMed

    Knoppers, Bartha M; Isasi, Rosario

    2010-10-05

    Stem cell banks are increasingly seen as an essential resource of biological materials for both basic and translational research. Stem cell banks support transnational access to quality-controlled and ethically sourced stem cell lines from different origins and of varying grades. According to the Organisation for Economic Co-operation and Development, advances in regenerative medicine are leading to the development of a bioeconomy, 'a world where biotechnology contributes to a significant share of economic output'. Consequently, stem cell banks are destined to constitute a pillar of the bioeconomy in many countries. While certain ethical and legal concerns are specific to the nature of stem cells, stem cell banking could do well to examine the approaches fostered by tissue banking generally. Indeed, the past decade has seen a move to simplify and harmonize biological tissue and data banking so as to foster international interoperability. In particular, the issues of consent and of traceability illustrate not only commonalities but the opportunity for stem cell banking to appreciate the lessons learned in biobanking generally. This paper analyzes convergence and divergence in issues surrounding policy harmonization, transnational sharing, informed consent, traceability and return of results in the context of stem cell banks.

  16. Ultrastructure and lipid composition of detergent-resistant membranes derived from mammalian sperm and two types of epithelial cells.

    PubMed

    van Gestel, Renske A; Brouwers, Jos F; Ultee, Anton; Helms, J Bernd; Gadella, Bart M

    2016-01-01

    Lipid rafts are micro-domains of ordered lipids (Lo phase) in biological membranes. The Lo phase of cellular membranes can be isolated from disordered lipids (Ld phase) after treatment with 1 % Triton  X-100 at 4 °C in which the Lo phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and porcine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with <5 mM methyl-ß-cyclodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein-cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the Lo phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.

  17. Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons.

    PubMed

    Jeger, Rina; Lichtenfeld, Yona; Peretz, Hagit; Shany, Boaz; Vago, Razi; Baranes, Danny

    2009-04-01

    Crystalline, porous biomaterials, such as marine invertebrate skeletons, have been widely used for functional reconstruction of human tissues like bone and dental implants. Since in such an abrasive microenvironment adequate cell-material interactions are crucial for a successful treatment, it is of great importance to improve the means to examine these interactions. We developed a method that reveals the ultrastructure of the interface between coral skeletons and cultured neural cells to a higher quality than do traditional methods as it does not include damaging procedures like decalcification or sectioning non-decalcified skeletons. It is rather based on generating two electron opacity distinct Araldite masks, of the skeleton and its surrounding, by polymerizing them to different durations. The contrast created at the border of the two masks outlined the fine and fragile crystals of the coral skeleton's outer and inner surfaces and their contact sites with the cells. The skeleton's internal structure contains a mesh of narrow (few microns wide) and large channel-shaped gaps interrupted by irregular-shaped crystalline material. Neural cells grew on the skeleton surface by stretching between crystal tips, with occasional rearrangements of cytoskeletal fibers located near the anchorage focal adherence points. Cell processes infiltrated the skeleton interior by stretching between inter-surface crystals and by adjusting their volume to the space of the conduits they grew into. The technique advances the study of coral biology and of neural cells-hard biomaterial interaction; it can be applied to other biomaterials and cell types and open new ways for studying tissue development and engineering.

  18. Effects of water turbulence on variations in cell ultrastructure and metabolism of amino acids in the submersed macrophyte, Elodea nuttallii (Planch.) H. St. John.

    PubMed

    Atapaththu, K S S; Miyagi, A; Atsuzawa, K; Kaneko, Y; Kawai-Yamada, M; Asaeda, T

    2015-09-01

    The interactions between macrophytes and water movement are not yet fully understood, and the causes responsible for the metabolic and ultrastructural variations in plant cells as a consequence of turbulence are largely unknown. In the present study, growth, metabolism and ultrastructural changes were evaluated in the aquatic macrophyte Elodea nuttallii, after exposure to turbulence for 30 days. The turbulence was generated with a vertically oscillating horizontal grid. The turbulence reduced plant growth, plasmolysed leaf cells and strengthened cell walls, and plants exposed to turbulence accumulated starch granules in stem chloroplasts. The size of the starch granules increased with the magnitude of the turbulence. Using capillary electrophoresis-mass spectrometry (CE-MS), analysis of the metabolome found metabolite accumulation in response to the turbulence. Asparagine was the dominant amino acid that was concentrated in stressed plants, and organic acids such as citrate, ascorbate, oxalate and γ-amino butyric acid (GABA) also accumulated in response to turbulence. These results indicate that turbulence caused severe stress that affected plant growth, cell ultrastructure and some metabolic functions of E. nuttallii. Our findings offer insights to explain the effects of water movement on the functions of aquatic plants.

  19. Identifying ECM Mediators of Tumor Cell Dormancy

    DTIC Science & Technology

    2009-05-01

    determined by vaginal smear so that inter- animal variation due to estrous can be controlled for across groups. Further, cervical samples will be... peptides identified for each protein divided by the total number of peptides identified in the two hour run are displayed as the peptide count ratio...Using this peptide counting label-free quantification approach, surprisingly, suggested that the three fibrillar collagen chains were at lower levels in

  20. Ultrastructural analysis of contractile cell development in lung microvessels in hyperoxic pulmonary hypertension. Fibroblasts and intermediate cells selectively reorganize nonmuscular segments.

    PubMed Central

    Jones, R.

    1992-01-01

    The current study traces the development of contractile cells in the nonmuscular segments of rat lung microvessels in hyperoxic pulmonary hypertension. New intimal cells first develop into a well-defined layer beneath the endothelium and internal to an elastic lamina. Ultrastructurally, these cells are found to be 1) fibroblasts recruited to the vessel wall from the interstitium and 2) intermediate cells, a population of preexisting vascular cells (structurally between a smooth muscle cell and a pericyte). Early in hyperoxia (days 3 through 7), interstitial fibroblasts migrate and align around the smallest vessels in which an elastic lamina is either absent or fragmentary. These cells then are incorporated into the vessel wall by tropoelastin secretion and the formation of an elastic lamina along their abluminal margin. After day 7, the new mural fibroblasts acquire the features of contractile cells, namely a basal lamina, extensive microfilaments, and dense bodies. In other vessels, as early as day 3 of hyperoxia, intermediate cells within the vessel intima begin to acquire the additional filaments and dense bodies of contractile cells. As hyperoxia continues, each cell pathway gives rise to vessels with distinct intimal or medial layers of contractile cells. In this way, thick-walled 'newly muscularized' vessel segments form adjacent to the capillary bed. Images Figure 1 Figure 5 Figure 6 Figure 7 p1500-a Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:1466406

  1. Ultrastructural distribution of the death-domain-containing MyD88 protein in HeLa cells.

    PubMed

    Jaunin, F; Burns, K; Tschopp, J; Martin, T E; Fakan, S

    1998-08-25

    MyD88, a protein implicated in interleukin-1 signaling, was localized in HeLa cells transiently transfected with an epitope-tagged (flag) version of MyD88. Overexpression of MyD88 can induce apoptosis. We have analyzed the fine structural intracellular distribution of MyD88 using immunoelectron microscopy. MyD88 is localized to the nucleus and to the cytoplasm as revealed by immunofluorescence visualization. Ultrastructural immunocytochemistry shows that, in the cytoplasm, this protein is associated with fibrillar aggregates containing beta-actin. In the nucleus, MyD88 was found in fibrillar domains present only in cells not yet displaying morphological signs of apoptosis. These domains are not derived from nucleoli and do not constitute an accumulation site of splicing factors. We suggest that such structures could be involved in the formation of the apoptotic bodies and/or in the modification of the nuclear structure and of nucleocytoplasmic trafficking during apoptosis.

  2. The influence of microgravity and spaceflight on columella cell ultrastructure in starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Guisinger, M. M.; Kiss, J. Z.

    1999-01-01

    The ultrastructure of root cap columella cells was studied by morphometric analysis in wild-type, a reduced-starch mutant, and a starchless mutant of Arabidopsis grown in microgravity (F-microgravity) and compared to ground 1g (G-1g) and flight 1g (F-1g) controls. Seedlings of the wild-type and reduced-starch mutant that developed during an experiment on the Space Shuttle (both the F-microgravity samples and the F-lg control) exhibited a decreased starch content in comparison to the G-1g control. These results suggest that some factor associated with spaceflight (and not microgravity per se) affects starch metabolism. Elevated levels of ethylene were found during the experiments on the Space Shuttle, and analysis of ground controls with added ethylene demonstrated that this gas was responsible for decreased starch levels in the columella cells. This is the first study to use an on-board centrifuge as a control when quantifying starch in spaceflight-grown plants. Furthermore, our results show that ethylene levels must be carefully considered and controlled when designing experiments with plants for the International Space Station.

  3. Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components.

    PubMed

    Koga, Daisuke; Kusumi, Satoshi; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Ushiki, Tatsuo

    2015-12-01

    Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.

  4. Identify multiple myeloma stem cells: Utopia?

    PubMed

    Saltarella, Ilaria; Lamanuzzi, Aurelia; Reale, Antonia; Vacca, Angelo; Ria, Roberto

    2015-01-26

    Multiple myeloma (MM) is a hematologic malignancy of monoclonal plasma cells which remains incurable despite recent advances in therapies. The presence of cancer stem cells (CSCs) has been demonstrated in many solid and hematologic tumors, so the idea of CSCs has been proposed for MM, even if MM CSCs have not been define yet. The existence of myeloma CSCs with clonotypic B and clonotypic non B cells was postulated by many groups. This review aims to focus on these distinct clonotypic subpopulations and on their ability to develop and sustain MM. The bone marrow microenvironment provides to MM CSCs self-renewal, survival and drug resistance thanks to the presence of normal and cancer stem cell niches. The niches and CSCs interact each other through adhesion molecules and the interplay between ligands and receptors activates stemness signaling (Hedgehog, Wnt and Notch pathways). MM CSCs are also supposed to be responsible for drug resistance that happens in three steps from the initial cancer cell homing microenvironment-mediated to development of microenvironment-independent drug resistance. In this review, we will underline all these aspects of MM CSCs.

  5. Microstructure Features of Proventriculus and Ultrastructure of the Gastric Gland Cells in Chinese Taihe Black-bone Silky Fowl (Gallus gallus domesticus Brisson).

    PubMed

    Zhang, H; Ge, T; Peng, S; Zhong, S; Zhou, Z

    2016-02-01

    To investigate microstructure of proventriculus and ultrastructure of the gastric gland cells from Chinese Taihe black-bone silky fowl (BSF), the proventriculus of 4-week-old BSF was sampled. Conventional histological and transmission electron microscope (TEM) methods were used in this study. The wall of the Taihe BSF proventriculus was consisted of four layers, the mucous, submucosa, muscularis externa and the serosa as others birds. The muscularis externa of the birds' proventriculus contained three layers. Much of the melanin was present in loose connective tissue of lamina propria, submucosa, and muscularis externa unlike others. In addition, the ultrastructure of the gastric gland cells was observed by TEM. There was only one kind of gland cell, for example oxynticopeptic cell in proventriculus of Taihe BSF. The oxynticopeptic cells contained numerous mitochondria, cisternae of rough endoplasmic reticulum (CRER), intracellular canaliculi (IC) that secrete hydrochloric acid and small amounts of pepsinogen granules. The rough endoplasmic reticulum (RER) was irregular cisternae with ribosomes and surrounded tightly the mitochondria along their configuration. The electron-dense pepsinogen granules were round with various sizes. The neighbouring oxynticopeptic cells were closed up with tight junction and gap junction. The inter-space between the neighbouring oxynticopeptic cells was stenosis or was filled with electron-dense extracellular substance. In conclusion, the gastric gland cells of Chinese Taihe BSF proventriculus were only oxynticopeptic cells that secrete hydrochloric acid and pepsinogen, but no parietal cells and chief cells of mammal. The gastric gland cells of proventriculus were underdeveloped compared with those of mammals.

  6. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

    PubMed Central

    Spiegelhalter, Coralie; Tosch, Valérie; Hentsch, Didier; Koch, Marc; Kessler, Pascal; Schwab, Yannick; Laporte, Jocelyn

    2010-01-01

    Background In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM. PMID:20140253

  7. Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826).

    PubMed

    dos Santos, Daiane Cristina Marques; Cupertino, Marli do Carmo; Fialho, Maria do Carmo Queiroz; Barbosa, Alfredo Jose Afonso; Fonseca, Cláudio Cesar; Sartori, Sirlene Souza Rodrigues; da Matta, Sérgio Luis Pinto

    2014-02-01

    This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.

  8. Novel Identified Receptors on Mast Cells

    PubMed Central

    Migalovich-Sheikhet, Helena; Friedman, Sheli; Mankuta, David; Levi-Schaffer, Francesca

    2012-01-01

    Mast cells (MC) are major participants in the allergic reaction. In addition they possess immunomodulatory roles in the innate and adaptive immune reactions. Their functions are modulated through a number of activating and inhibitory receptors expressed on their surface. This review deals with some of the most recently described receptors, their expression patterns, ligand(s), signal transduction mechanisms, possible cross-talk with other receptors and, last but not least, regulatory functions that the MC can perform based on their receptor expression in health or in disease. Where the receptor role on MC is still not clear, evidences from other hematopoietic cells expressing them is provided as a possible insight for their function on MC. Suggested strategies to modulate these receptors’ activity for the purpose of therapeutic intervention are also discussed. PMID:22876248

  9. Identifying ECM Mediators of Tumor Cell Dormancy

    DTIC Science & Technology

    2007-05-01

    oil). d) At end of 4 week treatment (Gp2) and after 1 month post-weaning (Gp3), all groups will be euthanized. e) Two hours prior to euthanasia ...inject rats with 50 mg/BrdU i.p. for incorporation into actively proliferating cells. f) At time of euthanasia , stage of estrous will be determined...of New Mexico , ‘Plasticity of the mammary gland extracellular matrix and breast cancer progression’. Albuquerque, New Mexico , Sept 22, 2006

  10. Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): steroidogenic and ultrastructural properties.

    PubMed

    Carsia, R V; Scanes, C G; Malamed, S

    1985-02-01

    Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.

  11. Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method

    PubMed Central

    1984-01-01

    Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system. PMID:6207180

  12. Impact of cadmium on early stages of flax fibre differentiation: ultrastructural aspects and pectic features of cell walls.

    PubMed

    Douchiche, Olfa; Driouich, Azeddine; Morvan, Claudine

    2011-06-01

    The effect of 0.5mM cadmium (Cd) was studied on the ultrastructural aspects and pectin features of the walls of flax cellulosic fibres when the thickening of secondary wall had just started in the hypocotyl of 10-day old seedlings. As seen by PATAg staining in controls, cell-wall formation displayed two distinct steps, secretion and remodelling, which did not occur simultaneously for all the neighbouring fibres. The inner part of the secondary wall, where the cellulose molecules had just been synthesized, appeared very reactive to PATAg. The outer part, where the cellulose fibrils associated in larger microfibril complexes, became non-reactive to PATAg. Under Cd treatment, we noticed some acceleration of fibre differentiation in terms of fibre number, wall thickness and yield. As revealed by PATAg staining, treated fibres exhibited a disturbed cell-wall texture, indicating a modified adhesion between the matrix polysaccharides and the cellulose microfibrils. The Cd impact on the distribution of highly methylesterified homogalacturonans (recognized by JIM7 antibody) was moderate in the cell junctions and low in the primary wall and outer part of secondary wall. The data meant that no early deesterification occurred in these domains, a behaviour related to the specificity of the CW-II metabolism. No large redistribution of low esterified homogalacturonans (recognized by JIM5 antibody) happened either. In parallel, the amount of uronic acid significantly increased in the so-called H(2)SO(4) cell-wall extract, indicating a Cd impact on pectin structure not detected by JIM5 or JIM7 antibodies.

  13. Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush.

    PubMed

    Barron, K D; Dentinger, M P; Krohel, G; Easton, S K; Mankes, R

    1986-06-01

    Rat retinal ganglion cell layer (GCL) was examined ultrastructurally 1-180 days after intraorbital crushing of one optic nerve. It was confirmed quantitatively that axotomized ganglion cells lost cisternal membranes of the rough endoplasmic reticulum (RER) and showed disintegration of Nissl bodies and ribosomal rosettes 3 days postoperatively. Between 60 and 180 days after neurotomy there was partial reversion of the RER towards normal. At postoperative intervals of 14-60 days, chromatin aggregation became conspicuous and some nuclei were prominently furrowed and contained electron-dense inclusions. Concurrently, profiles of dead ganglion cells were encountered. Mean mitochondrial area increased in axotomized neurons but mitochondrial density declined, while the Golgi apparatus, lamellar specializations of the RER and the size of nuclei did not change significantly. Cytoplasmic atrophy was profound, however. Small nerve cells of the GCL appeared morphologically distinct from ganglion cells and did not undergo appreciable alteration. A decline in neuronal density, approximating 35%, occurred between the third and seventh postoperative day and progressed slowly thereafter. Neuronal density was 32% of normal 180 days postoperatively. A temporary increase in glial density 3-28 days after operation was due to microglial hyperplasia. Müller cell and astrocytic processes hypertrophied, infiltrated nerve fibre bundles, and surrounded and intruded into neuronal somata. Bundles of unmyelinated small axons, invested by astrocytes and basal lamina, were present within the necrotic cavity of the lesioned nerve 28-90 days postoperatively and had cytologic features of regenerative axonal sprouts. We conclude that intraorbital optic nerve crush is followed by a noteworthy degree of regenerative axonal sprouting which occurs and persists against a background of slow but relentless decline in the retinal ganglion cell population. This slow decline follows a rapidly-sustained loss

  14. What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy.

    PubMed

    Kondo, Hisatake

    2008-06-01

    soluble proteins; the lattice-compactness indicates the concentration of soluble proteins in the domain, and the aqueous cytoplasm is equivalent to the aqueous solution. Further, the appearance of two contiguous lattice domains exhibiting differing degrees of compactness in a given cell indicates that cytoplasmic proteins are solated in a domain with less compact lattices, whereas they are gelated in the other domain. These proposed interpretations need to be confirmed by further studies. If confirmed, the control mechanisms of the localization and movement of intracellular organelles could then be understood on the basis not only of information about the cytoskeletons but also of cell ultrastructure-related information on the concentration and sol-gel states of intracellular proteins. In addition, possible interpretations of the significance of strand-lattices in PEG-EM are also applicable to the nucleoplasm, especially extra-heterochromatin (euchromatin) areas. Finally, several potential uses/advantages of PEG-EM in the cell-ultrastructure have also been demonstrated, especially in three-dimensional reconstructions of nonmembranous structures including stereo-viewing using a pair of EM images with appropriate tilting as well as electron microscopic tomography.

  15. Structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines.

    PubMed

    Martínez-Rodríguez, A J; Polo, M C; Carrascosa, A V

    2001-12-04

    This study shows the changes that occur during the autolysis of yeast in a model wine medium and in a sparkling wine after 12 months of aging, using Nomarsky Light Microscopy and Low Temperature Scanning Electron Microscopy (LTSEM). The size of the yeasts after 24 h of autolysis in a model medium is much smaller than when they are in the growth stage. With LTSEM. a large number of folds can be observed on the surface of the yeast and practically empty cells. Greater morphological changes, both structural and ultrastructural, can be observed in the yeast after 12 months of aging in wine than in the yeast after 24 h of induced autolysis. However, less of the cytoplasmic content of the yeast that has undergone autolysis in the wine was solubilized than that of the yeast after 24 h of autolysis in the model wine system. These findings indicate that autolysis of yeast in wine is a long-lasting process, which continues for at least 12 months.

  16. Ultrastructure of the ciliated cells of the free-swimming larva, and sessile stages, of the marine sponge Haliclona indistincta (Demospongiae: Haplosclerida).

    PubMed

    Stephens, Kelly M; Ereskovsky, Alexander; Lalor, Pierce; McCormack, Grace P

    2013-11-01

    We provide a detailed, comparative study of the ciliated cells of the marine haplosclerid sponge Haliclona indistincta, in order to make data available for future phylogenetic comparisons at the ultrastructural level. Our study focuses on the description and analysis of the larval epithelial cells, and choanocytes of the metamorphosed juvenile sponge. The ultrastructure of the two cell types is sufficiently different to prevent our ability to conclusively determine the origin of the choanocytes from the larval ciliated cells. However, ciliated, epithelial cells were observed in a migratory position within the inner cell mass of the larval stages. Some cilia were observed within the cell's cytoplasm, which is indicative of the ciliated epithelial cell undergoing transdifferentiation into a choanocyte; while traces of other ciliated epithelial cells were contained within phagosomes, suggesting they are phagocytosed. We compared our data with other species described in the literature. However, any phylogenetic inference must wait until further detailed comparisons can be made with species whose phylogenetic position has been determined by other means, such as phylogenomics, in order to more closely link genomic, and morphological information.

  17. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    DTIC Science & Technology

    2014-09-01

    AD_________________ Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma ...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT We identified 14 microRNA candidates that induce neuroblastoma cell differentiation based on a high...content screening of neurite outgrowth — the morphological differentiation marker of neuroblastoma cells. We further validated that the identified

  18. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

    NASA Technical Reports Server (NTRS)

    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  19. Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification.

    PubMed

    Cavusoglu, T; Popken, J; Guengoer, T; Yilmaz, O; Uyanikgil, Y; Ates, U; Baka, M; Oztas, E; Zakhartchenko, V

    2016-08-01

    Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four-cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow-freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra-structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.

  20. An ultrastructural study of sinuatrial node cells in the embryonic rat heart.

    PubMed Central

    Domenech-Mateu, J M; Boya-Vegué, J

    1975-01-01

    Sinuatrial nodal tissue, obtained from rat embryos of 15, 16 and 17 days, was examined with the electron microscope. Embryonic nodal cells were generally similar to adult cells except that (1) they showed thick prolongations of the cytoplasm which insinuated themselves between neighbouring cells; (2) they possessed osmiophilic granules with a predeliction for the region of the Golgi complex; (3) they exhibited a lesser and variable degree of pinocytosis. Images Fig. 1 Fig. 2 Fig. 3 PMID:1133091

  1. Angiolymphoid hyperplasia with eosinophilia presenting multinucleated cells in histology: an ultrastructural study.

    PubMed

    Sakamoto, F; Hashimoto, T; Takenouchi, T; Ito, M; Nitto, H

    1998-07-01

    A case of angiolymphoid hyperplasia with eosinophilia arising on the face of a woman is reported. Histologically, the uniqueness of this case is the presence of multinucleated cells (MNCs), besides the conventional dermal changes. Electron microscopy showed that some of the apparent MNCs are clusters of endothelial cells forming immature vascular lumens with numerous microvilli, and the other MNCs displayed the recognized features of fibrohistiocytic or myofibroblastic cells. Immunohistochemically, some MNCs were positive for Ulex europaeus agglutinin and Factor VIII-related antigen. From these findings, some of the MNCs are histologically endothelial sprouts, and the others are fibrohistiocytic cells in the present case.

  2. Evolution of acute chest syndrome in sickle cell trait: an ultrastructural and light microscopic study.

    PubMed Central

    Hasleton, P S; Orr, K; Webster, A; Lawson, R A

    1989-01-01

    Light and electron microscopic studies of a patient with sickle cell trait who had an episode of sickling during coronary artery surgery, from which he died, showed fibrin thrombi, focal alveolar wall necrosis, and epithelial cell damage. It is suggested that in cases of sickle trait full precautionary measures should be taken to prevent sickling in these circumstances. Images PMID:2617448

  3. The ultrastructure of bovine ileal follicle-associated epithelial (FAE) cells during the perinatal period.

    PubMed Central

    Asari, M; Kano, Y; Wakui, S; Nishita, T; Matsushita, H; Oshige, H

    1989-01-01

    The ileal follicle-associated epithelial (FAE) cells in bovine fetuses and neonates were examined by light and electron microscopy. In 7-9 months old fetuses (68, 82 and 86 cm CRL) the dome epithelium was usually a little thinner than elsewhere and contained more intra-epithelial leucocytes. FAE cells were already distinguishable by their being more cuboidal and eosinophilic than the other epithelial cells. The cytoplasm of the FAE cells bulged noticeably into the lumen and contained numerous mitochondria and vacuoles. At 18 hours and 21 hours after birth, the dome epithelium was more columnar and eosinophilic than previously and contained more intra-epithelial leucocytes. The FAE cells showed characteristic bulging of large cytoplasmic processes into the lumen, as seen in the previous stage. In the cytoplasm, moderate numbers of mitochondria, numerous vesicles and microtubules could be seen. Frequently degenerated FAE cells could also be found among normal FAE cells in the epithelium. After this stage the cytoplasmic processes almost disappeared but distribution of the other organelles was similar to that seen at the previous stage except that multivesicular bodies were frequently seen in the apical cytoplasm. These histological results suggest that bovine ileal FAE cells are histologically and functionally mature by birth and that at birth they seem to be able to react against the penetration of pathogenic substances from the extrauterine environment. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:2606783

  4. Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung

    PubMed Central

    Galiger, Celimene; Kostin, Sawa; Golec, Anita; Ahlbrecht, Katrin; Becker, Sven; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Morty, Rory E; Seeger, Werner; Voswinckel, Robert

    2014-01-01

    Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAMneg/CD45neg/Oct4-GFPpos that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration. PMID:24889158

  5. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

    PubMed Central

    Dashtdar, Havva; Selvaratnam, Lakshmi; Balaji Raghavendran, Hanumantharao; Suhaeb, Abdulrazzaq Mahmod; Ahmad, Tunku Sara

    2016-01-01

    Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis. PMID:26966647

  6. Ultrastructural study of the morphogenesis of human herpesvirus 6 type B in human T-lymphotropic virus type I-producing lymphoid cells.

    PubMed

    Ohtsuki, Yuji; Daibata, Masanori; Bandobashi, Kentaro; Lee, Gang-Hong; Furihata, Mutsuo; Yokoyama, Akihito; Miyoshi, Isao

    2008-12-01

    A few studies of the morphogenesis of human herpesvirus (HHV) 6 type A and B (HHV-6A, -6B) have been performed using neurogenic, lymphoid, or epithelial cells. When human MT-4 T-lymphotropic virus type I (HTLV-I)-producing lymphoid cells were coinfected with HHV-6B in vitro, viral-specific proteins were clearly detected. We therefore attempted to detect virus particles at the ultrastructural level, focusing on the morphogenesis of such particles. Ultrastructurally, HHV-6B virus particles could be observed in the nuclei, cytoplasm, and extracellular spaces of MT-4 cells, in addition to extracellular HTLV-I particles of C type. In the nuclei, dense-cored or doughnut-shaped viral capsids were found, as well as peculiar tubular rods. When budding to perinuclear spaces, these intranuclear capsids exhibited a thin tegument on their surfaces. Distinct teguments were found in the intracytoplasmic particles, which budded into cytoplasmic vacuoles during the process of maturation. The mature particles were detected in the extracellular spaces and the intracytoplasmic vacuoles, with a distinct tegument and surface spikes. An electron-dense layer in the outer part of the tegument was found in some mature particles located in the extracellular space, but no such layer was detected in mature particles in intracytoplasmic vacuoles. No annulate lamellae, but intranuclear tubular rods, were found in the cytoplasm of MT-4 cells. These observations indicate that HHV-6B in MT-4 cells is similar to HHV-6A in fine structure, but differs from HHV-7 and HHV-8 in ultrastructural characteristics. Further comparisons of HHV-6B with HHV-6A, HHV-7, and HHV-8 are needed with regard to functional activity.

  7. Changes in mouse Leydig cells ultrastructure and testosterone secretion after diethylcarbamazine administration.

    PubMed

    Saraiva, Karina Lidianne Alcântara; Silva, Valdemiro Amaro Da; Torres, Dilênia De Oliveira Cipriano; Donato, Mariana Aragão Matos; Peres, Newton Gil; Souza, José Roberto Botelho De; Peixoto, Christina Alves

    2008-07-01

    Diethylcarbamazine (DEC) has been proven to be highly effective against lymphatic filariasis, although its effect on vertebrate cells remains uncertain. Mice Leydig cells after treatment with 200mg/kg of DEC for 12 days showed numerous lipid droplets, degenerated mitochondria, residual bodies and several giant whorl-like smooth endoplasmic reticulum, some of them encircling large lipids droplets. Treatment with lower dosages showed similar alterations on Leydig cells and the morphological effects decreased directly proportional to the drug concentration. Serum testosterone levels were significantly lower only in 200 mg/kg DEC-treated group when compared to the controls. However, no significant changes were observed in the pregnancy rates and offspring number of DEC-treated male-mated female mice in any doses studied. The results obtained in the present study are consistent with the hypothesis that DEC has some effects on mice Leydig cells, although they were not sufficient enough to interfere with the rodent fertility.

  8. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans.

    PubMed Central

    Brawner, D L; Cutler, J E

    1986-01-01

    Variability in the expression of two different cell surface carbohydrate determinants was examined with two agglutinating immunoglobulin M monoclonal antibodies (H9 and C6) and immunoelectron microscopy during growth of three strains of Candida albicans. A single strain of Candida parapsilosis did not express either antigen at any time during growth. Antigens were detected on the surface of C. albicans by agglutination tests with either H9 or C6 over a 48-h growth period. The difference in specificities of the monoclonal antibodies was demonstrated by Ouchterlony double-diffusion tests with solubilized antigens and by variabilities in the reactivity of the agglutinins among yeast strains. The antigenic determinants were isolated by specific immunoprecipitation and protease digestion and characterized by methods including high-pressure liquid chromatography, gas-liquid chromatography, and mass spectroscopy with both chemical and electron ionization. These determinants both contain mannose and glucose. In the case of antigen H9, an additional carbohydrate was detected with gas chromatography and mass spectroscopy. The location of antigens on individual cells was determined by indirect labeling of the determinants, first reacting cells with H9 or C6 followed by goat anti-mouse antibody conjugated with 20-nm colloidal gold particles. Transmission electron microscopy was used to examine cells. The antigens that were reactive with the monoclonal antibodies were associated with a flocculent surface layer. Expression of this layer and expression of the antigens is a dynamic process which is growth phase and strain dependent. The antigens were not expressed on very young cells and disappeared from the cell surface of most C. albicans strains with age. The use of monoclonal antibody to cell surface determinants may allow characterization of cell surface antigens of C. albicans and be helpful in establishing receptors which mediate adherence. Images PMID:3510174

  9. Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Takagi, Tomoko; Ishijima, Sanae A; Ochi, Hisako; Osumi, Masako

    2003-01-01

    Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.

  10. AN ULTRASTRUCTURAL STUDY OF VEGETATIVE CELL DIVISION IN OEDOGONIUM BORISIANUM(1) (2).

    PubMed

    Hill, G J; Machlis, L

    1968-12-01

    Vegetative cell division in Oedogonium borisianum is initiated by the formation of a 3-layered ring adjacent to the wall in the upper portion of the cell. This structure enlarges by the coalescence of vesicles. When the ring is fully developed, the parent wall splits adjacent to the ring, and the ring expands into a cylinder, which becomes the cuticle of the upper daughter cell. The lateral wall then forms between this cuticle and the plasmalemma of the cell. Concurrent with ring development and expansion, the nucleus migrates to a position in the center of the cell and karyokinesis occurs. Commencing with late telophase, evidence of transverse wall formation becomes apparent. The zone between the daughter nuclei contains a layer of microtubules in a plane parallel to the plane in which the transverse wall will develop. Subsequently a random coalescence of vesicles occurs along this plane. During the latter stages of this process, the ring expands and the plane of the transverse wall moves upward to the base of the ring cylinder. The completed transverse wall then fuses at is periphery with the newly formed lateral wall.

  11. Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia.

    PubMed

    Sekot, Gerhard; Posch, Gerald; Oh, Yoo Jin; Zayni, Sonja; Mayer, Harald F; Pum, Dietmar; Messner, Paul; Hinterdorfer, Peter; Schäffer, Christina

    2012-06-01

    The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.

  12. Ultrastructural pathology of a Chilean case of tropical spastic paraparesis/human T-cell lymphotropic type I-associated myelopathy (TSP/HAM).

    PubMed

    Liberski, P P; Buczyński, J; Yanagihara, R; Mora, C; Gibbs, C J; Gajdusek, C; Cartier, L; Verdugo, A; Araya, F; Castillo, L

    1999-01-01

    Human T-cell lymphotropic virus type I (HTLV-I), is the cause of endemic tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). Because TSP/HAM is not a fatal disease, the neuropathology of this disease, albeit relatively well understood, is based on the examination of just a few incidental cases. Previously, we demonstrated peculiar lamellated structures, called "multilamellar bodies" (MLB). In this report, we present the ultrastructural neuropathology of a TSP/HAM case from Chile, with further detailed descriptions of MLB. It is tempting to suggest that MLB may represent specific ultrastructural markers of TSP/HAM. The pathology of the anterior and posterior horns was similar and was comprised of axonal degeneration, accompanied by extensive astrocytic gliosis. Lymphocytic infiltration, particularly observed as "cuffs" around blood vessels, was scattered among other cellular elements. Ultrastructurally, myelin sheaths were relatively well preserved, and some demyelinated but not remyelinated fibers were observed. Moreover, axons with abnormal accumulations of neurofilaments, suggestive of axonal degeneration, were detected. Several axons contained Hirano bodies. In many samples, glial processes replaced most of the remaining neuropil. In a few specimens of the anterior and posterior horns of the spinal cord, MLB were observed. These structures consisted of stacks of 30 to 40 electron-dense lamellae, which were interrupted by narrow electron-lucent spaces. All of the lamellae were immersed within an amorphous substance of intermediate density. Neurons of the dorsal root ganglia were basically normal except for increased lipofuscin accumulation. As in the spinal cord, myelinated axons were well preserved, but a few were demyelinated and surrounded by concentric arrays of Schwann cell membranes. Also, axons of the dorsal roots accumulated increased number of neurofilaments. Mast cells and Schwann cells were increased in number, the latter

  13. Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

    PubMed

    Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

    2014-03-01

    One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

  14. Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

    PubMed

    Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

    2012-07-01

    Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

  15. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure.

    PubMed

    Coleman, Heather D; Yan, Jimmy; Mansfield, Shawn D

    2009-08-04

    Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba x grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in all lines, with increases of 2% to 6% over control levels, without influencing plant growth. The elevated concentration of cellulose was associated with an increase in cell wall crystallinity but did not alter secondary wall microfibril angle. This finding suggests that the observed increase in crystallinity is a function of altered carbon partitioning to cellulose biosynthesis rather than the result of tension wood formation. Furthermore, the augmented deposition of cellulose in the transgenic lines resulted in thicker xylem secondary cell wall and consequently improved wood density. These findings clearly implicate SuSy as a key regulator of sink strength in poplar trees and demonstrate the tight association of SuSy with cellulose synthesis and secondary wall formation.

  16. THE ULTRASTRUCTURE OF MAUTHNER CELL SYNAPSES AND NODES IN GOLDFISH BRAINS

    PubMed Central

    Robertson, J. David; Bodenheimer, Thomas S.; Stage, David E.

    1963-01-01

    An electron microscope study of goldfish Mauthner cells is reported.1 The cell is covered by a synaptic bed ∼ 5 µ thick containing unusual amounts of extracellular matrix material in which synapses and clear glia processes are implanted. The preterminal synaptic neurites are closely invested by an interwoven layer of filament-containing satellite cell processes. The axoplasm of the club endings contains oriented mitochondria, neurofilaments, neurotubules, and relatively few synaptic vesicles. That of the boutons terminaux contains many unoriented mitochondria and is packed with synaptic vesicles and some glycogen but no neurofilaments or neurotubules. The bare axons of club endings are surrounded by a moderately abundant layer of matrix material. The synaptic membrane complex (SMC) in cross-section shows segments of closure of the synaptic cleft ∼ 0.2 to 0.5 µ long. These alternate with desmosome-like regions of about the same length in which the gap widens to ∼ 150 A and contains a condensed central stratum of dense material. Here, there are also accumulations of dense material in pre- and postsynaptic neuroplasm. The boutons show no such differentiation and the extracellular matrix is largely excluded around them. The axon cap is a dense neuropil of interwoven neural and glial elements free of myelin. It is covered by a closely packed layer of glia cells. The findings are interpreted as suggestive of electrical transmission in the club endings. PMID:14069792

  17. Ultrastructural and Functional Effects of Lipopolysaccharide and Interleukin-2 on Human NK Cells

    DTIC Science & Technology

    1988-01-01

    various tii--e-periods. Eremin et al., 1980), felines (Herberman et al., 1980). Sorted Leu-ila+ NK cells were incubated with LPS canines (Krakowka, 1983...anti-Leu-11a and biotinyla- interferon treatment on vesicular stomatitis virus ted anti-Leu-7 antibodies. J Immunol Methods, 84: (vsv): release of

  18. Ablation of lens epithelial cells with a laser photolysis system: Histopathology, ultrastructure, and immunochemistry

    PubMed Central

    Mamalis, Nick; Grossniklaus, Hans E.; Waring, George O.; Werner, Liliana; Brubaker, Jacob; Davis, Don; Espandar, Ladan; Walker, Rudolf; Thyzel, Reinhardt

    2010-01-01

    PURPOSE To evaluate efficacy of a neodymium:YAG (Nd:YAG) laser photolysis system in removing lens epithelial cells (LECs) and characterize the effect of the laser on laminin and fibronectin involved in LEC adhesion and migration. METHODS Cadaver eyes were evaluated using the Miyake technique. The lenses were removed with phacoemulsification. The modified Nd:YAG laser was used to clean the LECs from the capsule. Only the fornix was cleaned in some eyes and the anterior subcapsular area in other eyes. Some areas were not treated and acted as controls. Standard irrigation/aspiration (I/A) removal of LECs was performed in additional eyes. The eyes were analyzed using light microscopy and immunohistochemical staining. RESULTS Histopathologic evaluation showed that the laser removed the LECs from the anterior lens capsule and from the fornix. Immunohistochemical staining showed fibronectin and laminin staining in the untreated areas that was absent in the treated areas. Standard I/A removal of the LECs showed absence of cells but persistent laminin and fibronectin. Electron microscopy showed epithelial cells in untreated areas with an absence of the LECs and debris in treated areas. CONCLUSIONS The laser photolysis system removed LECs from the anterior lens capsule and capsule fornix. Along with the cells, laminin, fibronectin, and cell debris remained in the untreated areas but were removed by the treatment. This treatment may be useful in preventing posterior capsule opacification. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes. PMID:20494774

  19. Comparison of the effects of difenacoum and brodifacoum on the ultrastructure of rat liver cells.

    PubMed

    Gül, Nursel; Yiğit, Nuri; Saygılı, Fulya; Demirel, Ebru; Geniş, Ceren

    2016-09-01

    We used transmission electron microscopy to examine the cytotoxic effects of the second-generation anticoagulant rodenticides difenacoum and brodifacoum on rat liver. A single dose of difenacoum or brodifacoum was administered to rats by gastric gavage and liver samples were taken after 24 h, four days or seven days. In the livers of rats treated with difenacoum for 24 h, hepatocytes typically showed increased numbers of lysosomes, as well as enlargement of both the perinuclear space and the cisternae of the rough endoplasmic reticulum (RER), while sinusoids were irregularly shaped and contained Kupffer cells. Similar irregularities occurred in brodifacoum-treated rats at the same time point, but additionally increased numbers of vacuoles, damaged mitochondrial cristae, and clumping of chromatin were observed in hepatocytes, and hemolysed erythrocytes were noted in the sinusoids. Comparable findings were made in each group of rats after four days. After seven days of difenacoum treatment, hepatocytes suffered loss of cytoplasmic material and mitochondrial shrinkage, while RER cisternae became discontinuous. In contrast, exposure to brodifacoum for seven days caused the formation of numerous vacuoles and lipid droplets, disordered mitochondrial morphology, chromatin clumping and invagination of the nuclear envelope in hepatocytes. Sinusoids in the livers of rodenticide-treated rats contained an accumulation of dense material, lipid droplets, cells with pycnotic nuclei and hemolysed erythrocytes. Overall, our results show that brodifacoum causes more severe effects in liver cells than difenacoum. Thus our microscopic data along with additional biochemical assays point to a severe effect of rodenticide on vertebrates.

  20. [The ultrastructure of Leydig cells under the influence of drinking mineral water and electromagnetic radiation under the stress conditions in the rats].

    PubMed

    Geniatulina, M S; Korolev, Yu N; Nikulina, L A

    2016-01-01

    The objective of the present study was elucidate the peculiar features of low-intensity electromagnetic radiation (LI EMR) and mineral water (MW) on the ultrastructure of rat Leydig cells under conditions of immobilization stress. The experiments were carried out on outbred male rats with the use of electron microscopy. It has been demonstrated that the prophylactic consumption of drinking sulfate-containing mineral water and the application low-intensity electromagnetic radiation (with the flow power density of 1 mcW/cm2 and frequency around 1,000 Hz) or the combination of these two modalities under conditions of immobilization stress reduced the degree of ultrastructural derangement in the rat Leydig cells and stimulated the development of regenerative processes. In the cases of the single-factor impact, drinking mineral water exerted more pronounced action than low-intensity electromagnetic radiation on mitochondrial regeneration. In case of the simultaneous application of the two factors their protective action on the Leydig cells was much more conspicuous than that of either of them applied alone. It is concluded that drinking sulfate-containing mineral water in combination with the application of low-intensity electromagnetic radiation enhances resistance of the rat Leydig cells to stress.

  1. Ultrastructural evidence for the presence of crystalline structures in pig vaginal epithelial cells.

    PubMed

    Gupta, P D; Sierralta, W D

    1994-01-01

    Terminally differentiated intermediate layers of vaginal epithelial cells from the late follicular phase of the pig show crystalline structures. Analysis of two dimensional images by transmission electron microscopy revealed that these structures are composed of rhomboid subunits. The longer side measures 26 nm whereas the smaller side measures 24 nm. The angles between the sides are 100 degrees and 80 degrees. These structures are not associated with any cellular structures and are not evident in other phases of the oestrous cycle. Structurally, they do not match with Reinke's crystals and are probably regulated by hormones.

  2. ARSENIC INDUCES SUSTAINED IMPAIRMENT OF SKELETAL MUSCLE AND MUSCLE PROGENITOR CELL ULTRASTRUCTURE AND BIOENERGETICS

    PubMed Central

    Fabrisia, Ambrosio; Elke, Brown; Donna, Stolz; Ricardo, Ferrari; Bret, Goodpaster; Bridget, Deasy; Giovanna, Distefano; Alexandra, Roperti; Amin, Cheikhi; Yesica, Garciafigueroa; Aaron, Barchowsky

    2014-01-01

    Over 4 million individuals in the US, and over 140 million individuals worldwide, are exposed daily to arsenic-contaminated drinking water. Human exposures can range from below the current limit of 10 µg/L to over 1 mg/L, with 100 µg/L promoting disease in a large portion of those exposed. Although increased attention has recently been paid to myopathy following arsenic exposure, the pathogenic mechanisms underlying clinical symptoms remain poorly understood. This study tested the hypothesis that arsenic induces lasting muscle mitochondrial dysfunction and impairs metabolism. When compared to non-exposed controls, mice exposed to drinking water containing 100µg/L arsenite for 5 weeks demonstrated impaired muscle function, mitochondrial myopathy, and altered oxygen consumption that were concomitant with increased mitochondrial fusion gene transcription. There was no difference in levels of inorganic arsenic or its mononomethyl- and dimethyl- metabolites between controls and exposed muscles, confirming that arsenic does not accumulate in muscle. Nevertheless, muscle progenitor cells isolated from exposed mice recapitulated the aberrant myofiber phenotype and were more resistant to oxidative stress, generated more reactive oxygen species, and displayed autophagic mitochondrial morphology, as compared to cells isolated from non-exposed mice. These pathological changes from a possible maladaptive oxidative stress response provide insight into declines in muscle functioning caused by exposure to this common environmental contaminant. PMID:24960579

  3. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    DTIC Science & Technology

    2015-10-01

    Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma Cell Differentiation PRINCIPAL INVESTIGATOR: Dr. Liqin Du...inducing miRNA, miR- 449a. We examined the differentiation-inducing function of miR-449a in multiple neuroblastoma cell lines. We have demonstrated that...miR-449a functions as an inducer of cell differentiation in neuroblastoma cell lines with distinct genetic backgrounds, including the MYCN

  4. Ultra-structure and histochemistry of digestive cells of Podisus nigrispinus (Hemiptera: Pentatomidae) fed with prey reared on bt-cotton.

    PubMed

    da Cunha, Franklin M; Caetano, Flávio H; Wanderley-Teixeira, Valéria; Torres, Jorge B; Teixeira, Alvaro A C; Alves, Luiz C

    2012-02-01

    The toxic effects of Bt proteins from the body of the prey/host to predators and parasitoids have been investigated as a route of impact on non-target insects of Bt crops. This research aimed to analyze ultrastructural changes and histochemistry in digestive cells of the middle region of the midgut of Podisus nigrispinus fed, since its second instar, with Spodoptera frugiperda reared on Bt cotton variety Acala90 B, which expresses the toxin Cry 1Ac, and its non-Bt isoline Acala 90. Fragments of the midgut of P. nigrispinus were analyzed by electron microscopy. For the histochemical analysis, Bromophenol Blue, Periodic Acid Schiff von Kossa, Alcian Blue pH 2.5 and Sudan Black were used. The Cry1Ac toxin of Bt cotton ingested by S. frugiperda promotes a disorganization in the perimicrovillar matrix of P. nigrispinus (third trophic level), thus generating ultrastructural changes in the digestive cells, as elongation of microvilli, presence of spherocrystals and granules of different electron densities, in addition to altering the distribution pattern of glycogen, lipids and calcium of these cells in the median region of the midgut. Thus, we conclude that species moderately susceptible to Cry1Ac toxin, such as S. frugiperda, can acquire this toxin and expose it to P. nigrispinus, which can interfere with your ability to predation.

  5. Development and ultrastructure of Cystoisospora canis Nemeséri, 1959 (syn, Isospora canis) monozoic cysts in two noncanine cell lines.

    PubMed

    Mitchell, Sheila M; Zajac, Anne M; Lindsay, David S

    2009-08-01

    Cystoisospora canis is a coccidial parasite of the intestinal tract that can cause severe disease in dogs. Clinical signs include watery diarrhea, vomiting, fever, and weight loss. Extraintestinal stages of Cystoisospora spp. have been demonstrated in the mesenteric lymph nodes of paratenic hosts. Information on the biology of extraintestinal stages of canine Cystoisospora species is limited. The current study examined the development of C. canis in 2 noncanine cell lines and the ultrastructure of the monozoic cysts that formed. Monolayers of bovine turbinate cells and African green monkey kidney cells were grown on coverslips and inoculated with excysted C. canis sporozoites. Coverslips were collected on various days and fixed and stained for light microscopy (LM) or transmission electron microscopy (TEM). A single, centrally located, slightly crescent-shaped sporozoite surrounded by a thick cyst wall within a parasitophorous vacuole was observed with the use of LM and TEM. No division and no multinucleated stages were observed with either LM or TEM. With TEM, typical organelles of sporozoites were observed, such as rhoptries, dense granules, a crystalloid body, polysaccharide granules, and a conoid. The structure and ultrastructure of C. canis monozoic cysts produced in vitro are similar to extraintestinal cysts of other Cystoisospora species in experimentally infected animals and those of Cystoisospora belli observed in immunocompromised humans. This is the first study that fully demonstrates in vitro the development of what structurally resemble extraintestinal cysts of a Cystoisospora spp.

  6. Effects of Electroacupuncture on Interstitial Cells of Cajal (ICC) Ultrastructure and Connexin 43 Protein Expression in the Gastrointestinal Tract of Functional Dyspepsia (FD) Rats

    PubMed Central

    Zhang, Guoshan; Xie, Shen; Hu, Wei; Liu, Yuer; Liu, Mailan; Liu, Mi; Chang, Xiaorong

    2016-01-01

    Background Gastrointestinal motility disorder is the main clinical manifestation in functional dyspepsia (FD) patients. Electroacupuncture is effective in improving gastrointestinal motility disorder in FD; however, the underlying mechanism remains unclear. It has been demonstrated that interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract, and the pacemaker potential is transmitted to nearby cells through gap junctions between ICC or ICC and the smooth muscle. Therefore, this study aimed to assess the effects of electroacupuncture on ICC ultrastructure and expression of the gap junction protein connexin 43 (Cx43) in FD rats. Material/Methods The animals were randomized into 3 groups: control, model, and electroacupuncture. Electroacupuncture was applied at Zusanli (ST36) in the electroacupuncture group daily for 10 days, while no electroacupuncture was applied to model group animals. Results Ultrastructure of ICC recovered normally in gastric antrum and small intestine specimens was improved, with Cx43 expression levels in these tissues significantly increased in the electroacupuncture group compared with the model group. Conclusions These findings indicated that electroacupuncture is effective in alleviating ICC damage and reduces Cx43 levels in FD rats, and suggest that ICC and Cx43 are involved in electroacupuncture treatment in rats with FD to improve gastrointestinal motility disorders. PMID:27297942

  7. Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, 'Candidatus Magnetoglobus multicellularis'.

    PubMed

    Silva, Karen Tavares; Abreu, Fernanda; Keim, Carolina N; Farina, Marcos; Lins, Ulysses

    2008-12-01

    Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named 'Candidatus Magnetoglobus multicellularis'. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the nature of the granules. Besides magnetosomes, three types of inclusions were observed: small (mean diameter=124 nm) polyhydroxyalkanoate-like (PHA) granules, large (diameters ranging from 0.11 to 2.5 microm) non-PHA lipid granules, and rare phosphorus-rich granules, which probably correspond to polyphosphate bodies. The PHA granules were rounded in projection, non-reactive with OsO(4), and suffered the typical plastic deformation of PHAs after freeze fracturing. The nature of the large granules, consisting of round globular structures (mean diameter=0.76 microm), was classified as non-PHA based on the following data: (a) multilayered structure in freeze-fracture electron microscopy, typical of non-PHA lipids; (b) Nile blue fluorescence imaging detected non-PHA lipids; (c) imidazole buffered osmium tetroxide and ruthenium red cytochemistry stained the globules, which appeared as electron-dense granules instead of electron lucent as PHAs do. Most likely, 'Candidatus Magnetoglobus multicellularis' stores carbon mainly as unusual lipid granules, together with smaller amounts of PHAs.

  8. Formation of Essential Ultrastructural Interface between Cultured Hippocampal Cells and Gold Mushroom-Shaped MEA- Toward "IN-CELL" Recordings from Vertebrate Neurons.

    PubMed

    Fendyur, Anna; Mazurski, Noa; Shappir, Joseph; Spira, Micha E

    2011-01-01

    Using cultured Aplysia neurons we recently reported on the development of a novel approach in which an extracellular, non-invasive multi-electrode-array system provides multisite, attenuated, intracellular recordings of subthreshold synaptic potentials, and action potentials (APs), the so called "IN-CELL" recording configuration (to differentiate it from intracellular recordings). Because of its non-invasive nature, the configuration can be used for long term semi intracellular electrophysiological monitoring of APs and synaptic potentials. Three principals converge to generate the IN-CELL configuration: (a) engulfment of approximately 1 μm size gold mushroom-shaped microelectrodes (gMμE) by the neurons, (b) formation of high seal resistance between the cell's plasma membrane and the engulfed gMμE, and (c), autonomous localized increased conductance of the membrane patch facing the gMμE. Using dissociated rat hippocampal cultures we report here that the necessary morphological and ultrastructural relationships to generate the IN-CELL recording configuration are formed between hippocampal cells and the gMμEs. Interestingly, even <1 μm thin branches expand and engulf the gMμE structures. Recordings of spontaneous electrical activity revealed fast ∼2 ms, 0.04-0.75 mV positive monophasic APs (FPMP). We propose that the FPMP are attenuated APs generated by neurons that engulf gMμEs. Computer simulations of analog electrical circuits depicting the cell-gMμE configuration point out the parameters that should be altered to improve the neuron-gMμE electrical coupling.

  9. Ultrastructure of germ cells, Sertoli cells and mitochondria during spermatogenesis in mature testis of the Chinese Taihang black goats (Capra hircus).

    PubMed

    Shi, Liguang; Xun, Wenjuan; Zhou, Hanlin; Hou, Guanyu; Yue, Wenbin; Zhang, Chunxiang; Ren, Youshe; Yang, Rujie

    2013-07-01

    The objective of this study was to describe the ultrastructure of germ cells, Sertoli cells and mitochondria in mature testis of the Chinese Taihang black goat. The characteristics of germ cell nucleus and mitochondria changing during spermatogenesis were investigated by transmission electron microscopy (TEM). The results showed that the spermatogonium was elliptical, and its nucleus was about 4-5 μm. The round mitochondria can be observed throughout the cytoplasm around the nucleus. Small patches of heterochromatin were distributed throughout the nucleus. Spermatocyte was oval-shaped with a nucleus of about 4-4.5 μm in diameter. The heterochromatin began to attach to the inner surface of the nuclear membrane. Spermatid was about 4 μm and oval in shape. Its nucleus was oval or round and approximately 2-3 μm in diameter. The borderline between nucleus membrane and karyoplasm was distinct. During spermiogenesis, spermatid nucleus was condensed and elongated, and chromatin reached the highest condensation in the mature spermatozoon. The mid-piece was surrounded by mitochondria at the neck region. The sperm tail showed the typical "9+2″ structure, contained axoneme and central singlet microtubules. The nuclei of the Sertoli cells were irregular shaped and showed indentations in the membrane. In the mature testes of goat bucks, abundant mitochondria were around the germ cells and Sertoli cells. The scattered mitochondria were aggregated around the base of the flagellum (axoneme) during the spermatid differentiation stage. In conclusion, the present study showed that the spermatogenic process of Taihang black goat followed the pattern of mammals with some specific.

  10. Some ultrastructural information on intact, living bacterial cells and related cell-wall fragments as given by FTIR

    NASA Astrophysics Data System (ADS)

    Naumann, D.

    1984-05-01

    Living bacterial cells of Staphylococcus aureus have been measured from aqueous suspensions taking advantage of the solvent subtraction capabilities of FTIR. All spectral features, between 1800-800 cm -1, of the intact cells could be measured with a reproducibility of better than ±5% when applying strict metabolic control of cell growth and a highly standardized experimental procedure prior to IR measurements. IR bands near 1745, 1656, 1547, 1240 and 1200-1000 cm -1were tentatively assigned to: CO stretching of ester groups, amide I and amide II bands of the various peptides and proteins, asymmetric stretching of phosphate groups and complex vibrational modes resulting from polysaccharidal compounds, respectively. Absorbance subtraction of IR spectra of different intact baterial cells and cell-wall preparations yielded reasonable results on structural variations accompanying: (i) cell growth, (ii) use of different growth media, (iii) chemical treatment of cells and (iv) biochemical isolation processes of cell walls from the intact cells.

  11. Functionally impaired, hypertrophic ECL cells accumulate vacuoles and lipofuscin bodies. An ultrastructural study of ECL cells isolated from hypergastrinemic rats.

    PubMed

    Zhao, C M; Bakke, I; Tostrup-Skogaker, N; Waldum, H L; Håkanson, R; Chen, D

    2001-03-01

    ECL cells in the oxyntic mucosa of stomach control gastric acid secretion by mobilizing histamine in response to gastrin. They respond to gastrin also with hypertrophy and hyperplasia. ECL cells exhibit functional impairment upon long-term gastrin stimulation. The impairment is manifested in a gradual decline of the activity of the histamine-forming enzyme per individual ECL cell and in a failure of gastrin to mobilize histamine. The mechanism behind this impairment is unknown. In the present study, rats were treated with the proton pump inhibitor pantoprazole for 45 days to induce sustained hypergastrinemia. The ECL cells were isolated from normogastrinemic and hypergastrinemic rats and size-separated from other mucosal cells by the elutriation technique. The total ECL cell number was twofold higher in hypergastrinemic rats than in normogastrinemic rats, and most of the cells appeared in elutriation fractions where large cells predominate. The ECL cells of the different fractions were analyzed by quantitative electron microscopy. Normal-sized ECL cells from hypergastrinemic rats displayed a reduced number of secretory vesicles (probably because of degranulation) compared with normal-sized ECL cells from normogastrinemic rats. Hypertrophic ECL cells from hypergastrinemic rats had an unchanged number of secretory vesicles, supporting the view that such cells fail to respond to gastrin with degranulation. Although both normal-sized and hypertrophic ECL cells from hypergastrinemic rats contained vacuoles, those in the hypertrophic ECL cells were larger and more numerous. In addition, hypertrophic ECL cells were found to contain numerous, prominent lipofuscin bodies which are the presumed end product of crinophagia. Conceivably therefore, large vacuoles and lipofuscin bodies cause functional impairment of the hypertrophic ECL cells.

  12. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

    PubMed

    Gupta, Prakash K; Godec, Jernej; Wolski, David; Adland, Emily; Yates, Kathleen; Pauken, Kristen E; Cosgrove, Cormac; Ledderose, Carola; Junger, Wolfgang G; Robson, Simon C; Wherry, E John; Alter, Galit; Goulder, Philip J R; Klenerman, Paul; Sharpe, Arlene H; Lauer, Georg M; Haining, W Nicholas

    2015-10-01

    Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

  13. Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reappraisal of clear cell sarcoma-like tumors of the gastrointestinal tract.

    PubMed

    Stockman, David L; Miettinen, Markku; Suster, Saul; Spagnolo, Dominic; Dominguez-Malagon, Hugo; Hornick, Jason L; Adsay, Volkan; Chou, Pauline M; Amanuel, Benhur; Vantuinen, Peter; Zambrano, Eduardo V

    2012-06-01

    The clinical, histologic, immunophenotypic, ultrastructural, and molecular features of a distinctive gastrointestinal tumor are described. Sixteen patients, 8 women and 8 men aged 17 to 77 years (mean age, 42 y; 63% less than 40 y) presented with abdominal pain, intestinal obstruction, and an abdominal mass. Mean tumor size was 5.2 cm (range, 2.4 to 15.0 cm). The tumors arose in the small bowel (10), stomach (4), and colon (2) and were histologically characterized by a sheet-like or nested population of epithelioid or oval-to-spindle cells with small nucleoli and scattered mitoses. Five cases showed focal clearing of the cytoplasm. Scattered osteoclast-type multinucleated giant cells were present in 8 cases. The tumor cells were positive for S-100 protein, SOX10, and vimentin in 100% of cases, for CD56 in 70%, for synaptophysin in 56%, for NB84 in 50%, for NSE in 45%, and for neurofilament protein in 14% of cases. All cases tested were negative for specific melanocytic, gastrointestinal stromal tumors, epithelial, and myoid markers. Ultrastructural examination of 5 cases showed features of primitive neuroectodermal cells with clear secretory vesicles, dense-core granules, occasional gap junctions, and no evidence of melanogenesis. EWSR1 gene rearrangement was assessed by fluorescence in situ hybridization in 14 cases. Twelve cases (86%) showed split EWSR1 signal consistent with a chromosomal translocation involving EWSR1. One case showed extra intact signals, indicating that the nuclei possessed either extra copies of the EWSR1 gene or chromosome 22 polysomy. Only 1 case showed no involvement of the EWSR1 gene. Six cases demonstrated rearrangement of the partner fusion gene ATF1 (46%), and 3 showed rearrangement of CREB1 (23%); 2 cases lacked rearrangement of either partner gene. Clinical follow-up was available in 12 patients and ranged from 1.5 to 106 months. Six patients died of their tumors (mean survival, 32 mo; 83% less than 24 mo). At last follow-up, 4

  14. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

    PubMed

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

    2016-10-31

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

  15. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

    PubMed Central

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-ichiro

    2016-01-01

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules. PMID:27796315

  16. The ultrastructure of the muscle coat of human gastro-oesophageal junction, with special reference to "interstitial cells of Cajal".

    PubMed

    Faussone-Pellegrini, Maria-Simonetta; Cortesini, Camillo; Romagnoli, Paolo

    2013-01-01

    innervated by varicose nerve fibers that were densely packed with synaptic vesicles; many close junctions to nerve endings were also detected. These morphological data lead us to assume that the interstitial cells demonstrated by the electron microscope do not correspond to the cells initially identified by Cajal and cannot even be considered connective tissue cells. We propose that they are specialized smooth muscle cells that are involved in generating spontaneous, myogenic electrical activity in the gastrointestinal tract.

  17. Comparative study of effects of magnesium and taurine on electrical parameters of natural and artificial membranes. VIII. Effect on the ultrastructure of human amniotic epithelial cells.

    PubMed

    Guiet-Bara, A; Bara, M; Durlach, J

    1991-03-01

    The ultrastructure of human amniotic epithelial cells from normal pregnancies, at term, was studied using transmission electron microscopy. The results were analysed by a stereological method which indicates the ratio between the volume of the intercellular space (R1, the microvilli (R2), and the podocytes (R3) versus the cell volume. At low concentration (2 mM), MgCl2 decreased R1 and R3 and had no significant effect on R2. In contrast, taurine (2 mM) increased R1 and had no significant effect on R2 and R3. There is no vicarious action between Mg and taurine. These data are in contrast to the results obtained after electrophysiological studies, which indicates that the structural targets for Mg and taurine are different from the targets responsible for ionic transfer.

  18. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

    SciTech Connect

    Kleinow, Tatjana; Tanwir, Fariha; Kocher, Cornelia; Krenz, Bjoern; Wege, Christina; Jeske, Holger

    2009-09-01

    The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

  19. Dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques.

    PubMed

    Gangodkar, Shobha; Jain, Preksha; Dixit, Nishikant; Ghosh, Kanjaksha; Basu, Atanu

    2010-01-01

    The biogenesis events and formation of dengue virus (DENV) in the infected host cells remain incompletely understood. In the present study, we examined the ultrastructural changes associated with DENV-2 replication in three susceptible host cells, C6/36, Vero and SK Hep1, a cell line of human endothelial origin, using transmission electron microscopy, whole-mount grid-cell culture techniques and electron tomography (ET). The prominent feature in C6/36 cells was the formation of large perinuclear vacuoles with mature DENV particles, and on-grid whole-mount examination of the infected Vero cells showed different forms of DENV core structures associated with cellular membranes within 48 h after infection. Distinct multivesicular structures and prominent autophagic vesicles were seen in the infected SK Hep1 cells when compared with the other two cell lines. ET showed the three-dimensional organization of these vesicles as a continuous system. This is the first report of ET-based analysis of DENV-2 replication in a human endothelial cell line. These results further emphasizes the strong role played by intracellular host membranes-virus interactions in the biogenesis of DENV and strongly argues for the possibility of targeting compounds to block such structure formation as key anti-dengue agents.

  20. Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

    PubMed

    Auer, George K; Lee, Timothy K; Rajendram, Manohary; Cesar, Spencer; Miguel, Amanda; Huang, Kerwyn Casey; Weibel, Douglas B

    2016-06-22

    Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

  1. Phenotypic Approaches to Identify Inhibitors of B Cell Activation

    PubMed Central

    Kim, Suzie; Wiener, Jake; Rao, Navin L.; Milla, Marcos E.; DiSepio, Daniel

    2015-01-01

    An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin’s lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton’s tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt’s lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation. PMID:25948491

  2. Ultrastructural studies of unstable angina in living man

    SciTech Connect

    Gotlieb, A.I.; Freeman, M.R.; Salerno, T.A.; Lichtenstein, S.V.; Armstrong, P.W. )

    1991-01-01

    Nineteen patients with refractory unstable angina who were undergoing aortocoronary bypass were studied to assess the extent of platelet aggregation present in the microvasculature. Ultrastructural findings on the morphology of cardiac muscle and microvasculature were correlated with the findings on coronary angiograms and thallium scans. There were no significant correlations. The presence of platelet aggregates was identified in four biopsies, two of which had thrombus by angiographic criteria. Biopsy in areas with thallium defects revealed an increased prevalence of white blood cells without acute myocardial infarction. This study confirms the presence of platelet aggregates in patients with unstable angina, albeit at a reduced frequency when compared with autopsy studies.

  3. Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation.

    PubMed

    Talbot, N C; Garrett, W M

    2001-09-01

    Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.

  4. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  5. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  6. Some physiological properties of identified mammalian neuroglial cells

    PubMed Central

    Dennis, M. J.; Gerschenfeld, H. M.

    1969-01-01

    Mammalian glial cells were identified and studied in the optic nerves of anaesthetized rats. Cells with membrane potentials of 77-85 mV were located in the optic nerve with capillary micropipettes. These were shown to be neuroglia by iontophoretic injection of a fluorescent dye through the recording electrode, followed by histological verification of the location of the dye. No distinction was made between astroglia and oligodendroglia. Neuroglial cells gave no impulse activity. Their membrane potential was studied in isolated optic nerves by varying the ionic composition of the bathing fluid. The glial membrane potential depends predominantly on a transmembrane gradient of potassium ions. ImagesFig. 1Fig. 2 PMID:5821876

  7. Reptilian spermatogenesis: A histological and ultrastructural perspective.

    PubMed

    Gribbins, Kevin M

    2011-07-01

    Until recently, the histology and ultrastructural events of spermatogenesis in reptiles were relatively unknown. Most of the available morphological information focuses on specific stages of spermatogenesis, spermiogenesis, and/or of the mature spermatozoa. No study to date has provided complete ultrastructural information on the early events of spermatogenesis, proliferation and meiosis in class Reptilia. Furthermore, no comprehensive data set exists that describes the ultrastructure of the entire ontogenic progression of germ cells through the phases of reptilian spermatogenesis (mitosis, meiosis and spermiogenesis). The purpose of this review is to provide an ultrastructural and histological atlas of spermatogenesis in reptiles. The morphological details provided here are the first of their kind and can hopefully provide histological information on spermatogenesis that can be compared to that already known for anamniotes (fish and amphibians), birds and mammals. The data supplied in this review will provide a basic model that can be utilized for the study of sperm development in other reptiles. The use of such an atlas will hopefully stimulate more interest in collecting histological and ultrastructural data sets on spermatogenesis that may play important roles in future nontraditional phylogenetic analyses and histopathological studies in reptiles.

  8. [Effect of fucoidan on the ultrastructure of mesophyll cells of Datura stramonium L. and accumulation of potato virus X in them].

    PubMed

    Lapshina, L A; Reunov, A V; Nagorskaia, V P; Zviagintseva, T N; Shevchenko, N M

    2009-01-01

    Influence of fucoidan from brown alga Fucus evanescens C. Ag. on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves was studied. It as been shown that 24 h after the treatment of the leaves with fucoidan and following infection of them with PVX the accumulation of virus particles in infected cells during early infection period was substantially less than that in untreated control. Using ultrastructure-morphometric analysis, it has been established that fucoidan treatment increases at protein-synthesizing capability of cells (nucleolus dimension, amount of mitochondria and rough endoplasmic reticulum membranes become increased). At the same time, the fucoidan treatment causes some activation of lytic compartment which leads to destruction of virus particles and, therefore, might be considered as one of fucoidan-dependent protective mechanisms limiting virus accumulation in cells. Fucoidan stimulation of the formation of PVX-specific laminated structures capable of virus particles binding is possibly another induced antiviral cell mechanism, preventing from virus reproduction and transposition.

  9. Annual changes in the number, testosterone content and ultrastructure of glandular tissue cells of the testis in the marbled newt Triturus marmoratus.

    PubMed Central

    Fraile, B; Paniagua, R; Rodríguez, M C; Sáez, F J; Jimenez, A

    1989-01-01

    The testes of 8 specimens of Triturus marmoratus were collected during each month of 1987 and processed for electron microscopy and light microscopy demonstration of testosterone (T) following the ABC (avidin-biotin peroxidase complex) method. According to their staining affinity for anti-T antibodies, the glandular tissue cells were classified as T-, T+, T++, and T and the annual changes in the numbers of these cell populations, as well as in the volume occupied by the glandular tissue, were calculated. The volume occupied by the glandular tissue increases from September to December; it begins to decrease in April and disappears from June to August. The glandular tissue is formed from the interstitial cells that surround the lobules containing differentiating germ cells. During the spermatogenic process, the interstitial cells do not show staining affinity for anti-T antibodies. In August-September, the interstitial cells around the lobules that have completed spermatogenesis become positively stained (T+) and form the glandular tissue when the spermatozoa leave the testis. The numbers of intensely stained cells in the glandular tissue (T++ and T ) increase from September to November; begin to decrease in December; disappear in January-February; increase again in March and decrease again in April until they disappear in June-September. The interstitial cells, before their transformation in glandular tissue, are ultrastructurally similar to fibroblasts. After their transformation these cells increase in size and develop abundant smooth endoplasmic reticulum, mitochondria with tubular cristae and lipid droplets. This morphological pattern is maintained in the glandular tissue from September to April in spite of the changes in staining affinity during this period. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 PMID:2630543

  10. The ultrastructural characterization of mitochondria-rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus).

    PubMed

    Yang, Sheng-Hui; Tsai, Jeng-Dau; Kang, Chao-Kai; Yang, Wen-Kai; Kung, Hsiu-Ni; Lee, Tsung-Han

    2017-03-01

    The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos) and the Mozambique tilapia (Oreochromis mossambicus), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae-free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)-rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt-rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)-acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron-dense, and were often vacuolated. The Mt in the PSCs of FW-acclimated milkfish and tilapia were larger and more electron-dense than those of their SW-acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo-osmotic environment. Conversely, the PSCs of tilapia were periodic acid-Schiff (PAS)-positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity-dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae-free and embedded pseudobranchs, whereas the PAS-positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol

  11. Alterations of leaf cell ultrastructures and AFLP DNA profiles in Earth-grown tomato plants propagated from long-term six years Mir-flown seeds

    NASA Astrophysics Data System (ADS)

    Liu, Min; Xue, Huai; Pan, Yi; Zhang, Chunhua; Lu, Jinying

    Leaf cell ultrastructures and DNA variations in the firstand the second-generation of Earthgrown tomato (Lycopersicon esculentun Mill) plants that had been endured a long-term six years spaceflight in the Mir were compared to their ground-based control plants, under observations with a Transmission Electron Microscope and the Amplification Fragment Length Polymorphism (AFLP) analysis. For alterations in the morphological ultrastructures, one plant among the 11 first-generation plants generated from 30 Mir-flown seeds had a three-layered palisade cell structure, while other 10 first-generation plants and all ground-based controls had one-layered palisade cell structure in leaves. Starch grains were larger and in clusters, numbers of starch grains increased in the chloroplasts in the Mir-flown plants. Leaf cells became contracted and deformed, and cell shape patterns were different in the Mir-flown plants. For the leaf genomic DNA alterations, 34 DNA bands were polymorphic with a 1.32% polymorphism among 2582 DNA bands in the first-generation Mir-flown plants. Band types in the spaceflight treated plants were also different from those in the ground-based control. Of 11 survived first-generation plants, 7 spaceflight treated plants (Plant Nos. 1-6 and No. 9) had a same 7 polymorphic bands and a same 0.27%DNA mutation. The DNA mutation rate was greatest in Plants No.10 and No.7 (0.90% and 0.94%), less in Plant No.11 (0.31%) and least in Plant No.8 (0.20%). For the 38 send-generation plants propagated from the No. 5 Mir-flown seed, 6 DNA bands were polymorphic with a 0.23% polymorphism among 2564 amplified DNA bands. Among those 38 second-generation plants amplified by primer pair (E4: ACC, M8: CTT), one DNA band disappeared in 29 second-generation plants and in the original Mir-flown No. 5 plant, compared to the ground-base controls. Among the 38 second-generation plants generated from the Mir-flown No. 5 seed, the DNA band types of 29 second-generation plants were

  12. The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy

    PubMed Central

    Vincent, Amy E.; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M.; McFarland, Robert; Gorman, Grainne S.; Taylor, Robert W.; Turnbull, Doug M.; Picard, Martin

    2016-01-01

    Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

  13. A pneumatically-driven microfluidic system for size-tunable generation of uniform cell-encapsulating collagen microbeads with the ultrastructure similar to native collagen.

    PubMed

    Huang, Song-Bin; Chang, Yu-Han; Lee, Hsin-Chieh; Tsai, Shiao-Wen; Wu, Min-Hsien

    2014-06-01

    This study reports a microfluidic system for high throughput, uniform, and size-tunable generation of cell-containing collagen microbeads. The principle is based on two pneumatically-driven mechanisms to achieve multi-channel mixture suspension transportation, and to actuate the spotting actions of micro-vibrators that continuously generate tiny collagen micro-droplets into a thin oil layer and then a sterile Pluronic® F127 surfactant solution located below. The temporarily formed collagen microdroplets are then thermally gelatinized. By regulating the feeding rate of cells/collagen suspension, and the spotting frequency of micro-vibrator, the size of the collagen microbeads can be manipulated. One of the key technical features is its capability to generate uniform collagen microbeads (coefficient of variation: 5.4-8.6 %) with sizes ranging from 73.9 to 349.3 μm in diameter. This is currently difficult to achieve using the existing methods particularly the generation of cell-encapsulating collagen microbeads with diameter less than 100 μm. Another advantageous trait is that the ultrastructure of the generated collagen microbeads is similar to that found in native collagen. In this study, moreover, the use of the proposed device for the microencapsulation of 3T3 cells in collagen microbeads has been successfully demonstrated showing that the encapsulated cells maintained high cell viability (96 ± 2 %). Furthermore, a reasonable proliferative capability of the encapsulated cells was observed during 7 days culture. As a whole, the proposed device has opened up a new route to generate cell-containing collagen microbeads, which is found particularly meaningful for biomedical applications.

  14. Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot.

    PubMed Central

    McCabe, P. F.; Valentine, T. A.; Forsberg, L. S.; Pennell, R. I.

    1997-01-01

    Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall. PMID:12237357

  15. [Effect of different water-soluble forms of the fullerene C60 on the metabolic activity and ultra-structure of cells in culture].

    PubMed

    Eropkina, E M; Il'inskaia, E V; Litasova, E V; Eropkin, M Iu; Piotrovskiĭ, L B; Dumpis, M A; Kiselev, O I

    2012-01-01

    In view of contradictory data on the toxicity of fullerenes for live organisms we studied the effect of water-soluble complexes of C60 with N-polyvivyl-pirrolidone (C60/PVP) and gamma-cyclodextrine (C60/gamma-CD) on MA-104 cells in culture. Both complexes proved to be non-toxic for cultured cells in the dark in wide range of concentrations. Both complexes provoke changes of cellular ultra-structure which reflect the enhancement of metabolic activity. At the same time only the exposition with the complex C60/PVP leads to the essential growth of number and size of mitochondria. However, the effect of two studied water-soluble forms of C60 under intensive UV-irradiation of cells proved to be opposite: C60/PVP had a cyto-protective action while C60/gamma-CD caused a significant growth of photo-toxicity. Possible reasons of the differences in the action of different forms of C60 on living organisms are discussed.

  16. Forecasting hailfall using parameters for convective cells identified by radar

    NASA Astrophysics Data System (ADS)

    Rigo, Tomeu; Carmen Llasat, M.

    2016-03-01

    The main goal of the present paper is to propose some new criteria that will improve the diagnosis for hail at the surface in real-time, so that they can be applied to surveillance tasks and for nowcasting purposes. The criteria are based on a better knowledge of convective cells that produce hail during their life cycle and better distinguishing between these cells and cells that do not produce hail on the surface. The work focused on a region in the northeast of the Iberian Peninsula, selecting hail events that occurred in the 2004-2012 period and using the information provided by the Meteorological Service of Catalonia's weather radar network. The methodology deals with the analysis of the level of reflectivity associated with the maximum values, which can be considered as the core of the convective vertical development. The chosen radar parameters are operative and they take into consideration the following: the reflectivity, the vertically integrated liquid, the highest altitude at which radar echoes have been observed over a determined reflectivity threshold, as well as the direction and the duration of the convective cells. This work aims to complement all the previous work carried out by different authors, in order to better identify hail in the chosen region.

  17. [Ultrastructural observation of morphologically abnormal sperm: Advances in studies and application].

    PubMed

    Wang, Jia-xiong; Shi, Yi-chao; Yang, Shen-min

    2016-01-01

    Sperm ultrastructural abnormalities are often associated with sperm motility, the integrity of genetic material, and the fertilization potential. The investigation of sperm ultrastructural abnormalities is based on the evolution of microscopy techniques. In his paper, we review the improvement of the microscopy techniques and the ultrastructure of several specific morphological defects and he apoptotic spermatogenic cells in order to expound the significance of sperm ultrastructural observation in clinical practice. We deem it necessary to analyze the sperm ultrastructure before exploring the pathology and adopting assisted reproductive technology for some special patients with teratozoospermia.

  18. Impact of drying on wood ultrastructure: similarities in cell wall alteration between native wood and isolated wood-based fibers.

    PubMed

    Suchy, Miro; Kontturi, Eero; Vuorinen, Tapani

    2010-08-09

    Ultrastructural alterations of fresh wood caused by initial drying were compared to changes incurred during drying of never-dried wood pulp fibers of different macromolecular composition. Drying induced inaccessibility of a native wood sample exhibited remarkable similarity to wood pulp samples of different lignin contents. The results suggest that the supramolecular rearrangements in native wood matrix upon dehydration are qualitatively identical to the well-known changes occurring in pulp fibers after drying, although the changes are considerably different in quantity. The alterations were observed and quantified by monitoring the conversion of accessible deuterium exchanged OH groups in fresh wood and wood pulp fibers to inaccessible, reprotonation resistant OD groups during drying. The deuteration/FT-IR measurements correlated well with the water retention measurement of the pulp samples. Irreversible reduction of water retention due to the supramolecular changes implies reduced accessibility of wood polymers in various chemical and mechanical treatments, such as enzymatic conversion of biomass or preparation of cellulosic nano-objects for diverse applications.

  19. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6

    PubMed Central

    Wang, Yanhua; Wang, Guangxiang; Cai, Jian Ping

    2016-01-01

    The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents. PMID:27658594

  20. Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry.

    PubMed

    Ozawa, Hitoshi; Han, Fang; Kawata, Mitsuhiro

    2004-12-01

    Growth hormone (GH) cells in the rat anterior pituitary have been morphologically classified into three subtypes: type I (mature) containing large secretory granules about 350 nm in diameter, type II (intermediate) containing a mixture of large and small granules, and type III (immature) containing small granules about 150 nm in diameter. However, the functional implications of morphological heterogeneity, especially the different sensitivities to growth hormone-releasing hormone (GRH) under different corticosteroid conditions have not been elucidated to date. In the present study, by application of microwave irradiation (MWI) for fixation and immunocytochemistry, new findings of the exocytotic response have been revealed among the subsets of GH cells following adrenalectomy (ADX), corticosterone treatment and/or GRH treatment. The MWI gave effective results for fixation, especially for the permeability of the fixative, and showed good results for immunoelectron microscopy using the protein-A gold method. Moreover, the use of MWI greatly shortened the fixation, processing and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The number of exocytotic figures was low in all subtypes of GH cells in the sham-operated control rats. GRH treatment induced a significant increase in exocytosis in each subtype of GH cells, particularly in type I (mature) and type II (intermediate) GH cells in the control rats. GRH injection to rats for 4 days after ADX also showed an increase in exocytosis, but the degree was significantly less in comparison with the GRH injection in the control group. Corticosterone replacement given to ADX rats induced a clear recovery of the exocytotic response to GRH to the control level. Serum GH content measured by radioimmunoassay correlated with these morphological results. These results suggest that the secretion of GH stimulated by GRH is closely related to corticosteroids, and

  1. Identifying states along the hematopoietic stem cell differentiation hierarchy with single cell specificity via Raman spectroscopy

    PubMed Central

    Ilin, Yelena; Choi, Ji Sun; Harley, Brendan A. C.; Kraft, Mary L.

    2015-01-01

    A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely-related short-term repopulating HSCs (ST-HSCs), and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four sub-populations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition, and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that

  2. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    PubMed Central

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  3. Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

    PubMed Central

    Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

  4. Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy.

    PubMed

    Chemes, Hector E

    2013-01-01

    Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

  5. Familial testicular germ cell tumor: no associated syndromic pattern identified

    PubMed Central

    2014-01-01

    Background Testicular germ cell tumor (TGCT) is the most common malignancy in young men. Familial clustering, epidemiologic evidence of increased risk with family or personal history, and the association of TGCT with genitourinary (GU) tract anomalies have suggested an underlying genetic predisposition. Linkage data have not identified a rare, highly-penetrant, single gene in familial TGCT (FTGCT) cases. Based on its association with congenital GU tract anomalies and suggestions that there is an intrauterine origin to TGCT, we hypothesized the existence of unrecognized dysmorphic features in FTGCT. Methods We evaluated 38 FTGCT individuals and 41 first-degree relatives from 22 multiple-case families with detailed dysmorphology examinations, physician-based medical history and physical examination, laboratory testing, and genitourinary imaging studies. Results The prevalence of major abnormalities and minor variants did not significantly differ between either FTGCT individuals or their first-degree relatives when compared with normal population controls, except for tall stature, macrocephaly, flat midface, and retro-/micrognathia. However, these four traits were not manifest as a constellation of features in any one individual or family. We did detect an excess prevalence of the genitourinary anomalies cryptorchidism and congenital inguinal hernia in our population, as previously described in sporadic TGCT, but no congenital renal, retroperitoneal or mediastinal anomalies were detected. Conclusions Overall, our study did not identify a constellation of dysmorphic features in FTGCT individuals, which is consistent with results of genetic studies suggesting that multiple low-penetrance genes are likely responsible for FTGCT susceptibility. PMID:24559313

  6. Single-cell RNA sequencing identifies distinct mouse medial ganglionic eminence cell types

    PubMed Central

    Chen, Ying-Jiun J.; Friedman, Brad A.; Ha, Connie; Durinck, Steffen; Liu, Jinfeng; Rubenstein, John L.; Seshagiri, Somasekar; Modrusan, Zora

    2017-01-01

    Many subtypes of cortical interneurons (CINs) are found in adult mouse cortices, but the mechanism generating their diversity remains elusive. We performed single-cell RNA sequencing on the mouse embryonic medial ganglionic eminence (MGE), the major birthplace for CINs, and on MGE-like cells differentiated from embryonic stem cells. Two distinct cell types were identified as proliferating neural progenitors and immature neurons, both of which comprised sub-populations. Although lineage development of MGE progenitors was reconstructed and immature neurons were characterized as GABAergic, cells that might correspond to precursors of different CINs were not identified. A few non-neuronal cell types were detected, including microglia. In vitro MGE-like cells resembled bona fide MGE cells but expressed lower levels of Foxg1 and Epha4. Together, our data provide detailed understanding of the embryonic MGE developmental program and suggest how CINs are specified. PMID:28361918

  7. A cell-based phenotypic assay to identify cardioprotective agents

    PubMed Central

    Guo, Stephanie; Olm-Shipman, Adam; Walters, Andrew; Urciuoli, William R.; Devito, Stefanie; Nadtochiy, Sergiy M.; Wojtovich, Andrew P.; Brookes, Paul S.

    2012-01-01

    Rationale Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). Objective To develop a model of IR injury that is both physiologically relevant and amenable to HTS. Methods and Results A micro-plate based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument’s mechanical systems, yielded a 24 well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. Following initial validation with known protective molecules, the model was used to screen a 2000 molecule library, with post IR cell death as an endpoint. pO2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental and inert molecules from the screen were subsequently tested in a Langendorff perfused heart model of IR injury, revealing strong correlations between the screening endpoint and both recovery of cardiac function (negative r2=0.66), and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics, and protection against IR injury, were also studied. Conclusion This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules. PMID:22394516

  8. Studies on batch and continuous cultures of Botryococcus braunii: hydrocarbon production in relation to physiological state, cell ultrastructure, and phosphate nutrition

    SciTech Connect

    Casadevall, E.; Dif, D.; Largeau, C.; Gudin, C.; Chaumont, D.; Desanti, O.

    1985-01-01

    The growth of the hydrocarbon-rich alga Botryococcus braunii was studied under air-lift conditions using batch and continuous cultures. Large variations in the physiological state of B. braunii were achieved in batch cultures and in continuous cultures with various dilution rates. The possible effects of these variations upon hydrocarbons (nature, relative abundance, location, level, productivity) and also on the production of exocellular polysaccharides were examined. The relationships between the physiological state of B. braunii and its hydrocarbon and polysaccharide production were discussed and compared with those generally observed in unicellular algae. The factors giving rise to the transition from high to low productivity stages were considered. To this end the authors examined, at first, the variations in cell ultrastructure and the resulting degeneration occurring during batch cultures. Afterward the parallel changes in some parameters of the medium (pH, phosphate level) were determined and their possible relationships with B. braunii growth and hydrocarbon production were discussed. The main features of phosphate nutrition in B. braunii and its effects on hydrocarbons were finally examined.

  9. IMMUNO-ELECTRON MICROSCOPE ANALYSIS OF THE SURFACE LAYERS OF THE UNFERTILISED SEA URCHIN EGG. I. EFFECTS OF THE ANTISERA ON THE CELL ULTRASTRUCTURE.

    PubMed

    BAXANDALL, J; PERLMANN, P; AFZELIUS, B A

    1964-12-01

    The response of unfertilised Paracentrotus lividus eggs to gamma-globulin fractions of antisera against isolated homologous jelly coat substance or homologous homogenates of jellyless eggs has been studied at the ultrastructural level. The antijelly gamma-globulin caused precipitation of the jelly layer, the density of precipitation varying between different eggs and being proportional to the gamma-globulin concentration. Agglutination of the jelly substance of adjacent eggs, which is species specific, occurred frequently with higher gamma-globulin concentrations. Antiegg gamma-globulins (from antiserum against total homogenates of jelly-free eggs or the heat-stable fraction thereof) did not produce these effects. Instead, these gamma-globulins caused various structural alterations mostly representing stages in parthenogenetic activation. This species-specific activation was induced by the reaction of antibodies with some heat-stable egg antigens different from those involved in jelly precipitation. Surface alterations included the formation of small papillae, membrane blisters, hyaline layer, and activation membrane, the release of material from the cell surface, and the breakdown of cortical granules. These alterations were dependent on both gamma-globulin concentration and the variable reactivity among different females. Aster formation, found intracellularly, verified that the surface responses represented real parthenogenetic activation and were not the result of immune lysis. No such alterations appeared in the controls.

  10. s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells

    PubMed Central

    Brocqueville, Guillaume; Chmelar, Renee S.; Bauderlique-Le Roy, Hélène; Deruy, Emeric; Tian, Lu; Vessella, Robert L.; Greenberg, Norman M.; Bourette, Roland P.

    2016-01-01

    Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin− CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells. PMID:27081082

  11. [The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 11. The ultrastructure of the bone marrow cells in different generations of rats].

    PubMed

    Afanas'eva, V V; Zak, K P; Indyk, V M; Serkiz, Ia I; Tron'ko, N D

    1991-01-01

    In studying the bone marrow cell ultrastructure in male rats (F0-F2 generations) aged 3 months, which were brought up within the thirty kilometer Chernobyl A.P.S. disaster area, considerable submicroscopic changes have been revealed in the cells at all stages of maturation, including undifferentiated blasts and mature forms of cells of the neutrophilic, eosinophilic, monocytic and erythroid haemopoiesis series, as well as stromal elements of the microenvironment, megakaryocytes and endothelium. The severity of these changes increases, as the number of generations grows, displaying frequently a destructive character.

  12. Formation of Essential Ultrastructural Interface between Cultured Hippocampal Cells and Gold Mushroom-Shaped MEA- Toward “IN-CELL” Recordings from Vertebrate Neurons

    PubMed Central

    Fendyur, Anna; Mazurski, Noa; Shappir, Joseph; Spira, Micha E.

    2011-01-01

    Using cultured Aplysia neurons we recently reported on the development of a novel approach in which an extracellular, non-invasive multi-electrode-array system provides multisite, attenuated, intracellular recordings of subthreshold synaptic potentials, and action potentials (APs), the so called “IN-CELL” recording configuration (to differentiate it from intracellular recordings). Because of its non-invasive nature, the configuration can be used for long term semi intracellular electrophysiological monitoring of APs and synaptic potentials. Three principals converge to generate the IN-CELL configuration: (a) engulfment of approximately 1 μm size gold mushroom-shaped microelectrodes (gMμE) by the neurons, (b) formation of high seal resistance between the cell’s plasma membrane and the engulfed gMμE, and (c), autonomous localized increased conductance of the membrane patch facing the gMμE. Using dissociated rat hippocampal cultures we report here that the necessary morphological and ultrastructural relationships to generate the IN-CELL recording configuration are formed between hippocampal cells and the gMμEs. Interestingly, even <1 μm thin branches expand and engulf the gMμE structures. Recordings of spontaneous electrical activity revealed fast ∼2 ms, 0.04–0.75 mV positive monophasic APs (FPMP). We propose that the FPMP are attenuated APs generated by neurons that engulf gMμEs. Computer simulations of analog electrical circuits depicting the cell–gMμE configuration point out the parameters that should be altered to improve the neuron–gMμE electrical coupling. PMID:22163219

  13. Thymosin Beta 4 May Translocate from the Cytoplasm in to the Nucleus in HepG2 Cells following Serum Starvation. An Ultrastructural Study

    PubMed Central

    Piludu, Marco; Piras, Monica; Pichiri, Giuseppina; Coni, Pierpaolo; Orrù, Germano; Cabras, Tiziana; Messana, Irene; Faa, Gavino; Castagnola, Massimo

    2015-01-01

    Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases. PMID:25835495

  14. Thymosin beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study.

    PubMed

    Piludu, Marco; Piras, Monica; Pichiri, Giuseppina; Coni, Pierpaolo; Orrù, Germano; Cabras, Tiziana; Messana, Irene; Faa, Gavino; Castagnola, Massimo

    2015-01-01

    Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases.

  15. [Changes in the ultrastructure of the stomach mucous membrane parietal cells caused by inhibitors of hydrochloric acid secretion].

    PubMed

    Dondukova, G V; Morozov, I A

    2002-01-01

    The study of the action of phamotidine and omeprazol on the stomach parietal cells in patients with duodenal ulcer has shown that phamotidin results in changes of secretory membrane of the parietal cells increasing its secretory potential while omeprazol reduces energetic metabolism of the lining cell by the impact on its mitochondrial apparatus. Both in children and adults with duodenal ulcer more developed mitochondrial cell activity was found after omeprazol treatment.

  16. Satellited 4q identified in amniotic fluid cells

    SciTech Connect

    Miller, I.; Hsieh, C.L.; Songster, G.

    1995-01-16

    Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father`s relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. 8 refs., 3 figs.

  17. Loss of Sleep Affects the Ultrastructure of Pyramidal Neurons in the Adolescent Mouse Frontal Cortex

    PubMed Central

    de Vivo, Luisa; Nelson, Aaron B.; Bellesi, Michele; Noguti, Juliana; Tononi, Giulio; Cirelli, Chiara

    2016-01-01

    Study Objective: The adolescent brain may be uniquely affected by acute sleep deprivation (ASD) and chronic sleep restriction (CSR), but direct evidence is lacking. We used electron microscopy to examine how ASD and CSR affect pyramidal neurons in the frontal cortex of adolescent mice, focusing on mitochondria, endosomes, and lysosomes that together perform most basic cellular functions, from nutrient intake to prevention of cellular stress. Methods: Adolescent (1-mo-old) mice slept (S) or were sleep deprived (ASD, with novel objects and running wheels) during the first 6–8 h of the light period, chronically sleep restricted (CSR) for > 4 days (using novel objects, running wheels, social interaction, forced locomotion, caffeinated water), or allowed to recover sleep (RS) for ∼32 h after CSR. Ultrastructural analysis of 350 pyramidal neurons was performed (S = 82; ASD = 86; CSR = 103; RS = 79; 4 to 5 mice/group). Results: Several ultrastructural parameters differed in S versus ASD, S versus CSR, CSR versus RS, and S versus RS, although the different methods used to enforce wake may have contributed to some of the differences between short and long sleep loss. Differences included larger cytoplasmic area occupied by mitochondria in CSR versus S, and higher number of secondary lysosomes in CSR versus S and RS. We also found that sleep loss may unmask interindividual differences not obvious during baseline sleep. Moreover, using a combination of 11 ultrastructural parameters, we could predict in up to 80% of cases whether sleep or wake occurred at the single cell level. Conclusions: Ultrastructural analysis may be a powerful tool to identify which cellular organelles, and thus which cellular functions, are most affected by sleep and sleep loss. Citation: de Vivo L, Nelson AB, Bellesi M, Noguti J, Tononi G, Cirelli C. Loss of sleep affects the ultrastructure of pyramidal neurons in the adolescent mouse frontal cortex. SLEEP 2016;39(4):861–874. PMID:26715225

  18. Harnessing single cell sorting to identify cell division genes and regulators in bacteria.

    PubMed

    Burke, Catherine; Liu, Michael; Britton, Warwick; Triccas, James A; Thomas, Torsten; Smith, Adrian L; Allen, Steven; Salomon, Robert; Harry, Elizabeth

    2013-01-01

    Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.

  19. Harnessing Single Cell Sorting to Identify Cell Division Genes and Regulators in Bacteria

    PubMed Central

    Burke, Catherine; Liu, Michael; Britton, Warwick; Triccas, James A.; Thomas, Torsten; Smith, Adrian L.; Allen, Steven; Salomon, Robert; Harry, Elizabeth

    2013-01-01

    Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development. PMID:23565292

  20. Ultrastructural changes in the ovary cells of engorged Rhipicephalus sanguineus female ticks treated with esters of ricinoleic acid from castor oil (Ricinus communis).

    PubMed

    Sampieri, Bruno Rodrigues; Arnosti, André; Nunes, Pablo Henrique; Furquim, Karim Christina Scopinho; Chierice, Gilberto Orivaldo; Mathias, Maria Izabel Camargo

    2012-05-01

    Rhipicephalus sanguineus is a widely distributed tick species that has adapted to the urban environment, and the dog is its main host. This species is also known as a vector and reservoir of diseases caused by bacteria, protozoa, and viruses. Currently, acaricides of synthetic chemical origin have been widely and indiscriminately used, leading to the development of resistance to these products by ticks and causing damage to the environment. Thus, these issues have made it necessary to seek other forms of controlling these ectoparasites. R. sanguineus was artificially infested in host New Zealand White rabbits, which were divided into four treatment groups: control (CG1 and CG2) and treatment (TG1 and TG2) groups. TG1 and TG2 hosts were provided with feed supplemented with esters of ricinoleic acid from castor oil at a concentration of 5 g/kg of feed for 7 and 15 days. Afterward, the ovaries of the female ticks were removed for analysis by transmission electron microscopy. The results showed ultrastructural changes in the somatic and germ cells of ovaries from TG1 and TG2 females, particularly with respect to chorion deposition, a protective membrane of the oocyte, as well as in the transport process of vitellogenic materials via the hemolymph and pedicel cells. Moreover, the mitochondria were less electron-dense and had cristae that were more disorganized than the mitochondria from CG1 and CG2 individuals. Thus, this study demonstrated the action of esters on the ovaries of R. sanguineus, signaling the prospect of a way to control this ectoparasite without affecting nontarget organisms or the environment.

  1. Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity.

    PubMed

    Wu, Gongqing; Liu, Yi; Ding, Ying; Yi, Yunhong

    2016-08-01

    Galleria mellonella larvae have been widely used as a model to study the virulence of various human pathogens. Hemocytes play important roles in the innate immune response of G. mellonella. In this study, the hemocytes of G. mellonella larvae were analyzed by transmission electron microscope, light microscope, and cytochemistry. The cytological and morphological analyses revealed four types of hemocytes; Plasmatocytes, granular cells, spherule cells and oenocytoids. Differential hemocyte counts showed that under our conditions plasmatocytes and granular cells were the most abundant circulating cell types in the hemolymph. We also investigated the role of different types of hemocytes in the cellular and humoral immune defenses. The in-vivo experiment showed that plasmatocytes, granular cells and oenocytoids phagocytized FITC-labelled Escherichia coli bacteria in larvae of G. mellonella, whereas the granular cells exhibited the strongest phagocytic ability against these microbial cells. After incubation with L-DOPA, plasmatocytes, granular cells and oenocytoids are stained brown, indicating the presence of phenoloxidase activity. These results shed new light on our understanding of the immune function of G. mellonella hemocytes.

  2. Cell wall ultrastructure of flocculent and non-flocculent Schizosaccharomyces pombe strains. Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure.

    PubMed

    Geleta, Anna; Kristóf, Z; Maráz, Anna

    2007-03-01

    Scanning and transmission electron microscopic studies revealed the presence of slime-like, amorphous material on the surface of Schizosaccahromyces pombe RIVE 4-2-1 cells, independently, whether they were in flocculated or in non-flocculated state. Close contact of the adjacent cells via the merging outermost cell wall layers was found, however, only in the case of floc formation, which was induced by cultivating the cells in the presence of 6% (v/v) ethanol. Irreversible loss of the flocculation ability of the cells by treatment with proteinases suggests that proteinaceous cell surface molecules as lectins contribute to the cell-to-cell interaction during flocculation. Both proteinase K and pronase treatments removed a distinct outer layer of the cell wall, which indicated that the protein moieties of the phosphogalactomannan outer surface layer has a crucial role in the maintenance of cell wall integrity. In the case of lysing enzyme treatment the removal of the outermost layer was also observed as the first step of the cell wall digestion, while driselase treatment resulted in almost complete digestion of the cell wall.

  3. Wishbone identifies bifurcating developmental trajectories from single-cell data

    PubMed Central

    Setty, Manu; Tadmor, Michelle D; Reich-Zeliger, Shlomit; Angel, Omer; Salame, Tomer Meir; Kathail, Pooja; Choi, Kristy; Bendall, Sean; Friedman, Nir; Pe’er, Dana

    2016-01-01

    Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-seq data, as input and orders cells according to their developmental progression by pinpointing bifurcation points and labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points. PMID:27136076

  4. Phenotypic and ultrastructural characteristics of bronchoalveolar lavage cells of lentivirus-infected lambs treated with recombinant ovine IFN-tau.

    PubMed

    Singh, B; Ott, T L; Bazer, F W; de la Concha-Bermejillo, A

    2001-09-01

    Ovine lentivirus (OvLV) belongs to the family Retroviridae and closely resembles the human immunodeficiency virus (HIV). Pulmonary lesions in OvLV-infected sheep consist of lymphoid interstitial pneumonia (LIP) and lymphocytic alveolitis. Similar pulmonary lesions occur in up to 40% of HIV-infected children and in some adults with AIDS. Interferon-tau (IFN-tau), a type I IFN, is produced by trophectoderm of ruminant conceptuses and is the pregnancy recognition signal in these species. To evaluate changes in phenotypes of bronchoalveolar lavage (BAL) cells of OvLV-infected lambs treated with recombinant ovine IFN-tau (rOvIFN-tau), 24 lambs were randomly allocated to one of four groups (n = 6 per group): 1, no virus + placebo (NVP); 2, no virus + rOvIFN-tau (NVI); 3, virus + placebo (VP); 4, virus + rOvIFN-tau (VI). The BAL cells from 3 lambs in each group were labeled with monoclonal antibodies (mAb) to cell surface markers at 16 weeks of treatment, and cells from the remaining 3 lambs in each group were labeled with mAb at 34 weeks of treatment. After labeling, BAL cells were analyzed by flow cytometry. The morphology of BAL cells from all experimental lambs was examined by transmission electron microscopy (TEM). At week 16, no differences in the relative proportions of BAL cell phenotypes were detected among the experimental groups. At week 34, VI lambs had higher proportions of CD8(+), gammadelta(+), MHC class II(+), and L-selectin (LS(+)) BAL cells compared with VP lambs. Higher proportions of CD14(+) and CD44(+) cells were found in VP lambs compared with NVP lambs at 34 weeks. OvLV-like particles were detected only in bronchoalveolar macrophages of VP lambs. In this study, rOvIFN-tau increased the proportions of primary antiviral gammadelta(+) and CD8(+) immune cells in OvLV-infected lambs. This may represent a cellular mechanism to explain the antiviral and therapeutic efficacy of this cytokine, in addition to its direct antiviral effect. However, because the

  5. [Changes in microstructure and ultrastructure between differentiation of cold and heat syndrome in chronic atrophied gastritis and exfoliative cells].

    PubMed

    Li, Y; Guo, Z Q

    1989-06-01

    In this paper, exfoliative cells of fur in 56 cases of chronic atrophied gastritis (CAG) with Cold or Heat syndrome was observed by means of microscopy and electron microscopy. With microscopy, the authors found that keratinization of epithelial cells of fur in Cold syndrome group of CAG were markedly fewer than those in Heat syndrome group (P less than 0.01); while pre-keratinization cells were much more than those in Heat syndrome group (P less than 0.01); the constituent ratio of complete keratinization cells of fur in the two groups were markedly different. With the electron microscopy, fibrosis changes was appeared in pre-keratinization cells of Cold syndrome patients with CAG; desmosome was disappeared; metachromasia was appeared in nucleus; fibrosis change in Heat syndrome group was not obvious. Cells were still joined to one another by fingered protrusion. There were bacterias in both Cold and Heat syndrome groups. The change of exfoliative cells of fur in Cold and Heat syndromes in CAG, probably, can offer us a microcosmic sign for its early differentiation or diagnosis.

  6. The interaction of microgravity and ethylene on the ultrastructure cell and Ca2+ localization in soybean hook hypocotyl

    NASA Technical Reports Server (NTRS)

    Nedukha, O. M.; Kordyum, E. L.; Brown, C.; Chapman, D.

    2001-01-01

    Calcium ions are secondary messenger in numerous cellular processes of plant grown at 1 g. Ca2+ are connected with oxygen atoms, of pectin carboxy groups and/or with H(+)-groups of protein (Roux and Slocum, 1982; Hepler and Wayne, 1985). The influence of altered gravity on the calcium balance in some cells is established. The increased synthesis of ethylene in plant grown in microgravity caused the change of the structural-functional organization of cell (Hensel and Iversen, 1980; Hilaire et al., 1996). Available data put the new question: how do high ethylene level and microgravity influence on the redistribution of Ca2+ in cell of seedling in early stage of growth? Therefore, the goal of our data was the comparable study of the cell ulltrastructure and localization of Ca2+ in hook hypocotyl of soybean seedling under interaction of microgravity and ethylene.

  7. Ultrastructural differences between diabetic and idiopathic gastroparesis

    PubMed Central

    Faussone-Pellegrini, Maria Simonetta; Grover, Madhusudan; Pasricha, Pankaj J; Bernard, Cheryl E; Lurken, Matthew S; Smyrk, Thomas C; Parkman, Henry P; Abell, Thomas L; Snape, William J; Hasler, William L; Ünalp-Arida, Aynur; Nguyen, Linda; Koch, Kenneth L; Calles, Jorges; Lee, Linda; Tonascia, James; Hamilton, Frank A; Farrugia, Gianrico

    2012-01-01

    Abstract The ultrastructural changes in diabetic and idiopathic gastroparesis are not well studied and it is not known whether there are different defects in the two disorders. As part of the Gastroparesis Clinical Research Consortium, full thickness gastric body biopsies from 20 diabetic and 20 idiopathic gastroparetics were studied by light microscopy. Abnormalities were found in many (83%) but not all patients. Among the common defects were loss of interstitial cells of Cajal (ICC) and neural abnormalities. No distinguishing features were seen between diabetic and idiopathic gastroparesis. Our aim was to provide a detailed description of the ultrastructural abnormalities, compare findings between diabetic and idiopathic gastroparesis and determine if patients with apparently normal immunohistological features have ultrastructural abnormalities. Tissues from 40 gastroparetic patients and 24 age- and sex-matched controls were examined by transmission electron microscopy (TEM). Interstitial cells of Cajal showing changes suggestive of injury, large and empty nerve endings, presence of lipofuscin and lamellar bodies in the smooth muscle cells were found in all patients. However, the ultrastructural changes in ICC and nerves differed between diabetic and idiopathic gastroparesis and were more severe in idiopathic gastroparesis. A thickened basal lamina around smooth muscle cells and nerves was characteristic of diabetic gastroparesis whereas idiopathic gastroparetics had fibrosis, especially around the nerves. In conclusion, in all the patients TEM showed abnormalities in ICC, nerves and smooth muscle consistent with the delay in gastric emptying. The significant differences found between diabetic and idiopathic gastroparesis offers insight into pathophysiology as well as into potential targeted therapies. PMID:21914127

  8. Ultrastructure of eccrine cystadenoma. A case report.

    PubMed

    Hassan, M O; Khan, M A

    1979-10-01

    A case of eccrine cystadenoma was studied by electron microscopy. The tumor showed two types of cells, luminal and basal cells. The cells lacked the characteristics of the secretory segment of the sweat glands. The features of the luminal cells are similar to those of the intradermal portion of the eccrine sweat duct. In some areas, the lesion showed features characteristic of apocrine gland structure. Nuclear bodies were very frequent. The ultrastructural findings of eccrine cystadenoma support an origin from the ductal portion of eccrine sweat glands.

  9. Effects of Stevia rebaudiana (Bertoni) extract and N-nitro-L-arginine on renal function and ultrastructure of kidney cells in experimental type 2 Diabetes.

    PubMed

    Ozbayer, Cansu; Kurt, Hulyam; Kalender, Suna; Ozden, Hilmi; Gunes, Hasan V; Basaran, Ayse; Cakmak, Ecir A; Civi, Kismet; Kalender, Yusuf; Degirmenci, Irfan

    2011-10-01

    Diabetes is the leading cause of chronic renal failure. Our purpose was to determine the effects of N-nitro-l-arginine (l-NNA) and an extract of Stevia rebaudiana (Bertoni) (SrB) leaves on renal function in streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats. Rats were divided into seven groups. Three of these groups were controls. Diabetes was induced by STZ-NA in the other four. Diabetic rats were treated with SrB (200 mg/kg), L-NNA (100 mg/kg), or SrB + L-NNA for 15 days after 5-8 weeks of diabetes. At the end of the experiments, urine and blood samples were collected from the rats, and kidney tissue samples were collected with the animals under ether anesthesia. Renal filtration changes were determined by measuring urine pH, urine volume, and serum and urine creatinine. Nitric oxide synthase (NOS) activity was measured in kidney homogenates. Alterations in kidney ultrastructure were determined by electron microscopy, and histological changes were examined by hematoxylin and eosin staining. No statistical differences were observed in urine creatinine or creatinine clearance. Even so, we observed higher NOS activity in SrB-treated diabetic rats. SrB-treated diabetic rats had less mitochondrial swelling and vacuolization in thin kidney sections than other diabetic groups. The control groups showed normal histological structure, whereas in the diabetic groups, membrane thickening, tubular epithelial cells, and cellular degeneration were observed. Thus, SrB has beneficial effects on diabetes compared with l-NNA. Our results support the validity of SrB for the management of diabetes as well as diabetes-induced renal disorders.

  10. Tendon’s ultrastructure

    PubMed Central

    Tresoldi, Ilaria; Oliva, Francesco; Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; Bei, Roberto; Modesti, Andrea

    2013-01-01

    Summary The structure of a tendon is an important example of complexity of ECM three-dimensional organization. The extracellular matrix (ECM) is a macromolecular network with both structural and regulatory functions. ECM components belong to four major types of macromolecules: the collagens, elastin, proteoglycans, and noncollagenous glycoproteins. Tendons are made by a fibrous, compact connective tissue that connect muscle to bone designed to transmit forces and withstand tension during muscle contraction. Here we show the ultrastructural features of tendon’s components. PMID:23885339

  11. Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis

    PubMed Central

    Kirshenbaum, Arnold S.; Cruse, Glenn; Desai, Avanti; Bandara, Geethani; Leerkes, Maarten; Lee, Chyi-Chia R.; Fischer, Elizabeth R.; O’Brien, Kevin J.; Gochuico, Bernadette R.; Stone, Kelly; Gahl, William A.; Metcalfe, Dean D.

    2016-01-01

    Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in HPS1 which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the HPS1 mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients’ serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with HPS1 rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and

  12. Identifying and removing the cell-cycle effect from single-cell RNA-Sequencing data

    PubMed Central

    Barron, Martin; Li, Jun

    2016-01-01

    Single-cell RNA-Sequencing (scRNA-Seq) is a revolutionary technique for discovering and describing cell types in heterogeneous tissues, yet its measurement of expression often suffers from large systematic bias. A major source of this bias is the cell cycle, which introduces large within-cell-type heterogeneity that can obscure the differences in expression between cell types. The current method for removing the cell-cycle effect is unable to effectively identify this effect and has a high risk of removing other biological components of interest, compromising downstream analysis. We present ccRemover, a new method that reliably identifies the cell-cycle effect and removes it. ccRemover preserves other biological signals of interest in the data and thus can serve as an important pre-processing step for many scRNA-Seq data analyses. The effectiveness of ccRemover is demonstrated using simulation data and three real scRNA-Seq datasets, where it boosts the performance of existing clustering algorithms in distinguishing between cell types. PMID:27670849

  13. COMPASS identifies T-cell subsets correlated with clinical outcomes.

    PubMed

    Lin, Lin; Finak, Greg; Ushey, Kevin; Seshadri, Chetan; Hawn, Thomas R; Frahm, Nicole; Scriba, Thomas J; Mahomed, Hassan; Hanekom, Willem; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; Tomaras, Georgia D; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Michael, Nelson L; Kim, Jerome H; Robb, Merlin L; O'Connell, Robert J; Karasavvas, Nicos; Gilbert, Peter; C De Rosa, Stephen; McElrath, M Juliana; Gottardo, Raphael

    2015-06-01

    Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells as well as the interrogation of cell population heterogeneity. However, in many instances, computational tools to analyze the wealth of data generated by these technologies are lacking. Here, we present a computational framework for unbiased combinatorial polyfunctionality analysis of antigen-specific T-cell subsets (COMPASS). COMPASS uses a Bayesian hierarchical framework to model all observed cell subsets and select those most likely to have antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, and human subject-level responses are quantified by two summary statistics that describe the quality of an individual's polyfunctional response and can be correlated directly with clinical outcome. Using three clinical data sets of cytokine production, we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals cellular 'correlates of protection/immunity' in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software.

  14. COMPASS identifies T-cell subsets correlated with clinical outcomes

    PubMed Central

    Lin, Lin; Finak, Greg; Ushey, Kevin; Seshadri, Chetan; Hawn, Thomas R.; Frahm, Nicole; Scriba, Thomas J.; Mahomed, Hassan; Hanekom, Willem; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; Tomaras, Georgia D.; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Michael, Nelson L.; Kim, Jerome H.; Robb, Merlin L.; O’Connell, Robert J.; Karasavvas, Nicos; Gilbert, Peter; DeRosa, Stephen; McElrath, M. Juliana

    2015-01-01

    Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells and allowed interrogation of cell population heterogeneity. Computational tools to take full advantage of these technologies are lacking. Here, we present COMPASS, a computational framework for unbiased polyfunctionality analysis of antigen-specific T-cell subsets. COMPASS uses a Bayesian hierarchical framework to model all observed functional cell subsets and select those most likely to exhibit antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, while subject-level responses are quantified by two novel summary statistics that can be correlated directly with clinical outcome, and describe the quality of an individual’s (poly)functional response. Using three clinical datasets of cytokine production we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals novel cellular correlates of protection in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software. PMID:26006008

  15. Ciliary ultrastructure of polyplacophorans (Mollusca, Amphineura, Polyplacophora).

    PubMed

    Lundin, K; Schander, C

    2001-01-01

    This study is part of a series of papers aiming to investigate the phylogenetic significance of ciliary ultrastructure among molluscs and to test the hypothesis of a relationship between Xenoturbella and the molluscs. The ultrastructure of the ciliary apparatus on the gills of the polyplacophorans Leptochiton asellus and Tonicella rubra was studied. The gill cilia of the two species are similar in shape. The free part of the cilium is long with a slender distal part. There are two ciliary rootlets. One of them is short, broad and placed on the anterior face of the basal body. The other rootlet is conical and has a vertical orientation. Among the mollusca, two ciliary rootlets in the ciliary apparatus of multiciliate ectodermal cells have only been reported from the Chaetodermomorpha and Neomeniomorpha. This character state is likely plesiomorphic for the Mollusca and indicates a basal (nonderived) position of these taxa among the molluscs. No possible synapomorphic character with Xenoturbella bocki was found.

  16. [Ultrastructural localization of lysosomal and nuclear beta-glycerophosphatase activity in the urethral discharge cells of gonorrhea].

    PubMed

    Delektorskiĭ, V V; Dmitriev, G A; Bukhvalov, I B

    1976-01-01

    Activity of acid phosphotase in the urethra discharge in patients with various forms of gonorrhea (acute, torpid, and chronic) was studied with the use of electron-microscopy and biochemical methods. A positive reaction of nuclei of the epithelial cells to acid phosphotase in lysosomes and perichromatin granules was demonstrated. In polymorphononuclear leucocytes the positive reaction to acid phosphotase could be also sometimes observed in granules of the cytoplasm. The electronograms presented testify to heterogeneity and high activity of acid phosphotase in lysosomes. Neither electron-microscopy nor biochemical methods could help reveal any differences in the activity of acid phosphotase in various forms of gonorrhea.

  17. Identifying Cell Types from Spatially Referenced Single-Cell Expression Datasets

    PubMed Central

    Achim, Kaia; Richardson, Sylvia; Azizi, Lamiae; Marioni, John

    2014-01-01

    Complex tissues, such as the brain, are composed of multiple different cell types, each of which have distinct and important roles, for example in neural function. Moreover, it has recently been appreciated that the cells that make up these sub-cell types themselves harbour significant cell-to-cell heterogeneity, in particular at the level of gene expression. The ability to study this heterogeneity has been revolutionised by advances in experimental technology, such as Wholemount in Situ Hybridizations (WiSH) and single-cell RNA-sequencing. Consequently, it is now possible to study gene expression levels in thousands of cells from the same tissue type. After generating such data one of the key goals is to cluster the cells into groups that correspond to both known and putatively novel cell types. Whilst many clustering algorithms exist, they are typically unable to incorporate information about the spatial dependence between cells within the tissue under study. When such information exists it provides important insights that should be directly included in the clustering scheme. To this end we have developed a clustering method that uses a Hidden Markov Random Field (HMRF) model to exploit both quantitative measures of expression and spatial information. To accurately reflect the underlying biology, we extend current HMRF approaches by allowing the degree of spatial coherency to differ between clusters. We demonstrate the utility of our method using simulated data before applying it to cluster single cell gene expression data generated by applying WiSH to study expression patterns in the brain of the marine annelid Platynereis dumereilii. Our approach allows known cell types to be identified as well as revealing new, previously unexplored cell types within the brain of this important model system. PMID:25254363

  18. Integrin-β4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells

    PubMed Central

    Bierie, Brian; Pierce, Sarah E.; Kroeger, Cornelia; Stover, Daniel G.; Pattabiraman, Diwakar R.; Thiru, Prathapan; Liu Donaher, Joana; Reinhardt, Ferenc; Chaffer, Christine L.; Keckesova, Zuzana; Weinberg, Robert A.

    2017-01-01

    Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-β4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC. PMID:28270621

  19. Identifiability and privacy in pluripotent stem cell research.

    PubMed

    Isasi, Rosario; Andrews, Peter W; Baltz, Jay M; Bredenoord, Annelien L; Burton, Paul; Chiu, Ing-Ming; Hull, Sara Chandros; Jung, Ji-Won; Kurtz, Andreas; Lomax, Geoffrey; Ludwig, Tenneille; McDonald, Michael; Morris, Clive; Ng, Huck Hui; Rooke, Heather; Sharma, Alka; Stacey, Glyn N; Williams, Clare; Zeng, Fanyi; Knoppers, Bartha Maria

    2014-04-03

    Data sharing is an essential element of research; however, recent scientific and social developments have challenged conventional methods for protecting privacy. Here we provide guidance for determining data sharing thresholds for human pluripotent stem cell research aimed at a wide range of stakeholders, including research consortia, biorepositories, policy-makers, and funders.

  20. Using isolated cell wall xylan to identify recalcitrant oligosaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Herbaceous biomass is a renewable source of carbohydrates with potential for use in microbial conversion to biofuels. Xylan comprises 20-40% of herbaceous biomass cell wall material and its full depolymerization benefits the economics of bioconversion. To understand the limitations of commercial enz...

  1. Using machine vision and data mining techniques to identify cell properties via microfluidic flow analysis

    NASA Astrophysics Data System (ADS)

    Horowitz, Geoffrey; Bowie, Samuel; Liu, Anna; Stone, Nicholas; Sulchek, Todd; Alexeev, Alexander

    2016-11-01

    In order to quickly identify the wide range of mechanistic properties that are seen in cell populations, a coupled machine vision and data mining analysis is developed to examine high speed videos of cells flowing through a microfluidic device. The microfluidic device contains a microchannel decorated with a periodical array of diagonal ridges. The ridges compress flowing cells that results in complex cell trajectory and induces cell cross-channel drift, both depend on the cell intrinsic mechanical properties that can be used to characterize specific cell lines. Thus, the cell trajectory analysis can yield a parameter set that can serve as a unique identifier of a cell's membership to a specific cell population. By using the correlations between the cell populations and measured cell trajectories in the ridged microchannel, mechanical properties of individual cells and their specific populations can be identified via only information captured using video analysis. Financial support provided by National Science Foundation (NSF) Grant No. CMMI 1538161.

  2. Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi).

    PubMed

    Martínez-Silvestre, A; Marco, I; Rodriguez-Dominguez, M A; Lavín, S; Cuenca, R

    2005-04-01

    The object of this study was to examine the erythrocytes, leukocytes and thrombocytes of the giant lizard of El Hierro (Gallotia simonyi) by light and electron (TEM) microscopy, and cytochemical staining. Smears were prepared from blood from the ventral coccygeal vein of 10 healthy adult lizards (five males and five females) from the Giant Lizard of El Hierro Reproduction and Research Centre, Canary Islands, Spain. The cytochemical stains used were: benzidine peroxidase (BP), chloroacetate esterase (CAE), alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), periodic acid-Schiff (PAS), toluidine blue (TB) and May-Grünwald-Giemsa (MGG). Electron microscopy was also performed on all samples. Heterophils had granules that were heterogeneous in both size and electron density, and stained with BP, PAS and ANAE. Eosinophil granules were homogeneously electron-dense and stained for AP, CAE and ANAE. Basophils had both highly and moderately electron-dense granules, and stained with TB and ANAE. Azurophil granules were of low electron-density and stained for AP, CAE and ANAE. Azurophil cytoplasm was vacuolated on TEM. The cytoplasm of lymphocytes contained many ribosomes and was positive for AP. Monocytes had a large nucleus and a vacuolated cytoplasm but did not stain by any of the cytochemical methods used. Thrombocytes had a relatively large nucleus but little cytoplasm; they did not stain cytochemically. The blood cells of the giant lizards of El Hierro differ from those of other members of the Order Squamata both morphologically and cytochemically. The variation in cytochemical responses in the blood of reptiles makes it necessary to study species individually if meaningful clinical decisions are to be made.

  3. Platelets: production, morphology and ultrastructure.

    PubMed

    Thon, Jonathan N; Italiano, Joseph E

    2012-01-01

    Platelets are anucleate, discoid cells, roughly 2-3 μm in diameter that function primarily as regulators of hemostasis, but also play secondary roles in angiogensis and innate immunity. Although human adults contain nearly one trillion platelets in circulation that are turned over every 8-10 days, our understanding of the mechanisms involved in platelet production is still incomplete. Platelets stem from large (30-100 μm) nucleated cells called megakaryocytes that reside primarily in the bone marrow. During maturation megakaryocytes extend long proplatelet elongations into sinusoidal blood vessels from which platelets ultimately release. During this process, platelets develop a number of distinguishable structural elements including: a delimited plasma membrane; invaginations of the surface membrane that form the open canalicular system (OCS); a closed-channel network of residual endoplasmic reticulum that form the dense tubular system (DTS); a spectrin-based membrane skeleton; an actin-based cytoskeletal network; a peripheral band of microtubules; and numerous organelles including α-granules, dense-granules, peroxisomes, lysosomes, and mitochondria. Proplatelet elongation and platelet production is an elaborate and complex process that defines the morphology and ultrastructure of circulating platelets, and is critical in understanding their increasingly numerous and varied biological functions.

  4. SILAC proteomics of planarians identifies Ncoa5 as a conserved component of pluripotent stem cells.

    PubMed

    Böser, Alexander; Drexler, Hannes C A; Reuter, Hanna; Schmitz, Henning; Wu, Guangming; Schöler, Hans R; Gentile, Luca; Bartscherer, Kerstin

    2013-11-27

    Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC) protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA), which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.

  5. Ultrastructure of the Subcutaneous Primo-Vascular System in Rat Abdomen.

    PubMed

    Lim, Chae Jeong; Lee, So Yeong; Ryu, Pan Dong

    2016-01-01

    Recently, we identified the primo-vascular system (PVS), a novel vascular network, in rat subcutaneous tissues. Little is known about the subcutaneous PVS (sc-PVS). Here, we examined the ultrastructure of the sc-PVS in the hypodermis at the rat abdominal midline by electron microscopy. On the surface of sc-PVS, we observed three types of cells: microcells (5-6 μm), large elliptical cells (>20 μm), and erythrocyte (3-4 μm). The inside of the sc-PVS was filled with numerous cells, which can be classified into three major groups: leucocytes, mast cells, and erythrocytes. The dense leucocytes and mast cells were easily noticed. The extracellular matrix of the sc-PVS was mainly composed of extensive fibers (79 ± 6.5 nm) tightly covered by micro- (0.5-1 μm) and nanoparticles (10-100 nm). In conclusion, the ultrastructural features, such as the resident cells on and in the sc-PVS and fiber meshwork covered by particles, indicate that sc-PVS might act as a circulatory channel for the flow and delivery of numerous cells and particles. Our findings will help understand the nature of various sc-PVS beneath-the-skin layers and how they relate to acupuncture meridians.

  6. Ultrastructural immunochemistry of J chains in chickens.

    PubMed

    Moriya, O; Ichikawa, Y

    1990-01-01

    Using ultrastructural immunochemistry, we have determined subcellular localization of J chain molecules in chicken splenic cells. The majority of J chain positive cells (JPC) were lymphoblast-like cells. The data show that J chains predominantly are localized in cytoplasm with a considerable amount distributed on the cell surface. In the cytoplasm, J chains were diffusely expressed. Furthermore, these J chain molecules were clearly seen as cluster type. In addition to the J chain localization of subcellular organelles as described above, J chains were partly found on perinuclear spaces. As J chains are key protein in B cell differentiation into immunoglobulin (Ig) producing cells, these findings might help for studying regulation of B cell differentiation in addition to revealing the molecular assembly of polymeric Ig.

  7. Sparse activity of identified dentate granule cells during spatial exploration

    PubMed Central

    Diamantaki, Maria; Frey, Markus; Berens, Philipp; Preston-Ferrer, Patricia; Burgalossi, Andrea

    2016-01-01

    In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population. DOI: http://dx.doi.org/10.7554/eLife.20252.001 PMID:27692065

  8. Cytological and ultrastructural studies on root tissues

    NASA Technical Reports Server (NTRS)

    Slocum, R. D.; Gaynor, J. J.; Galston, A. W.

    1984-01-01

    The anatomy and fine structure of roots from oat and mung bean seedlings, grown under microgravity conditions for 8 days aboard the Space Shuttle, was examined and compared to that of roots from ground control plants grown under similar conditions. Roots from both sets of oat seedlings exhibited characteristic monocotyledonous tissue organization and normal ultrastructural features, except for cortex cell mitochondria, which exhibited a 'swollen' morphology. Various stages of cell division were observed in the meristematic tissues of oat roots. Ground control and flight-grown mung bean roots also showed normal tissue organization, but root cap cells in the flight-grown roots were collapsed and degraded in appearance, especially at the cap periphery. At the ultrastructural level, these cells exhibited a loss of organelle integrity and a highly-condensed cytoplasm. This latter observation perhaps suggests a differing tissue sensitivity for the two species to growth conditions employed in space flight. The basis for abnormal root cap cell development is not understood, but the loss of these putative gravity-sensing cells holds potential significance for long term plant growth orientation during space flight.

  9. Basilar artery of the capybara (Hydrochaeris hydrochaeris): an ultrastructural study.

    PubMed

    Islam, S; Ribeiro, A A C M; Loesch, A

    2004-04-01

    The present study investigated the ultrastructural features of the basilar artery of the largest rodent species, the capybara. The study suggests that the general ultrastructural morphological organization of the basilar artery of the capybara is similar to that of small rodents. However, there are some exceptions. The basilar artery of the capybara contains a subpopulation of 'granular' vascular smooth muscle cells resembling monocytes and/or macrophages. The possibility cannot be excluded that the presence of these cells reflects the remodelling processes of the artery due to animal maturation and the regression of the internal carotid artery. To clarify this issue, more systemic studies are required involving capybaras of various ages.

  10. Studies on the ultrastructure of Yersinia pseudotuberculosis after treatment with some detergents and solvents.

    PubMed

    Cherepova, N; Baykousheva, S; Veljanov, D

    1977-06-01

    The ultrastructural changes in 3 strains Yersinia pseudotuberculosis with different virulence after treatment with sodium lauryl sulfate (SLS) and petroleum ether were studied. The ultrafine sections after treatment with SLS show heavy destructive changes, concerning the cell wall, the cytoplasmic membrane and the inner structure of the cell. It was established that the same cells Y. pseudotuberculosis after cultivation on a medium with glycerol show a tendency to recover their ultrastructure. The cells treated with petroleum ether did not exhibit any notable ultrastructural changes.

  11. Identifying the lineages of individual cells in cocultures by multivariate analysis of Raman spectra.

    PubMed

    Ilin, Yelena; Kraft, Mary L

    2014-05-07

    The cellular and matrix cues that induce stem cell differentiation into distinct cell lineages must be identified to permit the ex vivo expansion of desired cell populations for clinical applications. Combinatorial biomaterials enable screening multiple different microenvironments while using small numbers of rare stem cells. New methods to identify the phenotypes of individual cells in cocultures with location specificity would increase the efficiency and throughput of these screening platforms. Here, we demonstrate that partial least-squares discriminant analysis (PLS-DA) models of calibration Raman spectra from cells in pure cultures can be used to identify the lineages of individual cells in more complex culture environments. The calibration Raman spectra were collected from individual cells of four different lineages, and a PLS-DA model that captured the Raman spectral profiles characteristic of each cell line was created. The application of these models to Raman spectra from test sets of cells indicated individual, fixed and living cells in separate monocultures, as well as those in more complex culture environments, such as cocultures, could be identified with low error. Cells from populations with very similar biochemistries could also be identified with high accuracy. We show that these identifications are based on reproducible cell-related spectral features, and not spectral contributions from the culture environment. This work demonstrates that PLS-DA of Raman spectra acquired from pure monocultures provides an objective, noninvasive, and label-free approach for accurately identifying the lineages of individual, living cells in more complex coculture environments.

  12. Comparative proteomic profiling identifies potential prognostic factors for human clear cell renal cell carcinoma.

    PubMed

    Sun, Xiang; Zhang, Hongwei; Luo, Longhua; Zhong, Kezhao; Ma, Yushui; Fan, Linlin; Fu, Da; Wan, Lijuan

    2016-12-01

    The identification of markers for disease diagnostic, prognostic, or predictive purposes will have a great effect in improving patient management. Proteomic‑based approaches for biomarker discovery are promising strategies used in cancer research. In this study, we performed quantitative proteomic analysis on four patients including clear cell renal cell carcinoma (ccRCC) and paired adjacent non‑cancerous renal tissues using label‑free quantitative proteomics and liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) to identify differentially expressed proteins. Among 3,061 identified non‑redundant proteins, we found that 210 proteins were differentially expressed (83 overexpressed and 127 underexpressed) in ccRCC tissue when compared with normal kidney tissues. Two most significantly dysregulated proteins (PCK1 and SNRPF) were chosen to be confirmed by western blotting. Pathway analysis of 210 differentially expressed proteins showed that dysregulated proteins are related to many cancer‑related biological processes such as oxidative phosphorylation, glycolysis and amino acid synthetic pathways. Online survival analysis indicated the prognostic value of these dysregulated proteins. In conclusion, we identified some potential diagnostic biomarkers for ccRCC and an in‑depth understanding of their involved biological pathways may help pave the way to discover new therapeutic strategies for ccRCC.

  13. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    PubMed

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  14. Products of cells cultured from gliomas. VI. Immunofluorescent, morphometric, and ultrastructural characterization of two different cell types growing from explants of human gliomas.

    PubMed Central

    McKeever, P. E.; Smith, B. H.; Taren, J. A.; Wahl, R. L.; Kornblith, P. L.; Chronwall, B. M.

    1987-01-01

    Explants derived from human gliomas have been characterized with respect to their cellular outgrowth pattern after 1-22 weeks in culture. A mat of cells which were fibronectin (FN)-positive and glial fibrillary acidic protein (GFAP)-negative (hereafter designated FN+ cells) with a polygonal, flat morphology covered the growth substrate in a swirling pattern for a mean diameter of 9.2 mm around FN+ explants. FN+ cells showed ruffled plasmalemma, dilated rough endoplasmic reticulin (RDR), and extracellular filamentous strands. Rare desmosomes were compatible with at most minor leptomeningeal components or differentiation. FN+ cells predominated in six of seven cultures at passage 2, and their features were the same from various high-grade gliomas and gliosarcoma. Around other explants, elongated or stellate cells which were GFAP+ and FN- grew in a netlike pattern with little cell-to-cell contact. These GFAP+ cells surrounded explants at a mean diameter of 2 mm, substantially less than FN+ cells (P less than 0.005), and they grew more slowly than FN+ cells around explants. GFAP+ cells had an area/perimeter ratio which was less than that of FN+ cells. GFAP+ cells contained abundant intracellular filaments, rare desmosomes, and narrow RER cisternae. In mixed explants, GFAP+ cells often grew on top of FN+ cells. Individual cells which stained for both GFAP and FN were evident only from one glioma (8% doubly positive). Cells negative for both proteins resembled FN+ cells morphologically. Frozen sections of original glioma tissue showed FN+ vessel walls and GFAP+ parenchyma. Results are evidence for very early overgrowth of a preexistent FN+ cell type distinct from the GFAP+ parenchymal cell. The features of this distinct cell type are mesenchymal and resemble the proliferating vascular elements of gliomas in situ. The tendency for GFAP+ cells to grow on top of these FN+ cells suggests a feeder layer interaction. More knowledge of the origins and interactions of these two

  15. Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence

    PubMed Central

    2017-01-01

    Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface. PMID:28194280

  16. Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

    PubMed

    Gilloteaux, J; Jamison, J M; Arnold, D; Taper, H S; Summers, J L

    2001-01-01

    Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

  17. A functional genomic screen in planarians identifies novel regulators of germ cell development.

    PubMed

    Wang, Yuying; Stary, Joel M; Wilhelm, James E; Newmark, Phillip A

    2010-09-15

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms.

  18. Ultrastructural studies of the gray platelet syndrome.

    PubMed

    White, J G

    1979-05-01

    The gray platelet syndrome (GPS) is a rare inherited disorder in which peripheral blood platelets are relatively large, vacuolated, and almost devoid of cytoplasmic granulation. In the present study we have evaluated the ultrastructure and cytochemistry of platelets from 2 patients with the GPS to determine precisely which organelles are missing from their cells. The findings indicate that gray platelets contain normal numbers of mitochondria, dense bodies, peroxisomes, and lysosomes but specifically lack alpha-granules. Preliminary studies of megakaryocytes from 1 of the 2 patients suggest that the defect in granule formation may lie at the level of the Golgi zone.

  19. Functional genomics identifies novel genes essential for clear cell renal cell carcinoma tumor cell proliferation and migration

    PubMed Central

    von Roemeling, Christina A.; Marlow, Laura A.; Radisky, Derek C.; Rohl, Austin; Larsen, Hege E.; Wei, Johnny; Sasinowska, Heather; Zhu, Heng; Drake, Richard; Sasinowski, Maciek; Tun, Han W.; Copland, John A.

    2014-01-01

    Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 ‘hits’ that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC. PMID:24979721

  20. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity.

    PubMed

    D'Angelo, Rosemarie C; Ouzounova, Maria; Davis, April; Choi, Daejin; Tchuenkam, Stevie M; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A; Senbabaoglu, Yasin; Conley, Sarah J; Clouthier, Shawn G; Hassan, Khaled A; Wicha, Max S; Korkaya, Hasan

    2015-03-01

    Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch(+)) or reduced activity (Notch(-)) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch(+) cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch(+) cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch(-) cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch(+) cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics.

  1. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity

    PubMed Central

    Davis, April; Choi, Daejin; Tchuenkam, Stevie M.; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A.; Senbabaoglu, Yasin; Conley, Sarah J.; Clouthier, Shawn G.; Hassan, Khaled A.; Wicha, Max S.; Korkaya, Hasan

    2015-01-01

    Developmental pathways such as Notch play a pivotal role in tissue specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch-) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays we investigated the role of Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts while Notch- cells failed to generate tumors. Gamma-secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our studies reveal molecular mechanism for the role of Notch mediated regulation of breast CSCs and provide a compelling rationale for CSC targeted therapeutics. PMID:25673823

  2. An ultrastructural study of cell death in the CA1 pyramidal field of the hippocapmus in rats submitted to transient global ischemia followed by reperfusion

    PubMed Central

    de Souza Pagnussat, Aline; Faccioni-Heuser, Maria Cristina; Netto, Carlos Alexandre; Achaval, Matilde

    2007-01-01

    In the course of ischemia and reperfusion a disruption of release and uptake of excitatory neurotransmitters occurs. This excitotoxicity triggers delayed cell death, a process closely related to mitochondrial physiology and one that shows both apoptotic and necrotic features. The aim of the present study was to use electron microscopy to characterize the cell death of pyramidal cells from the CA1 field of the hippocampus after 10 min of transient global ischemia followed by short reperfusion periods. For this study 25 adult male Wistar rats were used, divided into six groups: 10 min of ischemia, 3, 6, 12 and 24 h of reperfusion and an untouched group. Transient forebrain ischemia was produced using the 4-vessel occlusion method. The pyramidal cells of the CA1 field from rat hippocampus submitted to ischemia exhibited intracellular alterations consistent with a process of degeneration, with varied intensities according to the reperfusion period and bearing both apoptotic and necrotic features. Gradual neuronal and glial modifications allowed for the classification of the degenerative process into three stages: initial, intermediate and final were found. With 3 and 6 h of reperfusion, slight and moderate morphological alterations were seen, such as organelle and cytoplasm edema. Within 12 h of reperfusion, there was an apparent recovery and more ‘intact’ cells could be identified, while 24 h after the event neuronal damage was more severe and cells with disrupted membranes and cell debris were identified. Necrotic-like neurons were found together with some apoptotic bodies with 24 h of reperfusion. Present results support the view that cell death in the CA1 field of rat hippocampus submitted to 10 min of global transient ischemia and early reperfusion times includes both apoptotic and necrotic features, a process referred to as parapoptosis. PMID:17784936

  3. Effect of low temperature on growth and ultra-structure of Staphylococcus spp.

    PubMed

    Onyango, Laura A; Dunstan, R Hugh; Gottfries, Johan; von Eiff, Christof; Roberts, Timothy K

    2012-01-01

    The effect of temperature fluctuation is an important factor in bacterial growth especially for pathogens such as the staphylococci that have to remain viable during potentially harsh and prolonged transfer conditions between hosts. The aim of this study was to investigate the response of S. aureus, S. epidermidis, and S. lugdunensis when exposed to low temperature (4°C) for prolonged periods, and how this factor affected their subsequent growth, colony morphology, cellular ultra-structure, and amino acid composition in the non-cytoplasmic hydrolysate fraction. Clinical isolates were grown under optimal conditions and then subjected to 4°C conditions for a period of 8 wks. Cold-stressed and reference control samples were assessed under transmission electron microscopy (TEM) to identify potential ultra-structural changes. To determine changes in amino acid composition, cells were fractured to remove the lipid and cytoplasmic components and the remaining structural components were hydrolysed. Amino acid profiles for the hydrolysis fraction were then analysed for changes by using principal component analysis (PCA). Exposure of the three staphylococci to prolonged low temperature stress resulted in the formation of increasing proportions of small colony variant (SCV) phenotypes. TEM revealed that SCV cells had significantly thicker and more diffuse cell-walls than their corresponding WT samples for both S. aureus and S. epidermidis, but the changes were not significant for S. lugdunensis. Substantial species-specific alterations in the amino acid composition of the structural hydrolysate fraction were also observed in the cold-treated cells. The data indicated that the staphylococci responded over prolonged periods of cold-stress treatment by transforming into SCV populations. The observed ultra-structural and amino acid changes were proposed to represent response mechanisms for staphylococcal survival amidst hostile conditions, thus maintaining the viability of the

  4. Structural and ultrastructural features of the agouti tongue (Dasyprocta aguti Linnaeus, 1766).

    PubMed

    Ciena, Adriano Polican; de Sousa Bolina, Cristina; de Almeida, Sonia Regina Yokomizo; Rici, Rose Eli Grassi; de Oliveira, Moacir Franco; da Silva, Marcelo Cavenaghi Pereira; Miglino, Maria Angélica; Watanabe, Ii-sei

    2013-08-01

    The agouti (Dasyprocta aguti Linnaeus, 1766) is a wild rodent belonging to the family Dasyproctidae that is found throughout Brazil and feeds on fruits and seeds. The aim of the present study was to describe the following features of the tongue of agouti: its morphological structures, the three-dimensional characteristics of the lingual papillae surface, the connective tissue cores (CTCs) and the epithelial cell ultrastructure. Four types of papillae were observed on the dorsal surface of the tongue with a triangular shape: filiform, fungiform, foliate and vallate. Filiform papillae were distributed throughout the tongue surface, and removal of the epithelial surface revealed conical CTCs and multifilaments. Fungiform papillae were observed in the rostral and middle regions, whereas foliate papillae developed in pairs on the lateral margin of the caudal region. Removal of the epithelium in these regions revealed CTCs with parallel laminar conformation. Vallate papillae were arranged in a V-shape in the caudal region, and their CTCs ranged in shape from elongate to ovoid. The ultrastructural components of the dorsal epithelium were the basal, spinous, granular and keratinised layers. A broad area with cytoplasmic projections was identified in the interface region between the lamina propria and the basal layer. Flattened cells with intermediate filaments were observed in the transitional region between spinous and granular layers. The keratinised layer was composed of superimposed epithelial cells where desmosomes and cell-surface microridges were observed. These structural features, including the three-dimensional aspects of the lingual papillae, the CTCs and the epithelial ultrastructure, indicate that when compared with other animals, particularly other rodent species, the morphological features of the tongue of agouti are relatively well developed, especially regarding foliate and vallate papillae.

  5. Structural and ultrastructural features of the agouti tongue (Dasyprocta aguti Linnaeus, 1766)

    PubMed Central

    Ciena, Adriano Polican; Bolina, Cristina de Sousa; de Almeida, Sonia Regina Yokomizo; Rici, Rose Eli Grassi; de Oliveira, Moacir Franco; da da Silva, Marcelo Cavenaghi Pereira; Miglino, Maria Angélica; Watanabe, Ii-sei

    2013-01-01

    The agouti (Dasyprocta aguti Linnaeus, 1766) is a wild rodent belonging to the family Dasyproctidae that is found throughout Brazil and feeds on fruits and seeds. The aim of the present study was to describe the following features of the tongue of agouti: its morphological structures, the three-dimensional characteristics of the lingual papillae surface, the connective tissue cores (CTCs) and the epithelial cell ultrastructure. Four types of papillae were observed on the dorsal surface of the tongue with a triangular shape: filiform, fungiform, foliate and vallate. Filiform papillae were distributed throughout the tongue surface, and removal of the epithelial surface revealed conical CTCs and multifilaments. Fungiform papillae were observed in the rostral and middle regions, whereas foliate papillae developed in pairs on the lateral margin of the caudal region. Removal of the epithelium in these regions revealed CTCs with parallel laminar conformation. Vallate papillae were arranged in a V-shape in the caudal region, and their CTCs ranged in shape from elongate to ovoid. The ultrastructural components of the dorsal epithelium were the basal, spinous, granular and keratinised layers. A broad area with cytoplasmic projections was identified in the interface region between the lamina propria and the basal layer. Flattened cells with intermediate filaments were observed in the transitional region between spinous and granular layers. The keratinised layer was composed of superimposed epithelial cells where desmosomes and cell-surface microridges were observed. These structural features, including the three-dimensional aspects of the lingual papillae, the CTCs and the epithelial ultrastructure, indicate that when compared with other animals, particularly other rodent species, the morphological features of the tongue of agouti are relatively well developed, especially regarding foliate and vallate papillae. PMID:23701183

  6. The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells).

    PubMed

    Armand, Ray; Channon, Jacqueline Y; Kintner, Jennifer; White, Kristina A; Miselis, Kristin A; Perez, Raymond P; Lewis, Lionel D

    2004-04-01

    Mitochondrial DNA-deficient (rho(0)) cells were generated following a 26-day incubation of MOLT-4 lymphoblastoid T cells in ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide). The absence of mitochondrial DNA (mtDNA) in the resultant MOLT-4 rho(0) cells was confirmed by Southern analysis and quantitative polymerase chain reaction (PCR). MOLT-4 rho(0) cells proliferated more slowly than parental cells (wild type) and produced significantly more lactate (approximately fourfold increase; P < 0.001) with concomitantly reduced oxygen consumption (12.3% vs. 100%; P < 0.001) compared with the wild type. MOLT-4 rho(0) cells also showed reduced cytochrome c oxidase activity and a reduced cytochrome c oxidase/citrate synthase activity ratio compared to parental wild-type MOLT-4 cells (P < 10(-11)). Electron microscopy showed elongated mitochondria with parallel cristae in MOLT-4 cells although the mitochondria in MOLT-4 rho(0) cells appeared enlarged, some were vacuolated with either an absent or a grossly distorted cristae pattern. Vital staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was used to image mitochondria in intact cells and study mitochondrial transmembrane potential (Deltapsi(m)). Flow cytometry using JC-1 indicated that MOLT-4 rho(0) had a lower Deltapsi(m) than MOLT-4. Sodium fluoride (an inhibitor of the glycolytic pathway) at a concentration of 20 mM further reduced the Deltapsi(m) in MOLT-4-rho(0) cells. This data suggested that a glycolytic pathway product, possibly ATP, was required for the maintenance of Deltapsi(m) in MOLT-4 rho(0) cells.

  7. Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways

    PubMed Central

    Coleman, Ilsa; Lakely, Bryce; Coleman, Roger; Larson, Sandy; Aguirre-Ghiso, Julio A.; Xia, Jing; Gulati, Roman; Nelson, Peter S.; Montgomery, Bruce; Lange, Paul; Snyder, Linda A.; Vessella, Robert L.; Morrissey, Colm

    2014-01-01

    Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45− cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention. PMID:25301725

  8. Hypertextual Ultrastructures: Movement and Containment in Texts and Hypertexts

    ERIC Educational Resources Information Center

    Coste, Rosemarie L.

    2009-01-01

    The surface-level experience of hypertextuality as formless and unbounded, blurring boundaries among texts and between readers and writers, is created by a deep structure which is not normally presented to readers and which, like the ultrastructure of living cells, defines and controls texts' nature and functions. Most readers, restricted to…

  9. Ultrastructural study of thyroid capillaries after IR laser radiation

    NASA Astrophysics Data System (ADS)

    Vidal, Lourdes; Perez de Vargas, I.; Carrillo, F.; Parrado, C.; Pelaez, A.

    1994-02-01

    Laser radiation causes microscopical changes in the follicular cells relative to dose intensity. So, we have observed focal degenerative phenomena, at maximal doses, and activation of cellular function similar to the ones observed after stimulation with TSH, at minimal doses. In order to evaluate the evolution of these changes we have planned an ultrastructural study of rats thyroid capillaries treated with IR laser radiation.

  10. Molecular and Ultrastructural Properties of Maize White Line Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reports the complete nucleotide sequence of the genome of Maize white line mosaic virus (MWLMV) and describes the ultrastructural features of infected maize cells. The viral genome is an RNA molecule 4293 nt in size with the same structural organization of members of the Aureusvirus and ...

  11. Three-dimensional reconstruction on cell level: case study elucidates the ultrastructure of the spinning apparatus of Embia sp. (Insecta: Embioptera)

    PubMed Central

    Hörnschemeyer, Thomas; Fischer, Christian

    2016-01-01

    Spinning is a phenomenon not only present in spiders, but also in many other arthropods. The functional morphology and complexity of spinning organs is often poorly understood. Their elements are minute and studying them poses substantial methodological difficulties. This study presents a three-dimensional reconstruction of a silk gland of Embia sp. on cellular level, based on serial sections acquired with serial block-face scanning electron microscopy (SBFSEM) to showcase the power of this method. Previous studies achieved either high resolution to elucidate the ultrastructure or satisfying three-dimensional representations. The high-resolution achieved by SBFSEM can be easily used to reconstruct the three-dimensional ultrastructural organization of cellular structures. The herein investigated spinning apparatus of Embioptera can be taken as an example demonstrating the potential of this method. It was possible to reconstruct a multinucleated silk gland containing 63 nuclei. We focused on the applicability of this method in the field of morphological research and provide a step-by-step guide to the methodology. This will help in applying the method to other arthropod taxa and will help significantly in adapting the method to other animals, animal parts and tissues. PMID:27853574

  12. The effect of age on mitochondrial ultrastructure.

    PubMed

    Wilson, P D; Franks, L M

    1975-01-01

    The ultrastructure of perfused livers and of mitochondrial fractions from 6-month-old and 30-month-old C57/BL mice were studied. In old mice the liver cell mitochondria were enlarged and rounded with a light 'foamy', vacuolated matrix, short cristae and a loss of dense granules. Quantitative studies showed a 60% increase in the mean size and an increased proportion of larger mitochondria in intact 30-month-old perfused livers. Endothelial and Kupffer cell mitochondria were smaller than those of the parenchymal cells. Mitochondria in pellets prepared from 6- and 30-month-old livers were rounded and condensed, although there were a few larger and 'foamy' mitochondria in the preparations from old mice. Up to 47% of large mitochondria in the old livers were lost during cell fractionation.

  13. Machine learning based methodology to identify cell shape phenotypes associated with microenvironmental cues.

    PubMed

    Chen, Desu; Sarkar, Sumona; Candia, Julián; Florczyk, Stephen J; Bodhak, Subhadip; Driscoll, Meghan K; Simon, Carl G; Dunkers, Joy P; Losert, Wolfgang

    2016-10-01

    Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a "supercell" method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment.

  14. Spontaneous activity of morphologically identified ganglion cells in the developing ferret retina.

    PubMed

    Liets, Lauren C; Olshausen, Bruno A; Wang, Guo-Yong; Chalupa, Leo M

    2003-08-13

    Whole-cell patch-clamp recordings were made from morphologically identified ganglion cells in the intact retina of developing ferrets. As early as 3 d after birth, all ganglion cells exhibited bursts of spontaneous activity, with the interval between bursts gradually decreasing with maturity. By 2 weeks after birth, ganglion cells could be morphologically differentiated into three major classes (alpha, beta, and gamma), and at this time each cell class was characterized by a distinct pattern of spontaneous activity. Dual patch-clamp recordings from pairs of neighboring cells revealed that cells of all morphological classes burst in a coordinated manner, regardless of cell type. These observations suggest that a common mechanism underlies the bursting patterns exhibited by all ganglion cell classes, and that class-specific firing patterns emerge coincident with retinal ganglion cell morphological differentiation.

  15. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

    PubMed

    Locard-Paulet, Marie; Lim, Lindsay; Veluscek, Giulia; McMahon, Kelly; Sinclair, John; van Weverwijk, Antoinette; Worboys, Jonathan D; Yuan, Yinyin; Isacke, Clare M; Jørgensen, Claus

    2016-02-09

    The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

  16. Stage-specific embryonic antigen-4 identifies human dental pulp stem cells.

    PubMed

    Kawanabe, Noriaki; Murata, Satoko; Fukushima, Hiroaki; Ishihara, Yoshihito; Yanagita, Takeshi; Yanagita, Emmy; Ono, Mitsuaki; Kurosaka, Hiroshi; Kamioka, Hiroshi; Itoh, Tomoo; Kuboki, Takuo; Yamashiro, Takashi

    2012-03-10

    Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.

  17. The defensive secretion of Carabus lefebvrei Dejean 1826 pupa (Coleoptera, Carabidae): gland ultrastructure and chemical identification.

    PubMed

    Giglio, Anita; Brandmayr, Pietro; Dalpozzo, Renato; Sindona, Giovanni; Tagarelli, Antonio; Talarico, Federica; Brandmayr, Tullia Zetto; Ferrero, Enrico A

    2009-05-01

    This study documents the defensive function of flavored humor secreted by the abdominal glands of Carabus lefebvrei pupae. The morphology and the ultrastructure of these glands were described and the volatile compounds of glands secretion were identified by gas chromatography/mass spectrometry. The ultrastructure analysis shows an acinose complex formed by about 50 clusters. Each cluster has 20 glandular units and the unit-composed of one secretory and one canal cell lying along a duct-belongs to the class 3 cell type of Quennedey (1998). In the cytoplasm, the secretory cell contains abundant rough endoplasmatic reticula, glycogen granules, numerous mitochondria, and many well-developed Golgi complexes producing electron-dense secretory granules. Mitochondria are large, elongated, and often adjoining electronlucent vesicles. The kind and the origin of secretory granules varying in size and density were discussed. The chemical analysis of the gland secretion revealed the presence of a mixture of low molecular weight terpenes, ketones, aldehydes, alcohols, esters, and carboxylic acids. Monoterpenes, especially linalool, were the major products. We supposed that ketones, aldehydes, alcohols, esters, and carboxylic acids have a deterrent function against the predators and monoterpenes provide a prophylaxis function against pathogens.

  18. Survey on Amacrine Cells Coupling to Retrograde-Identified Ganglion Cells in the Mouse Retina

    PubMed Central

    Pang, Ji-Jie; Paul, David L.; Wu, Samuel M.

    2013-01-01

    Purpose. Retinal amacrine cells (ACs) may make inhibitory chemical synapses and potentially excitatory gap junctions on ganglion cells (GCs). The total number and subtypes of ACs coupled to the entire GC population were investigated in wild-type and three lines of transgenic mice. Methods. GCs and GC-coupled ACs were identified by the previously established LY-NB (Lucifer yellow–Neurobiotin) retrograde double-labeling technique, in conjunction with specific antibodies and confocal microscopy. Results. GC-coupled ACs (NB-positive and LY-negative) comprised nearly 11% of displaced ACs and 4% of conventional ACs in wild-type mice, and were 9% and 4% of displaced ACs in Cx45−/− and Cx36/45−/− mice, respectively. Their somas were small in Cx36/45−/− mice, but variable in other strains. They were mostly γ-aminobutyric acid (GABA)-immunoreactive (IR) and located in the GC layer. They comprised only a small portion in the AC subpopulations, including GABA-IR, glycine-IR, calretinin-IR, 5-HT-accumulating, and ON-type choline acetyltransferase (ChAT) ACs in wild-type and ChAT transgenic mice (ChAT- tdTomato). In the distal 80% of the inner plexiform layer (IPL), dense GC dendrites coexisted with rich glycine-IR and GABA-IR. In the inner 20% of the IPL, sparse GC dendrites presented with a major GABA band and sparse glycine-IR. Conclusions. Various subtypes of ACs may couple to GCs. ACs of the same immunoreactivity may either couple or not couple to GCs. Cx36 and Cx45 dominate GC-AC coupling except for small ACs. The overall potency of GC-AC coupling is moderate, especially in the proximal 20% of the IPL, where inhibitory chemical signals are dominated by GABA ACs. PMID:23821205

  19. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    PubMed Central

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  20. How methylglyoxal kills bacteria: An ultrastructural study.

    PubMed

    Rabie, Erika; Serem, June Cheptoo; Oberholzer, Hester Magdalena; Gaspar, Anabella Regina Marques; Bester, Megan Jean

    2016-01-01

    Antibacterial activity of honey is due to the presence of methylglyoxal (MGO), H2O2, bee defensin as well as polyphenols. High MGO levels in manuka honey are the main source of antibacterial activity. Manuka honey has been reported to reduce the swarming and swimming motility of Pseudomonas aeruginosa due to de-flagellation. Due to the complexity of honey it is unknown if this effect is directly due to MGO. In this ultrastructural investigation the effects of MGO on the morphology of bacteria and specifically the structure of fimbriae and flagella were investigated. MGO effectively inhibited Gram positive (Bacillus subtilis; MIC 0.8 mM and Staphylococcus aureus; MIC 1.2 mM) and Gram negative (P. aeruginosa; MIC 1.0 mM and Escherichia coli; MIC 1.2 mM) bacteria growth. The ultrastructural effects of 0.5, 1.0 and 2 mM MGO on B. substilis and E. coli morphology was then evaluated. At 0.5 mM MGO, bacteria structure was unaltered. For both bacteria at 1 mM MGO fewer fimbriae were present and the flagella were less or absent. Identified structures appeared stunted and fragile. At 2 mM MGO fimbriae and flagella were absent while the bacteria were rounded with shrinkage and loss of membrane integrity. Antibacterial MGO causes alterations in the structure of bacterial fimbriae and flagella which would limit bacteria adherence and motility.

  1. Ultrastructural Morphology of Sperm from Human Globozoospermia.

    PubMed

    Ricci, Giuseppe; Andolfi, Laura; Zabucchi, Giuliano; Luppi, Stefania; Boscolo, Rita; Martinelli, Monica; Zweyer, Marina; Trevisan, Elisa

    2015-01-01

    Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

  2. Ultrastructural Morphology of Sperm from Human Globozoospermia

    PubMed Central

    Ricci, Giuseppe; Andolfi, Laura; Zabucchi, Giuliano; Luppi, Stefania; Boscolo, Rita; Martinelli, Monica; Zweyer, Marina; Trevisan, Elisa

    2015-01-01

    Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia. PMID:26436098

  3. Oscope identifies oscillatory genes in unsynchronized single-cell RNA-seq experiments.

    PubMed

    Leng, Ning; Chu, Li-Fang; Barry, Chris; Li, Yuan; Choi, Jeea; Li, Xiaomao; Jiang, Peng; Stewart, Ron M; Thomson, James A; Kendziorski, Christina

    2015-10-01

    Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform.

  4. Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying B | Division of Cancer Prevention

    Cancer.gov

    DESCRIPTION (provided by applicant): Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying Biomarkers for Early Detection and Risk Assessment. This application addresses Program Announcement PA-09-197: Biomarkers for Early Detection of Hematopoietic Malignancies (R01). The overall aim of this project is to identify novel biomarkers that may be used to diagnose and treat patients with Langerhans Cell Histiocytosis (LCH). LCH occurs with similar frequency as other rare malignancies including Hodgkin's lymphoma and AML. |

  5. Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

    PubMed Central

    George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

    2016-01-01

    The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

  6. Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors.

    PubMed

    Abbey, Janice L; Karsunky, Holger; Serwold, Thomas; Papathanasiou, Peter; Weissman, Irving L; O'Neill, Helen C

    2015-08-01

    Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2(+) Cβ(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-)) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vβ8.2(+) Cβ(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vβ8.2(+) Cβ(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

  7. Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

    PubMed Central

    Worthley, Daniel L.; Churchill, Michael; Compton, Jocelyn T.; Tailor, Yagnesh; Rao, Meenakshi; Si, Yiling; Levin, Daniel; Schwartz, Matthew G.; Uygur, Aysu; Hayakawa, Yoku; Gross, Stefanie; Renz, Bernhard W.; Setlik, Wanda; Martinez, Ashley N.; Chen, Xiaowei; Nizami, Saqib; Lee, Heon Goo; Kang, H. Paco; Caldwell, Jon-Michael; Asfaha, Samuel; Westphalen, C. Benedikt; Graham, Trevor; Jin, Guangchun; Nagar, Karan; Wang, Hongshan; Kheirbek, Mazen A.; Kolhe, Alka; Carpenter, Jared; Glaire, Mark; Nair, Abhinav; Renders, Simon; Manieri, Nicholas; Muthupalani, Sureshkumar; Fox, James G.; Reichert, Maximilian; Giraud, Andrew S.; Schwabe, Robert F.; Pradere, Jean-Phillipe; Walton, Katherine; Prakash, Ajay; Gumucio, Deborah; Rustgi, Anil K.; Stappenbeck, Thaddeus S.; Friedman, Richard A.; Gershon, Michael D.; Sims, Peter; Grikscheit, Tracy; Lee, Francis Y.; Karsenty, Gerard; Mukherjee, Siddhartha; Wang, Timothy C.

    2014-01-01

    The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs). PMID:25594183

  8. The mammalian tubuli recti: ultrastructural study.

    PubMed

    Osman, D I; Plöen, L

    1978-09-01

    The ultrastructure of the tubuli recti was studied in the testes of sexually mature bulls, boars, rams, goats, rabbits and rats fixed by vascular perfusion. The tubuli recti are lined with a simple epithelium that varies in height, from squamous to tall columnar according to the species and the region. The cells are characterized by extensive lateral and tortuous basal plasma membranes and a luminal border with microvilli. Tight junctions and desmosomes are found in the upper half of the lateral borders. The Golgi apparatus is sizable and associated with it are coated vesicles and many smooth vesicles concentrated towards the luminal border. A distal segment of the tubuli recti is found in bulls only and is characterized by a high epithelium which is thrown into folds giving the lumen a festooned appearance. It is suggested that the epithelial cells of the tubuli recti are involved in fluid exchange and in the removal of unwanted spermatozoa.

  9. Ultrastructure and phylogeny of Ustilago coicis *

    PubMed Central

    Zhang, Jing-ze; Guan, Pei-gang; Tao, Gang; Ojaghian, Mohammad Reza; Hyde, Kevin David

    2013-01-01

    Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town, Jinyun County, Zhejiang Province of China. In this paper, ultrastructural assessments on fungus-host interactions and teliospore development are presented, and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon. Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction, and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane. Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium. In addition, internal transcribed spacer (ITS) and large subunit (LSU) sequence data showed that U. coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa (Poaceae). PMID:23549851

  10. Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography

    PubMed Central

    Sherman, Shachar; Kirchenbuechler, David; Nachmias, Dikla; Tamir, Adi; Werner, Stephan; Elbaum, Michael; Elia, Natalie

    2016-01-01

    Mammalian cytokinetic abscission is mediated by the ESCRT membrane fission machinery. While much has been clarified on the topology and kinetics of abscission through high-resolution microscopy, key questions regarding the mechanism of abscission remain open. Here we apply cryogenic soft-X-ray tomography to elucidate new ultrastructural details in the intercellular membrane bridge connecting cells undergoing abscission. In particular, we resolve defined ring-like structures inside the midbody dark zone that have been inaccessible to EM, and identify membrane extrusions at the abscission sites. In cells at late stages of abscission we resolve a complex array of helical spirals, extending the structural information obtained by EM. Our results highlight the advantages of soft-X-ray tomography and emphasize the importance of using complementary approaches for characterizing cellular structures. Notably, by providing new structural data from intact cells we present a realistic view on the topology of abscission and suggest new mechanistic models for ESCRT mediated abscission. PMID:27282220

  11. Epigenetic landscapes explain partially reprogrammed cells and identify key reprogramming gene

    NASA Astrophysics Data System (ADS)

    Lang, Alex; Li, Hu; Collins, James; Mehta, Pankaj

    2013-03-01

    A common metaphor for describing development is a rugged epigenetic landscape where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from physics, specifically Hopfield neural networks. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells (cells found in reprogramming experiments but not in vivo) are a natural consequence of high-dimensional landscapes and predicts that partially reprogrammed cells should be hybrids that coexpress genes from multiple cell fates. We verify this prediction by reanalyzing existing data sets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity.

  12. Phenolic acid allelochemicals induced morphological, ultrastructural, and cytological modification on Cassia sophera L. and Allium cepa L.

    PubMed

    Gulzar, Aasifa; Siddiqui, M B; Bi, Shazia

    2016-09-01

    The allelopathic potential of leaf aqueous extract (LAE) of Calotropis procera on growth behavior, ultrastructural changes on Cassia sophera L., and cytological changes on Allium cepa L. was investigated. LAE at different concentrations (0.5, 1, 2, and 4 %) significantly reduced the root length, shoot length, and dry biomass of C. sophera. Besides, the ultrastructural changes (through scanning electron microscopy, SEM) induced in epidermal cells of 15-day-old seedlings of Cassia leaf were also noticed. The changes induced were shrinking and contraction of epidermal cells along with the formation of major grooves, canals, and cyst-like structures. The treated samples of epidermal cells no longer seem to be smooth as compared to control. LAE at different concentrations induces chromosomal aberrations and variation in shape of the interphase and prophase nucleus in A. cepa root tip cells when compared with control groups. The mitotic index in treated onion root tips decreased with increasing concentrations of the extracts. The most frequent aberrations were despiralization at prophase with the formation of micronuclei, sticky anaphase with bridges, sticky telophase, C-metaphase, etc. The results also show the induction of ghost cells, cells with membrane damage, and cells with heterochromatic nuclei by extract treatment. Upon HPLC analysis, nine phenolic acids (caffeic acid, gentisic acid, catechol, gallic acid, syringic acid, ellagic acid, resorcinol, p-coumaric acid, and p-hydroxy benzoic acid) were identified. Thus, the phenolic acids are mainly responsible for the allelopathic behavior of C. procera.

  13. Automated cell tracking identifies mechanically oriented cell divisions during Drosophila axis elongation.

    PubMed

    Wang, Michael F Z; Hunter, Miranda V; Wang, Gang; McFaul, Christopher; Yip, Christopher M; Fernandez-Gonzalez, Rodrigo

    2017-04-01

    Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.

  14. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.

    PubMed

    Berry, David; Mader, Esther; Lee, Tae Kwon; Woebken, Dagmar; Wang, Yun; Zhu, Di; Palatinszky, Marton; Schintlmeister, Arno; Schmid, Markus C; Hanson, Buck T; Shterzer, Naama; Mizrahi, Itzhak; Rauch, Isabella; Decker, Thomas; Bocklitz, Thomas; Popp, Jürgen; Gibson, Christopher M; Fowler, Patrick W; Huang, Wei E; Wagner, Michael

    2015-01-13

    Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

  15. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    NASA Astrophysics Data System (ADS)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  16. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability

    PubMed Central

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O.; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-01-01

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  17. The Effect of Spaceflight on the Ultrastructure of the Cerebellum

    NASA Technical Reports Server (NTRS)

    Holstein, Gay R.; Martinelli, Giorgio P.

    2003-01-01

    In weightlessness, astronauts and cosmonauts may experience postural illusions as well as motion sickness symptoms known as the space adaptation syndrome. Upon return to Earth, they have irregularities in posture and balance. The adaptation to microgravity and subsequent re-adaptation to Earth occurs over several days. At the cellular level, a process called neuronal plasticity may mediate this adaptation. The term plasticity refers to the flexibility and modifiability in the architecture and functions of the nervous system. In fact, plastic changes are thought to underlie not just behavioral adaptation, but also the more generalized phenomena of learning and memory. The goal of this experiment was to identify some of the structural alterations that occur in the rat brain during the sensory and motor adaptation to microgravity. One brain region where plasticity has been studied extensively is the cerebellar cortex-a structure thought to be critical for motor control, coordination, the timing of movements, and, most relevant to the present experiment, motor learning. Also, there are direct as well as indirect connections between projections from the gravity-sensing otolith organs and several subregions of the cerebellum. We tested the hypothesis that alterations in the ultrastructural (the structure within the cell) architecture of rat cerebellar cortex occur during the early period of adaptation to microgravity, as the cerebellum adapts to the absence of the usual gravitational inputs. The results show ultrastructural evidence for neuronal plasticity in the central nervous system of adult rats after 24 hours of spaceflight. Qualitative studies conducted on tissue from the cerebellar cortex (specifically, the nodulus of the cerebellum) indicate that ultrastructural signs of plasticity are present in the cerebellar zones that receive input from the gravity-sensing organs in the inner ear (the otoliths). These changes are not observed in this region in cagematched

  18. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells

    PubMed Central

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-01-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1–2+ MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8+ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8+ subsets we demonstrated that high expression of CD26 on CD8+ TRAV1–2+ cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161hi was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161dim. Using cell surface expression of CD8, TRAV1–2, and CD26hi in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis. PMID:25752900

  19. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells.

    PubMed

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-07-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1-2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1-2(+) MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8(+) T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8(+) subsets we demonstrated that high expression of CD26 on CD8(+)  TRAV1-2(+) cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161(hi) was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161(dim) . Using cell surface expression of CD8, TRAV1-2, and CD26(hi) in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.

  20. LGR5 and Nanog identify stem cell signature of pancreas beta cells which initiate pancreatic cancer.

    PubMed

    Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Polin, Nava; Givol, David

    2013-04-05

    Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer.

  1. Pulmonary ultrastructure of the late aspects of human paraquat poisoning.

    PubMed Central

    Dearden, L. C.; Fairshter, R. D.; McRae, D. M.; Smith, W. R.; Glauser, F. L.; Wilson, A. F.

    1978-01-01

    The pulmonary ultrastructure of the late aspects of a case of human paraquat poisoning is investigated and compared with normal human pulmonary ultrastructure. Alveoli in the paraquat patient are numerically reduced in comparison to the control. They are filled with edematous proteinaceous plasma-like fluid containing erythrocytes, macrophages, leukocytes, fibroblast-like cells, platelets, and fibrin. These alveoli are lined by granular pneumocytes. Interstitial areas in the paraquat patient are greatly expanded and there are no alveolar septums. Interstitial areas contain proteinaceous plasma-like material, collagen, fibrin, platelets, mature fibroblasts, plasma cells, many leukocytes, numerous erythrocytes, and capillaries. Capillary permeability seems to be enhanced in the paraquat patient either by vesicles forming transendothelial channels or pores or by disruption of endothelial cells. Images Figure 1 Figure 2 Figures 3-7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:213978

  2. Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion

    PubMed Central

    Giordano, Marilyn; Henin, Coralie; Maurizio, Julien; Imbratta, Claire; Bourdely, Pierre; Buferne, Michel; Baitsch, Lukas; Vanhille, Laurent; Sieweke, Michael H; Speiser, Daniel E; Auphan-Anezin, Nathalie; Schmitt-Verhulst, Anne-Marie; Verdeil, Grégory

    2015-01-01

    T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed “exhausted” T cells. We compared the transcriptome of “exhausted” CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFβ and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFβ/IL-6-mediated induction of Maf. PMID:26139534

  3. A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development

    PubMed Central

    Paige, Sharon L.; Thomas, Sean; Stoick-Cooper, Cristi L.; Wang, Hao; Maves, Lisa; Sandstrom, Richard; Pabon, Lil; Reinecke, Hans; Pratt, Gabriel; Keller, Gordon; Moon, Randall T.; Stamatoyannopoulos, John; Murry, Charles E.

    2012-01-01

    Summary Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. PMID:22981225

  4. Beta4 tubulin identifies a primitive cell source for oligodendrocytes in the mammalian brain.

    PubMed

    Wu, Chuanshen; Chang, Ansi; Smith, Maria C; Won, Roy; Yin, Xinghua; Staugaitis, Susan M; Agamanolis, Dimitri; Kidd, Grahame J; Miller, Robert H; Trapp, Bruce D

    2009-06-17

    We have identified a novel population of cells in the subventricular zone (SVZ) of the mammalian brain that expresses beta4 tubulin (betaT4) and has properties of primitive neuroectodermal cells. betaT4 cells are scattered throughout the SVZ of the lateral ventricles in adult human brain and are significantly increased in the SVZs bordering demyelinated white matter in multiple sclerosis brains. In human fetal brain, betaT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. betaT4 cells represent <2% of the cells present in neurospheres generated from postnatal rat brain but >95% of cells in neurospheres treated with the anti-mitotic agent Ara C. betaT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of betaT4-positive cells with A2B5-positive oligodendrocyte progenitor cells after transplantation (25,000 cells) into postnatal day 3 (P3) myelin-deficient rat brains. At P20, the progeny of betaT4 cells myelinated up to 4 mm of the external capsule, which significantly exceeded that of transplanted A2B5-positive progenitor cells. Such extensive and rapid mature CNS cell generation by a relatively small number of transplanted cells provides in vivo support for the therapeutic potential of betaT4 cells. We propose that betaT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.

  5. Epigenetic Landscapes Explain Partially Reprogrammed Cells and Identify Key Reprogramming Genes

    PubMed Central

    Lang, Alex H.; Li, Hu; Collins, James J.; Mehta, Pankaj

    2014-01-01

    A common metaphor for describing development is a rugged “epigenetic landscape” where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from spin-glass physics. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells are a natural consequence of high-dimensional landscapes, and predicts that partially reprogrammed cells should be hybrids that co-express genes from multiple cell fates. We verify this prediction by reanalyzing existing datasets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity. PMID:25122086

  6. Comparison of the effects of Origanum vulgare with LHRH-A2 and 17β-estradiol on the ultrastructure of gonadotroph cells and ovarian oogenesis in immature Trichogaster trichopterus.

    PubMed

    Bagheri Ziari, Sedigheh; Naji, Tahereh; Hosseinzadeh Sahafi, Homayoun

    2015-10-01

    Origanum vulgare is a plant of the mint family that contains phytoestrogens. This study compared the effects of O. vulgare, LHRH-A2, and 17β-estradiol on the ultrastructure of gonadotroph cells and ovarian oogenesis in immature Trichogaster trichopterus. Fish (5.1±0.032cm and 2.1±0.043g, n=150) were randomly divided into four treatment groups (three hormonal treatments and control) and treated intramuscularly at four levels with 17β-estradiol or O. vulgare at 10, 20, 30 and 50mg/kg body weight and with LHRH-A2 at 0.001, 0.002, 0.003, and 0.005mg/kg body weight. There were three control treatments: saline, ethanol and placebo. Fish were kept in 15 tanks, with 10 fish per tank, injected a total of seven doses over 13 days. Gonadosomatic index (GSI) and oocyte diameter were lower (P≤0.05) in the control than in the three hormonal treatments. The highest GSI and oocyte diameter responses were observed in fish treated with 17β-estradiol (2.76±0.23%, 149.8±15.43mm) followed by O. vulgare (1.86±0.18%, 104.3±11.5mm) and LHRH-A2 (1.52±0.12%, 91.75±9.02mm) (P≤0.05). Moreover, there was a significant effect of dose level within all the hormonal treatments (P≤0.05). The effect of treatment on the length and weight was likely GSI. Ovarian tissue results showed faster oogenesis of oocytes in fish treated with O. vulgare, after 17β-estradiol. Ultrastructure of gonadotroph cells demonstrated less stimulation by O. vulgare than by 17β-estradiol and LHRH-A2. This study suggests that compared with the two hormonal treatments, O. vulgare dose-dependently affects ovarian oogenesis and gonadotroph cells.

  7. Tim-3 identifies exhausted follicular helper T cells in breast cancer patients.

    PubMed

    Zhu, Shiguang; Lin, Jun; Qiao, Guangdong; Wang, Xingmiao; Xu, Yanping

    2016-09-01

    Breast cancer is the most common cancer diagnosed in women worldwide. Although a series of treatment options have improved the overall 5-year survival rate to 90%, individual responses still vary from patient to patient. New evidence suggested that the infiltration of CXCL13-expressing CD4(+) follicular helper cells (Tfh) in breast tumor predicted better survival. Here, we examined the regulation of Tfh function in breast cancer patients in depth. We found that the frequencies of circulating Tfh cells were not altered in breast cancer patients compared to healthy controls. However, the expression of PD-1 and Tim-3 in Tfh cells was significantly elevated in breast cancer patients. Interestingly, we observed a preferential upregulation of PD-1 in Tim-3(+) Tfh cells compared to Tim-3(-) Tfh cells. Coexpression of PD-1 and Tim-3 is typically a hallmark of functional exhaustion in chronic virus infections and tumor. To examine whether Tim-3(+) identifies exhausted Tfh cells, we stimulated Tfh cells with anti-CD3/CD28, and found that Tim-3(+) T cells expressed reduced frequencies of chemokine CXCL13 and cytokine interleukin 21 (IL-21), and contained fewer proliferating cells, than Tim-3(-) Tfh cells. Compared to those cocultured with Tim-3(-) Tfh cells, naive B cells cocultured with Tim-3(+) Tfh cells resulted in significantly less IgM, IgG and IgA production after 12 day incubation, demonstrating a reduction in Tim-3(+) Tfh-mediated B cell help. Moreover, the frequencies of Tim-3(+) Tfh cells in resected breast tumor were further upregulated than autologous blood, suggesting a participation of Tim-3(+) Tfh cells in tumor physiology. Overall, the data presented here provided new insight in the regulation of Tfh cells in breast cancer patients.

  8. Ultrastructural observations on feeding appendages and gills of Alvinella pompejana (Annelida, Polychaeta)

    NASA Astrophysics Data System (ADS)

    Storch, V.; Gaill, F.

    1986-09-01

    The feeding appendages of Alvinella pompejana obtained from a deep-sea hydrothermal vent environment are described. They are characterized by a ciliated groove, the cells of which have a very distinctive ultrastructure, by groups of bipolar receptor cells and by several kinds of gland cells. Among these, one cell type is in an upside down position suggesting a function completely different from other epidermal secretory cells. The gills differ considerably from the feeding appendages on the basis of their ultrastructure. Their epidermis is very irregular in height; basal infoldings give the blood access to a space coming very near to the external medium. The blood vascular system is open. On the other hand, the gills of Amphicteis gunneri are not effective sites of gas exchange, since their columnar epithelium is underlain with muscle cells. The cells composing the feeding appendages and gills of Alvinella pompejana are characterized by ultrastructurally very different mitochondria.

  9. Comparative Analysis of the Morphology, Ultrastructure, and Glycosylation Pattern of the Jejunum and Ileum of the Wild Rodent Lagostomus maximus.

    PubMed

    Tano De La Hoz, María Florencia; Flamini, Mirta Alicia; Díaz, Alcira Ofelia

    2016-05-01

    Morphological and histochemical analyses were performed to characterize the histology, ultrastructure, and glycosylation pattern of the jejunum and ileum of the wild rodent Lagostomus maximus. Enterocytes, goblet cells, Paneth cells, and enteroendocrine cells were identified in both intestinal epithelia. Two morphological types of enterocytes were identified only in the ileum based on their cytoplasm electron density. Although the histological and ultrastructural examination showed that the epithelia of both anatomical regions were morphologically similar, a certain specialization in their secretory products was evident. The glycosylation pattern of the jejunum and ileum was characterized in situ by histochemical and lectin histochemical methods. Histochemical results revealed the presence of carboxylated and sulfated gycoconjugates in both regions, although sulfomucins were clearly prevalent in the ileum. Sialic acid was highly O-acetylated and particularly abundant in the jejunum. The KOH/PA*/Bh/PAS technique evidenced a more intense histochemical reaction in the jejunal than in the ileum goblet cells, demonstrating a reduction of neutral mucin secretion in the distal small intestine. Further specific differences were revealed by lectin histochemistry. These data evidenced that the nature of mucus varies at different anatomical regions, probably adapted to physiological requirements.

  10. Bioorthogonal labeling cell-surface proteins expressed in pancreatic cancer cells to identify potential diagnostic/therapeutic biomarkers

    PubMed Central

    Haun, Randy S; Quick, Charles M; Siegel, Eric R; Raju, Ilangovan; Mackintosh, Samuel G; Tackett, Alan J

    2015-01-01

    To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, it is necessary to identify cell-surface proteins that may serve as potential tumor-specific targets. In this study we used an azido-labeled bioorthogonal chemical reporter to metabolically label N-linked glycoproteins on the surface of pancreatic cancer cell lines to identify potential targets that may be exploited for detection and/or treatment of pancreatic cancer. Labeled glycoproteins were tagged with biotin using click chemistry, purified by streptavidin-coupled magnetic beads, separated by gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (MS). MS/MS analysis of peptides from 3 cell lines revealed 954 unique proteins enriched in the azido sugar samples relative to control sugar samples. A comparison of the proteins identified in each sample indicated 20% of these proteins were present in 2 cell lines (193 of 954) and 17 of the proteins were found in all 3 cell lines. Five of the 17 proteins identified in all 3 cell lines have not been previously reported to be expressed in pancreatic cancer; thus indicating that novel cell-surface proteins can be revealed through glycoprotein profiling. Western analysis of one of these glycoproteins, ecto-5′-nucleotidase (NT5E), revealed it is expressed in 8 out of 8 pancreatic cancer cell lines examined. Further, immunohistochemical analysis of human pancreatic tissues indicates NT5E is significantly overexpressed in pancreatic tumors compared to normal pancreas. Thus, we have demonstrated that metabolic labeling with bioorthogonal chemical reporters can be used to selectively enrich and identify novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas. PMID:26176765

  11. Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation

    PubMed Central

    Sokol, Ethan S.; Miller, Daniel H.; Mathis, Robert A.; Gupta, Piyush B.

    2015-01-01

    The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available. PMID:25894653

  12. Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

    PubMed Central

    Maestre-Batlle, Danay; Pena, Olga M.; Hirota, Jeremy A.; Gunawan, Evelyn; Rider, Christopher F.; Sutherland, Darren; Alexis, Neil E.; Carlsten, Chris

    2017-01-01

    Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. PMID:28165060

  13. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds.

    PubMed

    Holt, David; Parthasarathy, Ashwin B; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  14. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    NASA Astrophysics Data System (ADS)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  15. TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

    PubMed Central

    O'Malley, John T.; Williamson, David W.; Scott, Laura-Louise; Elco, Christopher P.; Teague, Jessica E.; Gehad, Ahmed; Lowry, Elizabeth L.; LeBoeuf, Nicole R.; Krueger, James G.; Robins, Harlan S.; Kupper, Thomas S.; Clark, Rachael A.

    2016-01-01

    Early diagnosis of CTCL is difficult and takes on average six years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL but the TCRγ PCR analysis in current clinical use detect clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46/46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting a niche of finite size may exist for benign T cells in skin. Lastly, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases and provided insights into the cell of origin and location of malignant CTCL cells in skin. PMID:26446955

  16. Ultrastructural Analysis of Myoblast Fusion in Drosophila

    PubMed Central

    Zhang, Shiliang; Chen, Elizabeth H.

    2015-01-01

    Summary Myoblast fusion in Drosophila has become a powerful genetic system with which to unravel the mechanisms underlying cell fusion. The identification of important components of myoblast fusion by genetic analysis has led to a molecular pathway toward our understanding of this cellular process. In addition to the application of immunohistochemistry and live imaging techniques to visualize myoblast fusion at the light microscopic level, ultrastructural analysis using electron microscopy remains an indispensable tool to reveal fusion intermediates and specific membrane events at sites of fusion. In this chapter, we describe conventional chemical fixation and high-pressure freezing/freeze substitution methods for visualizing fusion intermediates during Drosophila myoblast fusion. Furthermore, we describe an immunoelectron microscopic method for localizing specific proteins relative to the fusion apparatus. PMID:18979250

  17. Using Heterologous COS-7 Cells to Identify Semaphorin-Signaling Components.

    PubMed

    Sakurai, Atsuko; Doçi, Colleen L; Gutkind, J Silvio

    2017-01-01

    Semaphorins are a family of membrane-bound and secreted type of proteins which were initially identified as chemorepulsive axon guidance molecules. Plexins and neuropilins are two major receptor families of semaphorins, and their common downstream targets are the actin cytoskeleton and cell-to-extracellular matrix adhesions. Semaphorins promote the collapse of growth cones by inducing rapid changes in the cytoskeleton and disassembly of focal adhesion structures. When transfected with appropriate receptors, non-neuronal COS-7 cells exhibit a similar cell collapse phenotype upon semaphorin stimulation. This heterologous system using COS-7 cells has been developed and widely used to investigate semaphorin-signaling pathways. In this chapter, we describe a COS-7 collapse assay protocol used to identify semaphorin-signaling components and a method to produce recombinant class 3 semaphorin proteins.

  18. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    PubMed Central

    Fox, Jennifer T.; Sakamuru, Srilatha; Huang, Ruili; Teneva, Nedelina; Simmons, Steven O.; Xia, Menghang; Tice, Raymond R.; Austin, Christopher P.; Myung, Kyungjae

    2012-01-01

    Human ATAD5 is a biomarker for identifying genotoxic compounds because ATAD5 protein levels increase posttranscriptionally in response to DNA damage. We screened over 4,000 compounds with a cell-based quantitative high-throughput ATAD5-luciferase assay detecting genotoxic compounds. We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, as well as for antiaging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anticancer drugs. Furthermore, resveratrol and genistein killed multidrug-resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. PMID:22431602

  19. Ultrastructural morphogenesis of salmonid alphavirus 1.

    PubMed

    Herath, T K; Ferguson, H W; Thompson, K D; Adams, A; Richards, R H

    2012-11-01

    Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE-214). Different stages of viral replication were observed under electron microscopy. Virus-like particles were observed inside membrane-bound vesicles as early as 1 h following contact of the virus with the cells. Membrane-dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome-like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive-sense single-stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by 'fuzzy'-coated membranes was greater in virus-infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post-infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.

  20. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells*

    PubMed Central

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-01-01

    results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick–pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181. PMID:26424601

  1. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    PubMed

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181.

  2. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    PubMed

    Dahlman, Kimberly Brown; Parker, Joel S; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J; Sawyers, Charles L

    2012-01-01

    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  3. Ultrastructure of the membrana limitans interna after dye-assisted membrane peeling.

    PubMed

    Brockmann, Tobias; Steger, Claudia; Westermann, Martin; Nietzsche, Sandor; Koenigsdoerffer, Ekkehart; Strobel, Juergen; Dawczynski, Jens

    2011-01-01

    The purpose of this study was to investigate the ultrastructure of the membrana limitans interna (internal limiting membrane, ILM) and to evaluate alterations to the retinal cell layers after membrane peeling with vital dyes. Twenty-five patients (25 eyes) who underwent macular hole surgery were included, whereby 12 indocyanine green (ICG)- and 13 brilliant blue G (BBG)-stained ILM were analyzed using light, transmission electron and scanning electron microscopy. Retinal cell fragments on the ILM were identified in both groups using immunohistochemistry. Comparing ICG- and BBG-stained membranes, larger cellular fragments were observed at a higher frequency in the BBG group. Thereby, the findings indicate that ICG permits an enhanced separation of the ILM from the underlying retina with less mechanical destruction. A possible explanation might be seen in the known photosensitivity of ICG, which induces a stiffening and shrinkage of the ILM but also generates retinal toxic metabolites.

  4. Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila.

    PubMed

    Zeng, Xiankun; Han, Lili; Singh, Shree Ram; Liu, Hanhan; Neumüller, Ralph A; Yan, Dong; Hu, Yanhui; Liu, Ying; Liu, Wei; Lin, Xinhua; Hou, Steven X

    2015-02-24

    The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

  5. Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer

    PubMed Central

    Wong, Jocelyn P.; Natrajan, Rachael C.; Yuan, Yinyin; Tan, Aik-Choon; Huang, Paul H.

    2016-01-01

    Tumour cell-extracellular matrix (ECM) interactions are fundamental for discrete steps in breast cancer progression. In particular, cancer cell adhesion to ECM proteins present in the microenvironment is critical for accelerating tumour growth and facilitating metastatic spread. To assess the utility of tumour cell-ECM adhesion as a means for discovering prognostic factors in breast cancer survival, here we perform a systematic phenotypic screen and characterise the adhesion properties of a panel of human HER2 amplified breast cancer cell lines across six ECM proteins commonly deregulated in breast cancer. We determine a gene expression signature that defines a subset of cell lines displaying impaired adhesion to laminin. Cells with impaired laminin adhesion showed an enrichment in genes associated with cell motility and molecular pathways linked to cytokine signalling and inflammation. Evaluation of this gene set in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of 1,964 patients identifies the F12 and STC2 genes as independent prognostic factors for overall survival in breast cancer. Our study demonstrates the potential of in vitro cell adhesion screens as a novel approach for identifying prognostic factors for disease outcome. PMID:27556857

  6. Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis

    PubMed Central

    Alonso-Martin, Sonia; Rochat, Anne; Mademtzoglou, Despoina; Morais, Jessica; de Reyniès, Aurélien; Auradé, Frédéric; Chang, Ted Hung-Tse; Zammit, Peter S.; Relaix, Frédéric

    2016-01-01

    Skeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies. PMID:27446912

  7. Density-based separation in multiphase systems provides a simple method to identify sickle cell disease

    PubMed Central

    Kumar, Ashok A.; Patton, Matthew R.; Hennek, Jonathan W.; Lee, Si Yi Ryan; D’Alesio-Spina, Gaetana; Yang, Xiaoxi; Kanter, Julie; Shevkoplyas, Sergey S.; Brugnara, Carlo; Whitesides, George M.

    2014-01-01

    Although effective low-cost interventions exist, child mortality attributable to sickle cell disease (SCD) remains high in low-resource areas due, in large part, to the lack of accessible diagnostic methods. The presence of dense (ρ > 1.120 g/cm3) cells is characteristic of SCD. The fluid, self-assembling step-gradients in density created by aqueous multiphase systems (AMPSs) identifies SCD by detecting dense cells. AMPSs separate different forms of red blood cells by density in a microhematocrit centrifuge and provide a visual means to distinguish individuals with SCD from those with normal hemoglobin or with nondisease, sickle-cell trait in under 12 min. Visual evaluation of a simple two-phase system identified the two main subclasses of SCD [homozygous (Hb SS) and heterozygous (Hb SC)] with a sensitivity of 90% (73–98%) and a specificity of 97% (86–100%). A three-phase system identified these two types of SCD with a sensitivity of 91% (78–98%) and a specificity of 88% (74–98%). This system could also distinguish between Hb SS and Hb SC. To the authors’ knowledge, this test demonstrates the first separation of cells by density with AMPSs, and the usefulness of AMPSs in point-of-care diagnostic hematology. PMID:25197072

  8. Immunofluorescence identifies distinct subsets of endothelial cells in the human liver

    PubMed Central

    Strauss, Otto; Phillips, Anthony; Ruggiero, Katya; Bartlett, Adam; Dunbar, P. Rod

    2017-01-01

    As well as systemic vascular endothelial cells, the liver has specialised sinusoidal endothelial cells (LSEC). LSEC dysfunction has been documented in many diseased states yet their phenotype in normal human liver has not been comprehensively assessed. Our aim was to improve characterisation of subsets of endothelial cells and associated pericytes in the human liver. Immunofluorescence microscopy was performed on normal human liver tissue samples to assess endothelial and structural proteins in a minimum of three donors. LSEC are distributed in an acinar pattern and universally express CD36, but two distinctive subsets of LSEC can be identified in different acinar zones. Type 1 LSEC are CD36hiCD32−CD14−LYVE-1− and are located in acinar zone 1 of the lobule, while Type 2 LSEC are LYVE-1+CD32hiCD14+CD54+CD36mid-lo and are located in acinar zones 2 and 3 of the lobule. Portal tracts and central veins can be identified using markers for systemic vascular endothelia and pericytes, none of which are expressed by LSEC. In areas of low hydrostatic pressure LSEC are lined by stellate cells that express the pericyte marker CD146. Our findings identify distinctive populations of LSEC and distinguish these cells from adjacent stellate cells, systemic vasculature and pericytes in different zones of the liver acinus. PMID:28287163

  9. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    PubMed Central

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  10. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

    PubMed

    Guo, Ge; Wang, Wei; Bradley, Allan

    2004-06-24

    Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

  11. Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

    PubMed

    Sukumar, Madhusudhanan; Liu, Jie; Mehta, Gautam U; Patel, Shashank J; Roychoudhuri, Rahul; Crompton, Joseph G; Klebanoff, Christopher A; Ji, Yun; Li, Peng; Yu, Zhiya; Whitehill, Greg D; Clever, David; Eil, Robert L; Palmer, Douglas C; Mitra, Suman; Rao, Mahadev; Keyvanfar, Keyvan; Schrump, David S; Wang, Ena; Marincola, Francesco M; Gattinoni, Luca; Leonard, Warren J; Muranski, Pawel; Finkel, Toren; Restifo, Nicholas P

    2016-01-12

    Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

  12. CD87-positive tumor cells in bone marrow aspirates identified by confocal laser scanning fluorescence microscopy.

    PubMed

    Noack, F; Helmecke, D; Rosenberg, R; Thorban, S; Nekarda, H; Fink, U; Lewald, J; Stich, M; Schutze, K; Harbeck, N; Magdolen, V; Graeff, H; Schmitt, M

    1999-10-01

    Dissemination of single tumor cells to the bone marrow is a common event in cancer. The clinical significance of cytokeratin-positive cells detected in the bone marrow of cancer patients is still a matter of debate. In gastric cancer, overexpression of the receptor (uPAR or CD87) for the serine protease urokinase-type plasminogen activator (uPA) in disseminated cancer cells indicates shorter survival of cancer patients. A new immunofluorescence approach, applying confocal laser scanning microscopy, is introduced to locate CD87 antigen in cytokeratin-positive tumor cells and to quantify the CD87 antigen by consecutive scanning. At first, cytokeratin 8/18/19-positive carcinoma cells are identified at excitation wavelength 488 nm using monoclonal antibody A45B/B3 to the cytokeratins and goat anti-mouse IgG labeled with the fluorochrome Alexa488. Next, CD87 in tumor cells is identified by chicken antibody HU277 to the uPA-receptor and goat anti-chicken IgY labeled with fluorochrome Alexa568 (excitation wavelength 568 nm) and the fluorescence signal quantified on a single cell basis using fluorescently labeled latex beads as the fluorescence reference. From 16 patients with gastric or esophageal carcinoma, bone marrow aspirates were obtained, stained for cytokeratins and CD87 and then subjected to laser scanning fluorescence microscopy. Three of six gastric cancer patients had tumor cells present in the bone marrow of which 2 stained for CD87. Three of ten esophageal carcinoma patients had tumor cells in the bone marrow, all three samples stained for CD87. CD87-positive tumor cells were also dissected from stained bone marrow aspirates by laser microdissection microscope to allow analysis of single cells at the gene level.

  13. Ultrastructure damage of oviduct telocytes in rat model of acute salpingitis

    PubMed Central

    Yang, Jian; Chi, Chi; Liu, Zhen; Yang, Gang; Shen, Zong-Ji; Yang, Xiao-Jun

    2015-01-01

    Acute salpingitis (AS) is an inflammatory disease which causes severe damage to a subset of classically described cells lining in oviduct wall and contributes to interstitial fibrosis and fertility problems. Telocytes (TCs), a newly discovered peculiar type of stromal cells, have been identified in many organs, including oviduct, with proposed multiple potential bio-functions. However, with recent increasing reports regarding TCs alterations in disease-affected tissues, there is still lack of evidence about TCs involvement in AS-affected oviduct tissues and potential pathophysiological roles. We presently identified normal TCs by their characteristic ultrastructural features and immunophenotype. However, in AS-affected oviduct tissues, TCs displayed multiple ultrastructural damage both in cellular body and prolongations, with obvious loss of TCs and development of tissue fibrosis. Furthermore, TCs lose their interstitial 3-D network connected by homocellular or heterocellular junctions between TCs and adjacent cells. And especially, TCs connected to the activated immunocytes (mononuclear cells, eosinophils) and affected local immune state (repression or activation). Meanwhile, massive neutrophils infiltration and overproduced Inducible Nitric Oxide Synthase (iNOS), COX-2, suggested mechanism of inflammatory-induced TCs damage. Consequently, TCs damage might contribute to AS-induced structural and reproductive functional abnormalities of oviduct, probably via: (i) substances, energy and functional insufficiency, presumably, e.g. TC-specific genetic material profiles, ion channels, cytoskeletal elements, Tps dynamics, etc., (ii) impaired TCs-mediated multicellular signalling, such as homeostasis/angiogenesis, tissue repair/regeneration, neurotransmission, (iii) derangement of 3-D network and impaired mechanical support for TCs-mediated multicellular signals within the stromal compartment, consequently induced interstitial fibrosis, (iv) involvement in local

  14. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  15. Ultrastructural alterations in skeletal muscle fibers of rats after exercise

    NASA Technical Reports Server (NTRS)

    Akuzawa, M.; Hataya, M.

    1982-01-01

    Ultrastructural alterations in skeletal muscle fibers were electron microscopically studied in rats forced to run on the treadmill until all-out. When they were mild and limited to relatively small areas, the reconstruction of filaments ensued within 10 days without infiltration of cells. When they were severe and extensive, phagocytes infiltrated in the lesions and removed degenerative sacroplasmic debris from muscle fibers. A little later, myoblasts appeared and regeneration was accomplished in 30 days in much the same manner as in myogenesis.

  16. Amphiesmal ultrastructure in Noctiluca miliaris Suriray (Dinophyceae)

    NASA Astrophysics Data System (ADS)

    Melkonian, M.; Höhfeld, I.

    1988-09-01

    The ultrastructure of the cell covering (amphiesma) of vegetative cells of Noctiluca miliaris (Dinophyceae) was studied in detail using thin sections. The amphiesma is typically amphidinoid and contains the following components (starting from the outside): (a) a continuous outer membrane (plasmamembrane) surrounding the cell; (b) a layer of contiguous vesicles (amphiesmal vesicles) that contain a thin “honeycomb-patterned” layer of material appressed mainly to the outer portion of the vesicle membrane; (c) a finely granular pellicular layer that lies beneath the amphiesmal vesicles and (d) groups of cortical microtubules (only present in certain regions of the cell). The pellicular layer is always present but its thickness is highly variable (20 800 nm) depending on regional specializations of the amphiesma. Trichocysts and mucocysts project through the pellicular layer and amphiesmal vesicles, the apical portion of their limiting membrane docks at the plasmamembrane. Small vesicles that presumably contain material for the “honeycomb-patterned” layer traverse the pellicular layer through discontinuities and presumably fuse with the amphiesmal vesicles. We conclude that Noctiluca has a typical dinophycean (i.e. amphidinoid) cell covering, and that the most recent proposal for the developmental origin of the dinoflagellate pellicle should be revised.

  17. Ultrastructural Characterization of Zika Virus Replication Factories.

    PubMed

    Cortese, Mirko; Goellner, Sarah; Acosta, Eliana Gisela; Neufeldt, Christopher John; Oleksiuk, Olga; Lampe, Marko; Haselmann, Uta; Funaya, Charlotta; Schieber, Nicole; Ronchi, Paolo; Schorb, Martin; Pruunsild, Priit; Schwab, Yannick; Chatel-Chaix, Laurent; Ruggieri, Alessia; Bartenschlager, Ralf

    2017-02-28

    A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

  18. NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

    PubMed Central

    Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

    2016-01-01

    Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

  19. Identifying Students Difficulties in Understanding Concepts Pertaining to Cell Water Relations: An Exploratory Study.

    ERIC Educational Resources Information Center

    Friedler, Y.; And Others

    This study identified students' conceptual difficulties in understanding concepts and processes associated with cell water relationships (osmosis), determined possible reasons for these difficulties, and pilot-tested instruments and research strategies for a large scale comprehensive study. Research strategies used included content analysis of…

  20. A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Perrimon, Norbert; Brückner, Katja

    2015-01-01

    In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems. PMID:25749252

  1. Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.

    PubMed

    Xie, Sheila Q; Pombo, Ana

    2006-01-01

    The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

  2. Identification and Phylogeny of the First T Cell Epitope Identified from a Human Gut Bacteroides Species

    PubMed Central

    Perez-Muñoz, Maria Elisa; Joglekar, Payal; Shen, Yi-Ji; Chang, Kuan Y.; Peterson, Daniel A.

    2015-01-01

    Host T cell reactivity toward gut bacterial epitopes has been recognized as part of disease pathogenesis. However, the specificity of T cells that recognize this vast number of epitopes has not yet been well described. After colonizing a C57BL/6J germ-free mouse with the human gut symbiotic bacteria Bacteroides thetaiotaomicron, we isolated a T cell that recognized these bacteria in vitro. Using this T cell, we mapped the first known non-carbohydrate T cell epitope within the phylum Bacteroidetes. The T cell also reacted to two other additional Bacteroides species. We identified the peptide that stimulated the T cell by using a genetic approach. Genomic data from the epitope-positive and epitope-negative bacteria explain the cross-reactivity of the T cell to multiple species. This epitope degeneracy should shape our understanding of the T cell repertoire stimulated by the complex microbiome residing in the gastrointestinal tract in both healthy and disease states. PMID:26637014

  3. CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

    PubMed Central

    Álvarez-Viejo, María; Menéndez-Menéndez, Yolanda; Otero-Hernández, Jesús

    2015-01-01

    Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization and purification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others. PMID:25815130

  4. Transcriptome sequencing of HER2-positive breast cancer stem cells identifies potential prognostic marker.

    PubMed

    Lei, Bo; Zhang, Xian-Yu; Zhou, Jia-Peng; Mu, Guan-Nan; Li, Yi-Wen; Zhang, You-Xue; Pang, Da

    2016-11-01

    In cancer stem cell theory, breast cancer stem cells (BCSCs) are postulated to be the root cause of recurrence and metastasis in breast cancer. Discovery of new biomarkers and development of BCSC-targeted therapy are practical issues that urgently need to be addressed in the clinic. However, few breast cancer stem cell targets are known. Given that there are few BCSCs, performing transcriptome sequencing on them thus far has not been possible. With the emergence of single-cell sequencing technology, we have now undertaken such a study. We prepared single-cell suspensions, which were sorted using flow cytometry from breast tumor tissue and adjacent normal breast tissue from two HER2-positive patients. We obtained BCSCs, breast cancer cells, mammary cells, and CD44(+) mammary cells. Transcriptome sequencing was then performed on these four cell types. Using bioinformatics, we identified 404 differentially expressed BCSC genes from the HER2-positive tumors and preliminary explored transcriptome characteristics of BCSCs. Finally, by querying a public database, we found that CA12 was a novel prognostic biomarker in HER2-positive breast cancer, which also had prognostic value in all breast cancer types. In conclusion, our results suggest that CA12 may be associated with BCSCs, especially HER2-positive BCSCs, and is a potential novel therapeutic target and biomarker.

  5. Cell Type-Specific Expression Analysis to Identify Putative Cellular Mechanisms for Neurogenetic Disorders

    PubMed Central

    Xu, Xiaoxiao; Wells, Alan B.; O'Brien, David R.; Nehorai, Arye

    2014-01-01

    Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions. PMID:24453331

  6. Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

    PubMed Central

    Klima, Stefanie; Karreman, Christiaan; Grinberg, Marianna; Meisig, Johannes; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Blüthgen, Nils; Sachinidis, Agapios; Waldmann, Tanja; Leist, Marcel

    2016-01-01

    Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates. Significance The generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as

  7. Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation

    PubMed Central

    Tosaki, Asako; Bauer, Peter O.; Wada, Koji; Kurosawa, Masaru; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2014-01-01

    In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms. PMID:24705917

  8. Ultrastructure of the root cap of Arabidopsis Thaliana L. Heynh under spaceflight conditions

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Peculiarities of the ultrastructural organization of Arabidopsis root cap cells grown from the stage of two cotyledonous leaves in the Svetoblok-1 apparatus aboard the Salyut 6 research orbital station and in the laboratory are assessed. It is established that under conditions of real space flight vacuolization of the root cap cells increses considerably compared to the control variant. Changes in the topography and ulstrastructure of amyloplasts as well as lysis of cell walls are observed in the material under study. An assumption is advanced on analogous cell responses observed at the ultrastructural level to weightlessness and clinostatic conditions.

  9. eFORGE: A Tool for Identifying Cell Type-Specific Signal in Epigenomic Data.

    PubMed

    Breeze, Charles E; Paul, Dirk S; van Dongen, Jenny; Butcher, Lee M; Ambrose, John C; Barrett, James E; Lowe, Robert; Rakyan, Vardhman K; Iotchkova, Valentina; Frontini, Mattia; Downes, Kate; Ouwehand, Willem H; Laperle, Jonathan; Jacques, Pierre-Étienne; Bourque, Guillaume; Bergmann, Anke K; Siebert, Reiner; Vellenga, Edo; Saeed, Sadia; Matarese, Filomena; Martens, Joost H A; Stunnenberg, Hendrik G; Teschendorff, Andrew E; Herrero, Javier; Birney, Ewan; Dunham, Ian; Beck, Stephan

    2016-11-15

    Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology.

  10. Ultrastructural studies on Maedi-Visna virus.

    PubMed

    Weiland, F; Bruns, M

    1980-01-01

    Ultrastructural studies of Maedi-Visna virus (MVV) particles isolated from tissue culture fluids of MVV-infected cells as well as cultured cells infected with MVV were performed. MVV particles aree bounded by an envelope with projections loosely attached to its surface. Virions contain a core (sometimes two or more) of conical or ovoid shape enclosing an electron-dense nucleoid which is much smaller in diameter than the core and which can only be seen in ultrathin sections. A distinct core shell is to be found in most of the ultrasectioned particles. Cores, liberated from the virions by detergent treatment, exhibited the same shape as their enveloped counterparts. Budding structures with crescents underlying the cell membrane without an intermediate space seem to be bordered on their cell side by an electron-dense thin layer. Particles obviously representing intervenig stages of viral maturation showing parts of the crescents at the viral membrane and empty core shells could be found in single cases.

  11. Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant.

    PubMed

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M; Look, Maxime P; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A C 't; Martens, John W M; Foekens, John A; Geiger, Benjamin; van de Water, Bob

    2015-04-01

    Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3-binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

  12. Marker genes identify three somatic cell types in the fetal mouse ovary.

    PubMed

    Rastetter, Raphael H; Bernard, Pascal; Palmer, James S; Chassot, Anne-Amandine; Chen, Huijun; Western, Patrick S; Ramsay, Robert G; Chaboissier, Marie-Christine; Wilhelm, Dagmar

    2014-10-15

    The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.

  13. Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

    PubMed Central

    van Roosmalen, Wies; Le Dévédec, Sylvia E.; Golani, Ofra; Smid, Marcel; Pulyakhina, Irina; Timmermans, Annemieke M.; Look, Maxime P.; Zi, Di; Pont, Chantal; de Graauw, Marjo; Naffar-Abu-Amara, Suha; Kirsanova, Catherine; Rustici, Gabriella; Hoen, Peter A.C. ‘t; Martens, John W.M.; Foekens, John A.; Geiger, Benjamin; van de Water, Bob

    2015-01-01

    Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3–binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis. PMID:25774502

  14. Heterogeneous vesicles in mucous epithelial cells of posterior esophagus of Chinese giant salamander (Andrias davidianus).

    PubMed

    Zhang, H; Guo, X; Zhong, S; Ge, T; Peng, S; Yu, P; Zhou, Z

    2015-08-25

    The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E). Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM). The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i) electron-lucent vesicles mucous cell (ELV-MC); ii) electron-dense vesicles mucous cell (EDV-MC); and iii) mixed vesicles mucous cell (MV-MC).

  15. Monocyte-derived dendritic cells identified as booster of T follicular helper cell differentiation

    PubMed Central

    Fillatreau, Simon

    2014-01-01

    Adjuvants play an essential role in the induction of acquired immunity upon vaccination with protein antigen. In this issue of EMBO Molecular Medicine, a classical type of adjuvant made of DNA oligonucleotide containing CpG motifs, which has already been used in humans, is shown to boost humoral immunity primarily by acting on monocyte-derived dendritic cells. This study provides novel insight on the mode of action of adjuvant targeting Toll-like receptors. PMID:24803394

  16. An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis.

    PubMed

    Busser, Brian W; Lin, Yongshun; Yang, Yanqin; Zhu, Jun; Chen, Guokai; Michelson, Alan M

    2015-01-01

    Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways.

  17. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation

    PubMed Central

    Yang, Zijiang; Concannon, John; Ng, Kelvin S.; Seyb, Kathleen; Mortensen, Luke J.; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P.; Glicksman, Marcie A.; Karp, Jeffrey M.

    2016-01-01

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy. PMID:27457881

  18. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    PubMed

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  19. Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

    PubMed Central

    Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

    2012-01-01

    Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

  20. Effects of methylphenidate (Ritalin) on mammalian myocardial ultrastructure.

    PubMed

    Henderson, T A; Fischer, V W

    1995-01-01

    Previous observations have indicated lamellated ultrastructural lesions in the myocardium of a patient treated with methylphenidate (Ritalin) hydrochloride (MPH). A causal relationship between MPH exposure and these membranous changes was tested in the myocardium of rats and mice. Following injection of varying doses of MPH for different periods, myocardial ultrastructure was examined and lesions were quantified by stereological techniques. Myocardial tissue also was stained using techniques selective for acid phosphatase and for sarcoplasmic reticulum to identify possible pathogenetic mechanisms. MPH induced membrane accumulations and lamellations which were not membrane-bound and did not react for acid phosphatase, but stained positively for sarcoplasmic reticulum. Both lesions were highly focal, surrounded by normal appearing myocardial tissue. Lamellations were evident at the earliest timepoints examined and appeared to occur without lysosomal involvement. Lesions were still apparent 12 weeks after terminating MPH. These data suggest that MPH may have persistent, cumulative effects on the myocardium.

  1. Bacillus atrophaeus Outer Spore Coat Assembly and Ultrastructure

    SciTech Connect

    Plomp, M; Leighton, T J; Wheeler, K E; Pitesky, M E; Malkin, A J

    2005-11-21

    Our previous atomic force microscopy (AFM) studies successfully visualized native Bacillus atrophaeus spore coat ultrastructure and surface morphology. We have shown that the outer spore coat surface is formed by a crystalline array of {approx}11 nm thick rodlets, having a periodicity of {approx}8 nm. We present here further AFM ultrastructural investigations of air-dried and fully hydrated spore surface architecture. In the rodlet layer, planar and point defects, as well as domain boundaries, similar to those described for inorganic and macromolecular crystals, were identified. For several Bacillus species, rodlet structure assembly and architectural variation appear to be a consequence of species-specific nucleation and crystallization mechanisms that regulate the formation of the outer spore coat. We propose a unifying mechanism for nucleation and self-assembly of this crystalline layer on the outer spore coat surface.

  2. Bacillus atrophaeus outer spore coat assembly and ultrastructure.

    PubMed

    Plomp, Marco; Leighton, Terrance J; Wheeler, Katherine E; Pitesky, Maurice E; Malkin, Alexander J

    2005-11-08

    Our previous atomic force microscopy (AFM) studies successfully visualized native Bacillus atrophaeus spore coat ultrastructure and surface morphology. We have shown that the outer spore coat surface is formed by a crystalline array of approximately 11 nm thick rodlets, having a periodicity of approximately 8 nm. We present here further AFM ultrastructural investigations of air-dried and fully hydrated spore surface architecture. In the rodlet layer planar and point defects as well as domain boundaries similar to those described for inorganic and macromolecular crystals were identified. For several Bacillus species rodlet structure assembly and architectural variation appear to be a consequence of species-specific nucleation and crystallization mechanisms that regulate the formation of the outer spore coat. We propose a unifying mechanism for nucleation and self-assembly of this crystalline layer on the outer spore coat surface.

  3. A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation.

    PubMed

    Zhao, Zhenze; Ma, Xiuye; Hsiao, Tzu-Hung; Lin, Gregory; Kosti, Adam; Yu, Xiaojie; Suresh, Uthra; Chen, Yidong; Tomlinson, Gail E; Pertsemlidis, Alexander; Du, Liqin

    2014-05-15

    Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.

  4. HEPATOBLASTOMAS IN THE MUMMICHOG, FUNDULUS HETEROCLITUS (L.), FROM A CREOSOTE-CONTAMINATED ENVIRONMENT: A HISTOLOGIC, ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL STUDY

    EPA Science Inventory

    A detailed histologic and ultrastructural description of two cases of hepatoblastoma, a primitive liver cell neoplasm, is provided from mummichog, Fundulus heteroclitus(L.), inhabiting a creosote-contaminated site in the Elizabeth River, Virginia, USA. Both neoplasms were multifo...

  5. [Ultrastructural features of femoral artery myocytes during experimental leg lengthening].

    PubMed

    Ir'ianov, Iu M; Migalkin, N S; Kniazeva, L M

    1984-11-01

    Femoral arteries in mature dogs have been studied electron microscopically at various stages of the shin lengthening performed after G. A. Ilizarov method. Certain ultrastructural signs demonstrating biosynthetic and secretory activation of myocytes directed to intensification of elastogenetic processes have been revealed. Immature elastic fibers are forming around myocytes as aggregations of microfibrils, later accumulations of amorphous material appear in them. On the 28th, 42d days of distraction, hyperproduction of intra- and extracellular vesicles is noted, as well as that of intracellular matrix. Cytoplasmic islets of myocytes and intercellular connections increase in number. In the subintimal layer, of the tunica media and at its border with adventitium, longitudinally situating fasciculi of smooth muscle cells are forming. The myocytic ultrastructural peculiarities noted, the new formations of elastic elements depend, at early stages of the experiment, on changes of regional hemodynamics, and at advanced stages - also on the effect of longitudinally acting tension stress.

  6. Epidermal ultrastructure of the southern right whale calf (Eubalaena australis).

    PubMed

    Pfeiffer, C J; Rowntree, V J

    1996-04-01

    An ultrastructural analysis by transmission and scanning electron microscopy was carried out on normal epidermis of six southern right whale (Eubalaena australis) calves which stranded over a period of several months at Peninsula Valdes, Argentina. This was undertaken to 1) provide the first normal skin ultrastructural data on this highly endangered species which is known to display skin pathology in some instances, and 2) to elucidate further the integumentary specializations which have developed in diving marine mammals. Southern right whale lipokeratinocytes demonstrated parakeratosis and numerous intracellular lipid bodies, keratin and melanosomes, as reported for other cetacean species, but showed several unique ultrastructural features as well. These included a high prevalence of intranuclear inclusion bodies resembling small fragments of cytoplasmic keratin, and close structural relationship between cytoplasmic lipid droplets and the nucleus. The subcellular morphology supported the concept of possible nuclear import of cytoplasmic keratin and lipid metabolites through enlargements of the nuclear pore complex or other disruptions of the nuclear envelope. The light microscopy and scanning electron microscopy also revealed an irregular contour of the lipokeratinocytes which comprised the thick stratum externum, and surface flaking of the outermost cells which were covered by stubby microvillous-like remnants of intercellular junctions. These results thus suggest that the long-tem aquatic evolution of this cetacean species has resulted in a number of integumentary specializations and that investigation of their submicroscopic cytology may help elucidate the general cell biology of nuclear-cytoplasmic interactions.

  7. Quantitative proteomic analysis identifies new effectors of FOXM1 involved in breast cancer cell migration

    PubMed Central

    Ye, Xiaojuan; Zhang, Yi; He, Bin; Meng, Yuesheng; Li, Yandong; Gao, Yong

    2015-01-01

    The Forkhead Box M1 (FOXM1) transcription factor plays important roles in tumorigenesis and tumor metastasis in multiple human carcinomas. However, the underlying mechanisms for FOXM1 function remain to be classified. In the present study, we employed quantitative proteomic approach to search new downstream targets of FOXM1 in breast cancer MDA-MB-231 cells. A total of 4125 proteins were identified and quantified by label-free quantitation, of which 318 proteins were significantly changed (with P-value <0.05) between FOXM1 knockdown cells and control cells. Among them, three proteins ACSL4, CGGBP1 and PGRMC2 were significantly downregulated with FOXM1 reduction by western blot analysis. Further functional assays revealed that knockdown of the three proteins in MDA-MB-231 cells attenuated the ability of cell migration, consistent with the phenotype of FOXM1 knockdown. These results suggest that new potential downstream effectors of FOXM1 were identified by proteomic approach, and may provide new potential therapeutic targets in breast cancer. PMID:26884854

  8. High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

    PubMed Central

    Chng, Zhenzhi; Peh, Gary S. L.; Herath, Wishva B.; Cheng, Terence Y. D.; Ang, Heng-Pei; Toh, Kah-Peng; Robson, Paul; Mehta, Jodhbir S.; Colman, Alan

    2013-01-01

    Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. PMID:23844023

  9. Platelet satellitism: an ultrastructural study.

    PubMed Central

    Payne, C. M.

    1981-01-01

    The ultrastructural morphology of platelet-polymorph (platelet-polymorphonuclear leukocyte) rosettes was investigated in EDTA-anticoagulated blood obtained from two patients who exhibited the phenomenon of platelet satellitism. Most of the platelet profiles were attached to the polymorph surface by broad areas of contact. Examination of these broad areas of contact at high magnification revealed an intercellular material of low electron density. This material appeared to form strands, which bridged the intercellular space and spanned the entire area formed by the apposing plasma membranes. Phagocytosis of entire platelets was only observed in 1 case. The platelet profiles that participated in rosette formation revealed a large number of glycogen particles, compared with unattached platelets. Ultrastructural examination of "stress" platelets obtained from five normal subjects treated with steroids similarly showed a large number of glycogen particles, although no rosette formation or phagocytosis of platelets was observed. The etiology of platelet satellitism is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7223859

  10. Novel Macula-Like Virus Identified in Bombyx mori Cultured Cells

    PubMed Central

    Katsuma, Susumu; Tanaka, Shinichiro; Omuro, Naoko; Takabuchi, Lisa; Daimon, Takaaki; Imanishi, Shigeo; Yamashita, Shuichi; Iwanaga, Masashi; Mita, Kazuei; Maeda, Susumu; Kobayashi, Masahiko; Shimada, Toru

    2005-01-01

    We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells. PMID:15827172

  11. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

    PubMed Central

    Costigan, C; Gehrung, S; Snyder, M

    1992-01-01

    The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes. Images PMID:1545797

  12. Method for automatically identifying spectra of different wood cell wall layers in Raman imaging data set.

    PubMed

    Zhang, Xun; Ji, Zhe; Zhou, Xia; Ma, Jian-Feng; Hu, Ya-Hong; Xu, Feng

    2015-01-20

    The technique of Raman spectroscopic imaging is finding ever-increasing applications in the field of wood science for its ability to provide spatial and spectral information about the sample. On the basis of the acquired Raman imaging data set, it is possible to determine the distribution of chemical components in various wood cell wall layers. However, the Raman imaging data set often contains thousands of spectra measured at hundreds or even thousands of individual frequencies, which results in difficulties accurately and quickly extracting all of the spectra within a specific morphological region of wood cell walls. To address this issue, the authors propose a new method to automatically identify Raman spectra of different cell wall layers on the basis of principal component analysis (PCA) and cluster analysis. A Raman imaging data set collected from a 55.5 μm × 47.5 μm cross-section of poplar tension wood was analyzed. Several thousand spectra were successfully classified into five groups in accordance with different morphological regions, namely, cell corner (CC), compound middle lamella (CML), secondary wall (SW), gelatinous layer (G-layer), and cell lumen. Their corresponding average spectra were also calculated. In addition, the relationship between different characteristic peaks in the obtained Raman spectra was estimated and it was found that the peak at 1331 cm(-1) is more related to lignin rather than cellulose. Not only can this novel method provide a convenient and accurate procedure for identifying the spectra of different cell wall layers in a Raman imaging data set, but it also can bring new insights into studying the morphology and topochemistry in wood cell walls.

  13. Vulvar angiomyxoma, aggressive angiomyxoma, and angiomyofibroblastoma: an immunohistochemical and ultrastructural study.

    PubMed

    Alameda, Francesc; Munné, Assumpció; Baró, Teresa; Iglesias, Mar; Condom, Enric; Lloreta-Trull, Josep; Serrano, Sergi

    2006-01-01

    To investigate the histogenetical unifying theory of a single, pluripotential primitive cell for vulvar angiomyxoma, aggresive angiomyxoma, and angiomyofibroblastoma, an optical, immunohistochemical and ultrastructural study of a superficial angiomyxoma, aggressive angiomyxoma, and angiomyofibroblastoma was performed. These three tumors showed immunohistochemical and ultrastructural overlapping features. The results of the study suggest that these three tumor entities probably arise on a common pluripotential primitive cell located around the vessels of connective tissue, which could show the capacity for modulating its penotype toward similar but distinct mature cell types.

  14. High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

    PubMed

    Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

    2014-06-01

    The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

  15. Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

    PubMed

    Jarial, M S; Wilkins, J H

    2003-10-01

    The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

  16. Ultrastructure and morphogenesis of human immunodeficiency virus.

    PubMed

    Nakai, M; Goto, T

    1996-08-01

    The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.

  17. Nonspecific Cytotoxic Cell Antimicrobial Protein (NCAMP-1): A Novel Alarmin Ligand Identified in Zebrafish

    PubMed Central

    Monette, Margaret Mariscal; Evans, Donald Lee; Krunkosky, Thomas; Camus, Alvin; Jaso-Friedmann, Liliana

    2015-01-01

    Cells from the coelomic cavity of adult zebrafish (zf) were used to study the alarmin-like activities of nonspecific cytotoxic cell antimicrobial protein-1 (NCAMP-1). Immunohistochemistry studies using polyclonal anti-NCAMP-1 identified constitutive NCAMP-1 in epithelial cells of the zf anterior kidney, in liver parenchyma and in the lamina propria of the intestine. NCAMP-1 was also located in the cytosol of mononuclear cells in these tissues. Cytosolic NCAMP-1 was detected in a diverse population of coelomic cells (CC) using confocal microscopy and polyclonal anti-NCAMP-1 staining. Large mononuclear and heterophil-like CC had intracellular NCAMP-1. These studies indicated that NCAMP-1 is constitutively found in epithelial cells and in ZFCC. To establish a relationship between NCAMP-1 and the alarmin functions of ATP, a stimulation-secretion model was initiated using zf coelomic cells (ZFCC). ZFCCs treated with the alarmin ATP secreted NCAMP-1 into culture supernatants. Treatment of ZFCC with either ATP or NCAMP-1 activated purinergic receptor induced pore formation detected by the ZFCC uptake of the dye YO-PRO-1. ATP induced YO-PRO-1 uptake was inhibited by antagonists oxidized-ATP, KN62, or CBB. These antagonists did not compete with NCAMP-1 induced YO-PRO-1 uptake. Binding of ZFCC by both ATP and NCAMP-1 produced an influx of Ca2+. Combined treatment of ZFCC with ATP and NCAMP-1 increased target cell cytotoxicity. Individually NCAMP-1 or ATP treatment did not produce target cell damage. Similar to ATP, NCAMP-1 activates cellular pore formation, calcium influx and cytotoxicity. PMID:25689842

  18. Identifying same-cell contours in image stacks: a key step in making 3D reconstructions.

    PubMed

    Leung, Tony Kin Shun; Veldhuis, Jim H; Krens, S F Gabby; Heisenberg, C P; Brodland, G Wayne

    2011-02-01

    Identification of contours belonging to the same cell is a crucial step in the analysis of confocal stacks and other image sets in which cell outlines are visible, and it is central to the making of 3D cell reconstructions. When the cells are close packed, the contour grouping problem is more complex than that found in medical imaging, for example, because there are multiple regions of interest, the regions are not separable from each other by an identifiable background and regions cannot be distinguished by intensity differences. Here, we present an algorithm that uses three primary metrics-overlap of contour areas in adjacent images, co-linearity of the centroids of these areas across three images in a stack, and cell taper-to assign cells to groups. Decreasing thresholds are used to successively assign contours whose membership is less obvious. In a final step, remaining contours are assigned to existing groups by setting all thresholds to zero and groups having strong hour-glass shapes are partitioned. When applied to synthetic data from isotropic model aggregates, a curved model epithelium in which the long axes of the cells lie at all possible angles to the transection plane, and a confocal image stack, algorithm assignments were between 97 and 100% accurate in sets having at least four contours per cell. The algorithm is not particularly sensitive to the thresholds used, and a single set of parameters was used for all of the tests. The algorithm, which could be extended to time-lapse data, solves a key problem in the translation of image data into cell information.

  19. A strategy to identify genes associated with circulating solid tumor cell survival in peripheral blood.

    PubMed Central

    Fournier, M. V.; Carvalho, M. G.; Pardee, A. B.

    1999-01-01

    Efforts in metastasis research have centered on the phenotypic and genetic differences between primary site and metastatic site tumors. However, genes that may be used as molecular markers of metastasis in circulating tumor cells remain unidentified. Genes regulating the dissemination and survival of solid tumor cells in the blood, as well as their adaptation to new environments, could be candidates for unique metastatic tumor markers. Differential display (DD) was conducted to compare the blood of tumor-free individuals with the blood of patients with lung, breast, and colon cancers. Twenty-one up-expressed genes in the tumor patient blood samples but none in the tumor-free donor blood samples were identified. Nine of these samples were isolated, amplified, and directly sequenced. A gene AB-1 homologous to a Bcl-2 family member, which might function as an apoptosis inhibitor, was identified. The overexpression of an apoptosis inhibitor in blood from patients with metastatic tumors might be correlated with the capability of solid tumor cells to survive in peripheral blood. This is the first demonstration of the usefulness of comparing control and patient blood samples by DD to find novel potential genetic markers identifying metastasis in the blood. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p313.html Images Fig. 1 Fig. 2 Fig. 3 PMID:10390547

  20. Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

    PubMed

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.

  1. Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

    PubMed Central

    Justice, James F.; Morgan, Robin W.

    2015-01-01

    ABSTRACT Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. PMID:26670384

  2. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

  3. Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone

    PubMed Central

    Terayama, Hayato; Hirai, Shuichi; Naito, Munekazu; Qu, Ning; Katagiri, Chiaki; Nagahori, Kenta; Hayashi, Shogo; Sasaki, Hiraku; Moriya, Shota; Hiramoto, Masaki; Miyazawa, Keisuke; Hatayama, Naoyuki; Li, Zhong-Lian; Sakabe, Kou; Matsushita, Masayuki; Itoh, Masahiro

    2016-01-01

    There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body’s immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies. PMID:27752123

  4. Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma

    PubMed Central

    Li, C-Q; Huang, G-W; Wu, Z-Y; Xu, Y-J; Li, X-C; Xue, Y-J; Zhu, Y; Zhao, J-M; Li, M; Zhang, J; Wu, J-Y; Lei, F; Wang, Q-Y; Li, S; Zheng, C-P; Ai, B; Tang, Z-D; Feng, C-C; Liao, L-D; Wang, S-H; Shen, J-H; Liu, Y-J; Bai, X-F; He, J-Z; Cao, H-H; Wu, B-L; Wang, M-R; Lin, D-C; Koeffler, H P; Wang, L-D; Li, X; Li, E-M; Xu, L-Y

    2017-01-01

    Long non-coding RNAs (lncRNAs) have a critical role in cancer initiation and progression, and thus may mediate oncogenic or tumor suppressing effects, as well as be a new class of cancer therapeutic targets. We performed high-throughput sequencing of RNA (RNA-seq) to investigate the expression level of lncRNAs and protein-coding genes in 30 esophageal samples, comprised of 15 esophageal squamous cell carcinoma (ESCC) samples and their 15 paired non-tumor tissues. We further developed an integrative bioinformatics method, denoted URW-LPE, to identify key functional lncRNAs that regulate expression of downstream protein-coding genes in ESCC. A number of known onco-lncRNA and many putative novel ones were effectively identified by URW-LPE. Importantly, we identified lncRNA625 as a novel regulator of ESCC cell proliferation, invasion and migration. ESCC patients with high lncRNA625 expression had significantly shorter survival time than those with low expression. LncRNA625 also showed specific prognostic value for patients with metastatic ESCC. Finally, we identified E1A-binding protein p300 (EP300) as a downstream executor of lncRNA625-induced transcriptional responses. These findings establish a catalog of novel cancer-associated functional lncRNAs, which will promote our understanding of lncRNA-mediated regulation in this malignancy. PMID:28194033

  5. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    PubMed

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

  6. Use of anti-idiotypic antibodies to identify a receptor for the T-cell I-J determinant.

    PubMed Central

    Zupko, K; Waltenbaugh, C; Diamond, B

    1985-01-01

    In order to identify the molecule(s) interacting with the I-J determinant on suppressor T cells, we have generated two anti-idiotypic sera: one to monoclonal anti-I-Jd antibody and one to monoclonal anti-I-Jk antibody. These antisera specifically block suppressor T-cell function in a genetically restricted manner and have no effect on helper T-cell activation. Both recognize a marker on primary monocytes and B cells but not on T cells. A myeloma cell line bearing this marker has been identified. Therefore, these antisera may recognize a molecule on cells interacting with suppressor T cells that is involved in mediating suppressor T-cell activity. The relationship between the T-cell I-J determinant and the molecule identified by the anti-idiotype may be similar to the relationship between the receptor on helper T cells and Ia molecules. Images PMID:2932739

  7. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.

    PubMed

    Rodgers, Ursula R; Lanyon-Hogg, Thomas; Masumoto, Naoko; Ritzefeld, Markus; Burke, Rosemary; Blagg, Julian; Magee, Anthony I; Tate, Edward W

    2016-12-16

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.

  8. Cell-Based Screening Identifies Paroxetine as an Inhibitor of Diabetic Endothelial Dysfunction

    PubMed Central

    Gerö, Domokos; Szoleczky, Petra; Suzuki, Kunihiro; Módis, Katalin; Oláh, Gabor; Coletta, Ciro; Szabo, Csaba

    2013-01-01

    We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of <0.5%. Follow-up studies focused on paroxetine, a clinically used antidepressant compound that has not been previously implicated in the context of hyperglycemia or diabetes. Paroxetine reduced hyperglycemia-induced mitochondrial ROS formation, mitochondrial protein oxidation, and mitochondrial and nuclear DNA damage, without interfering with mitochondrial electron transport or cellular bioenergetics. The ability of paroxetine to improve hyperglycemic endothelial cell injury was unique among serotonin reuptake blockers and can be attributed to its antioxidant effect, which primarily resides within its sesamol moiety. Paroxetine maintained the ability of vascular rings to respond to the endothelium-dependent relaxant acetylcholine, both during in vitro hyperglycemia and ex vivo, in a rat model of streptozotocin-induced diabetes. Thus, the current work identifies a novel pharmacological action of paroxetine as a protector of endothelial cells against hyperglycemic injury and raises the potential of repurposing of this drug for the experimental therapy of diabetic cardiovascular complications. PMID:23223176

  9. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells

    PubMed Central

    2016-01-01

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function. PMID:27779865

  10. Gene trapping identifies a putative tumor suppressor and a new inducer of cell migration

    SciTech Connect

    Guardiola-Serrano, Francisca; Haendeler, Judith; Lukosz, Margarete; Sturm, Karsten; Melchner, Harald von; Altschmied, Joachim

    2008-11-28

    Tumor necrosis factor alpha (TNF{alpha}) is a pleiotropic cytokine involved in apoptotic cell death, cellular proliferation, differentiation, inflammation, and tumorigenesis. In tumors it is secreted by tumor associated macrophages and can have both pro- and anti-tumorigenic effects. To identify genes regulated by TNF{alpha}, we performed a gene trap screen in the mammary carcinoma cell line MCF-7 and recovered 64 unique, TNF{alpha}-induced gene trap integration sites. Among these were the genes coding for the zinc finger protein ZC3H10 and for the transcription factor grainyhead-like 3 (GRHL3). In line with the dual effects of TNF{alpha} on tumorigenesis, we found that ZC3H10 inhibits anchorage independent growth in soft agar suggesting a tumor suppressor function, whereas GRHL3 strongly stimulated the migration of endothelial cells which is consistent with an angiogenic, pro-tumorigenic function.

  11. Morphological, immunohistochemical and ultrastructural characterization of the skin of turbot (Psetta maxima L.).

    PubMed

    Faílde, L D; Bermúdez, R; Vigliano, F; Coscelli, G A; Quiroga, M I

    2014-10-01

    This study was undertaken to identify the normal morphologic, immunohistochemical and ultrastructural features of skin of the turbot (Psetta maxima L.). In the turbot skin, three morphologically distinct layers were identified: epidermis, dermis and hypodermis. The epidermis was non-keratinizing, stratified squamous epithelium that varies in thickness from 5 to 14 cells and 60 to 100μm in size. Goblet cells were seen randomly distributed between malpighian cells in the epidermal layer. These mucous cells were mainly located in the upper third of the epidermis and displayed a spherical to elongated morphology. Dermis was divided in two well-differentiated layers, the superficial stratum laxum and the deeper stratum compactum. Hypodermis was a loose layer mainly composed by adipocytes but we could observe variable amounts of fibroblast, collagen and blood vessels. In turbot two pigmentary layers could be identified: the pigmentary layer of dermis was located between basement membrane and dermis and the pigmentary layer of hypodermis immediately above the muscular layer. Three different types of chromatophores were present: melanophores, iridophores and xanthophores. The main differences observed between groups of fish with different colouration were in the amount of melanophores and xanthophores. The purpose of this article is to provide an overview of normal cutaneous biology prior to consideration of specific cutaneous alterations and diseases in turbot.

  12. Identifying microRNA-mRNA regulatory network in gemcitabine-resistant cells derived from human pancreatic cancer cells.

    PubMed

    Shen, Yehua; Pan, Yan; Xu, Litao; Chen, Lianyu; Liu, Luming; Chen, Hao; Chen, Zhen; Meng, Zhiqiang

    2015-06-01

    Pancreatic cancer is unresectable in over 80 % of patients owing to difficulty in early diagnosis. Chemotherapy is the most frequently adopted therapy for advanced pancreatic cancer. The development of drug resistance to gemcitabine (GEM), which is always used in standard chemotherapy, often results in therapeutic failure. However, the molecular mechanisms underlying the gemcitabine resistance remain unclear. Therefore, we sought to explore the microRNA-mRNA network that is associated with the development of gemcitabine resistance and to identify molecular targets for overcoming the gemcitabine resistance. By exposing SW1990 pancreatic cancer cells to long-term gemcitabine with increasing concentrations, we established a gemcitabine-resistant cell line (SW1990/GEM) with a high IC50 (the concentration needed for 50 % growth inhibition, 847.23 μM). The mRNA and microRNA expression profiles of SW1990 cells and SW1990/GEM cells were determined using RNA-seq analysis. By comparing the results in control SW1990 cells, 507 upregulated genes and 550 downregulated genes in SW1990/GEM cells were identified as differentially expressed genes correlated with gemcitabine sensitivity. Gene ontology (GO) analysis showed that the differentially expressed genes were related to diverse biological processes. The upregulated genes were mainly associated with drug response and apoptosis, and the downregulated genes were correlated with cell cycle progression and RNA splicing. Concurrently, the differentially expressed microRNAs, which are the important player in drug resistance development, were also examined in SW1990/GEM cells, and 56 differential microRNAs were identified. Additionally, the expression profiles of selected genes and microRNAs were confirmed by using Q-PCR assays. Furthermore, combining the differentially expressed microRNAs and mRNAs as well as the predicted targets for these microRNAs, a core microRNA-mRNA regulatory network was constructed, which included hub micro

  13. Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

    PubMed

    Bozzacco, Leonia; Yu, Haiqiang; Dengjel, Jörn; Trumpfheller, Christine; Zebroski, Henry A; Zhang, Nawei; Küttner, Victoria; Ueberheide, Beatrix M; Deng, Haiteng; Chait, Brian T; Steinman, Ralph M; Mojsov, Svetlana; Fenyö, David

    2012-01-01

    Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

  14. Lipid bodies in coral-dinoflagellate endosymbiosis: proteomic and ultrastructural studies.

    PubMed

    Peng, Shao-En; Chen, Wan-Nan U; Chen, Hung-Kai; Lu, Chi-Yu; Mayfield, Anderson B; Fang, Lee-Shing; Chen, Chii-Shiarng

    2011-09-01

    Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef-building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5-4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB-associated proteins were then purified, subjected to SDS-PAGE, and identified by MS using an LC-nano-ESI-MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high-electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron-transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.

  15. Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells.

    PubMed

    Wang, Yu-Chi; Chao, Tai-Kuang; Chang, Cheng-Chang; Yo, Yi-Te; Yu, Mu-Hsien; Lai, Hung-Cheng

    2013-01-01

    The primary cause of death from breast cancer is the progressive growth of tumors and resistance to conventional therapies. It is currently believed that recurrent cancer is repopulated according to a recently proposed cancer stem cell hypothesis. New therapeutic strategies that specifically target cancer stem-like cells may represent a new avenue of cancer therapy. We aimed to discover novel compounds that target breast cancer stem-like cells. We used a dye-exclusion method to isolate side population (SP) cancer cells and, subsequently, subjected these SP cells to a sphere formation assay to generate SP spheres (SPS) from breast cancer cell lines. Surface markers, stemness genes, and tumorigenicity were used to test stem properties. We performed a high-throughput drug screening using these SPS. The effects of candidate compounds were assessed in vitro and in vivo. We successfully generated breast cancer SPS with stem-like properties. These SPS were enriched for CD44(high) (2.8-fold) and CD24(low) (4-fold) cells. OCT4 and ABCG2 were overexpressed in SPS. Moreover, SPS grew tumors at a density of 10(3), whereas an equivalent number of parental cells did not initiate tumor formation. A clinically approved drug, niclosamide, was identified from the LOPAC chemical library of 1,258 compounds. Niclosamide downregulated stem pathways, inhibited the formation of spheroids, and induced apoptosis in breast cancer SPS. Animal studies also confirmed this therapeutic effect. The results of this proof-of-principle study may facilitate the development of new breast cancer therapies in the near future. The extension of niclosamide clinical trials is warranted.

  16. The ultrastructure of prolactin cells in the annual cyprinodont Cynolebias whitei during its life cycle. A morphometric study in freshwater- and saltwater-reared fish.

    PubMed

    Ruijter, J M; Creuwels, L A

    1988-08-01

    Prolactin (PRL) cells were studied electron-microscopically and morphometrically in the annual cyprinodont fish, Cynolebias whitei during its life cycle. In prehatching larvae, PRL cells possessed small secretory granules, giant mitochondria and a well-developed Golgi apparatus. During hatching, no changes were observed in the volume density of the secretory granules, indicating that no increased release of PRL occurs at hatching. A significant change in the composition of PRL cells, i.e., the volume densities per cytoplasm volume of the different organelles, occurred between one day and one week of age. Thereafter, only minor differences were observed between age groups, indicating that no major changes occur in PRL cell activity during the lifespan of C. whitei. However, the volume density per cell volume of the nucleus decreased steadily with age during the lifespan. A comparison of the PRL cells in young and adult fish reared in fresh water (FW) with siblings reared from hatching in diluted sea water (1/3 SW) did not reveal any differences with respect to the volume densities of the organelles, including the secretory granules. However, significant differences were observed with respect to the diameter, electron-dense content and affinity to anti-PRL serum of the secretory granules. These differences indicate that, despite the similar volumetric composition of the PRL cells, their secretory granules contain a substantially higher concentration of PRL in FW-reared fish than in 1/3 SW-reared fish.

  17. Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo.

    PubMed

    Newgreen, D F; Gibbins, I L; Sauter, J; Wallenfels, B; Wütz, R

    1982-01-01

    The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15-40nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (greater than 3nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (greater than 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen. Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates.

  18. Ultrastructural evidence for AMF mediated salt stress mitigation in Trigonella foenum-graecum.

    PubMed

    Evelin, Heikham; Giri, Bhoopander; Kapoor, Rupam

    2013-01-01

    The study unveils that inoculation with arbuscular mycorrhizal fungus (Glomus intraradices Schenck and Smith) prevents salt-induced ultrastructural alterations in fenugreek (Trigonella foenum-graecum L.) plants. Mycorrhizal (M) and non-mycorrhizal (NM) fenugreek plants were subjected to four levels of NaCl (0, 50, 100, and 200 mM NaCl). Salt-induced ultrastructural changes were captured using a Transmission Electron Microscope. Effects of salt on the ultrastructure of cells include shrinkage of protoplasm, widening apoplastic space between cell wall and cell membrane, disorganization of grana in chloroplast--swelling and reduction in the number of thylakoids, disintegration of chloroplast membrane, accumulation of plastoglobules, dilation of cristae and denser matrix in mitochondria, and aggregation of chromatin in nucleus. However, the extent of salt-induced ultrastructural damage was less in M plants as compared to NM plants. Lower lipid peroxidation and electrolyte leakage in M plants also indicated less membrane damage. This reduction of ultrastructure damage is a demonstration of enhanced tolerance in M plants to salt stress. The AMF-mediated lesser damage may be due to higher osmolyte (glycinebetaine, sugars) and polyamines concentration, and more and bigger plastoglobules (higher α-tocopherol concentration) in M plants as compared to NM plants. While lower Na(+) and Cl(-) ions assures less ionic toxicity, higher osmolytes and tocopherols ensure osmotic adjustment and better capacity to scavenge free radicals generated due to salt stress, respectively.

  19. Novel radiation response genes identified in gene-trapped MCF10A mammary epithelial cells.

    PubMed

    Malone, Jennifer; Ullrich, Robert

    2007-02-01

    We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2-30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

  20. Comparing plasma and X-ray exposure and identifying vulnerable cell parts

    NASA Astrophysics Data System (ADS)

    Graham, Bill

    2012-10-01

    Here two issues in plasma medicine that are being addressed in a collaboration between the Centre of Plasma Physics and the School of Pharmacy at Queen's University Belfast and the Plasma Institute at York University UK will be discussed. Recent measurements of the interaction of plasmas created directly in DMEM cell medium and MDAMB-231, a human breast cancer cell line, showed evidence of reduced cell viability and of DNA damage. The same set of experiments were undertaken but with X-ray exposure. A correlation of the dependence on plasma exposure time and X-ray dose was observed which might point the way to dose definition in plasma medicine. We have also been working to identify the cell parts most vulnerable to plasma exposure. In this study a 10 kHz atmospheric pressure non-thermal plasma jet, operating in He/0.5%O2 and characterized to determine the behavior of many of the plasma species, was incident onto the surface of media containing either bacterial strains, in their planktonic and biofilm forms, or isolated bacterial plasmid DNA. The results of measurements to look for changes in plasmid structural conformation, rates of single and double strand breaks, the catalytic activity of certain bacterial enzymes, the peroxidation of lipid content of the bacterial cells, the leakage of ATP and Scanning Electron Microscope (SEM) images will be discussed.

  1. Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

    NASA Astrophysics Data System (ADS)

    Cho, Edward H.; Wendel, Marco; Luttgen, Madelyn; Yoshioka, Craig; Marrinucci, Dena; Lazar, Daniel; Schram, Ethan; Nieva, Jorge; Bazhenova, Lyudmila; Morgan, Alison; Ko, Andrew H.; Korn, W. Michael; Kolatkar, Anand; Bethel, Kelly; Kuhn, Peter

    2012-02-01

    Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

  2. Genome-wide association study identifies novel susceptibility loci for cutaneous squamous cell carcinoma

    PubMed Central

    Chahal, Harvind S.; Lin, Yuan; Ransohoff, Katherine J.; Hinds, David A.; Wu, Wenting; Dai, Hong-Ji; Qureshi, Abrar A.; Li, Wen-Qing; Kraft, Peter; Tang, Jean Y.; Han, Jiali; Sarin, Kavita Y.

    2016-01-01

    Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7–11% of Caucasians in the United States. The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown. Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls. Eleven loci reached genome-wide significance (P<5 × 10−8) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2. We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation. These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma. PMID:27424798

  3. Vasa identifies germ cells and critical stages of oogenesis in the Asian seabass.

    PubMed

    Xu, Hongyan; Lim, Menghuat; Dwarakanath, Manali; Hong, Yunhan

    2014-01-01

    Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous m