scholarly journals Cubic membranes: a structure-based design for DNA uptake

2008 ◽  
Vol 5 (26) ◽  
pp. 1023-1029 ◽  
Author(s):  
Zakaria Almsherqi ◽  
Stephen Hyde ◽  
Malarmathy Ramachandran ◽  
Yuru Deng
Keyword(s):  
Mammalian Cells ◽  
Gene Transfection ◽  
Three Dimensional ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Dna Uptake ◽  
Cell Organelles ◽  
Electron Microscopic ◽  
Culture Cells ◽  
Cubic Membrane

Cubic membranes are soft three-dimensional crystals found within cell organelles in a variety of living systems, despite the aphorism of Fedorov: ‘crystallization is death’. They consist of multi-bilayer lipid–protein stacks, folded onto anticlastic surfaces that resemble triply periodic minimal surfaces, forming highly swollen crystalline sponges. Although cubic membranes have been observed in numerous cell types and under different pathophysiological conditions, knowledge about the formation and potential function(s) of non-lamellar, cubic structures in biological systems is scarce. We report that mitochondria with this cubic membrane organization isolated from starved amoeba Chaos carolinense interact sufficiently with short segments of phosphorothioate oligonucleotides (PS-ODNs) to give significant ODNs uptake. ODNs condensed within the convoluted channels of cubic membrane by an unknown passive targeting mechanism. Moreover, the interaction between ODNs and cubic membrane is sufficient to retard electrophoretic mobility of the ODN component in the gel matrix. These ODN–cubic membrane complexes are readily internalized within the cytoplasm of cultured mammalian cells. Transmission electron microscopic analysis confirms ODNs uptake by cubic membranes and internalization of ODN–cubic membrane complexes into the culture cells. Cubic membranes thus may offer a new, potentially benign medium for gene transfection.

scholarly journals Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids

2001 ◽  
Vol 75 (2) ◽  
pp. 961-970 ◽  
Author(s):  
Nico-Dirk van Loo ◽  
Elisabetta Fortunati ◽  
Erich Ehlert ◽  
Martijn Rabelink ◽  
Frank Grosveld ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Transport ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Nuclear Pore ◽  
Fusion Event ◽  
Electron Microscopic ◽  
Cytoplasmic Transport ◽  
Baculovirus Infection

ABSTRACT We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

scholarly journals Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

2002 ◽  
Vol 76 (15) ◽  
pp. 7672-7682 ◽  
Author(s):  
Reginald F. Clayton ◽  
Ania Owsianka ◽  
Jim Aitken ◽  
Susan Graham ◽  
David Bhella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Recombinant Baculovirus ◽  
Microscopic Analysis ◽  
Surface Topology ◽  
Viral Glycoprotein ◽  
Electron Microscopic ◽  
Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

All About Cells

Bioprinting ◽  
2021 ◽  
pp. 21-39
Author(s):  
Kenneth Douglas
Keyword(s):  
Stem Cells ◽  
Cell Membrane ◽  
Three Dimensional ◽  
Scuba Diving ◽  
Cell Types ◽  
Two Dimensional ◽  
Cell Organelles ◽  
Outer Skin ◽  
Induced Pluripotent ◽  
Selective Access

Abstract: This chapter takes the reader on an imaginary scuba diving tour of the watery world of the cell and its surroundings, pointing out features such as the cytoskeleton (that forms the equivalent of the bones and muscles of our cells), the cell membrane (the outer skin of the cell), and the cell membrane’s embedded proteins that provide selective access to the interior of the cell—organelles (elfin versions our own organs). The chapter stresses the tumultuous action that occurs non-stop within the cells as proteins are assembled for use within and outside the cells. The chapter discusses stem cells, including the discovery of induced pluripotent stem cells. The chapter relates how cells differentiate to become dissimilar cell types, stresses the importance of three-dimensional study of cells (rather than two-dimensional study), and explains the different ways in which cells talk to each other.

scholarly journals The neuronal endoplasmic reticulum: its cytochemistry and contribution to the endomembrane system. I. Cell bodies and dendrites.

1983 ◽  
Vol 31 (9) ◽  
pp. 1077-1088 ◽  
Author(s):  
R D Broadwell ◽  
A M Cataldo
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Types ◽  
Endomembrane System ◽  
Cell Organelles ◽  
Electron Microscopic ◽  
Enzyme Cytochemistry ◽  
G6pase Activity ◽  
Numerous Cell ◽  
Intracellular Compartments

The endoplasmic reticulum (ER) and its contribution to the endomembrane system (i.e., membranes of cell organelles) in the neuron have been investigated in brains of mice by applying electron microscopic enzyme cytochemistry for demonstration of glucose-6-phosphatase (G6Pase) activity. The phosphohydrolytic activity of G6Pase is a well-known cytochemical marker for the ER in numerous cell types. Of the different substrates employed, glucose-6-phosphate and mannose-6-phosphate were the only two with which G6Pase reaction product was seen in the neuronal ER and organelles related morphologically to the ER. G6Pase activity in cell bodies and dendrites was localized consistently within the lumen of the nuclear envelope, rough and smooth ER, lamellar bodies, hypolemmal and subsurface cisternae, and frequently in the cis saccules of the Golgi apparatus. The G6Pase reactive ER appeared as a network of saccules and tubules pervading the cell body and its dendrites. Possible membrane continuities were identified between the ER and the other reactive structures, including the cis half of the Golgi apparatus. Neither G6Pase activity nor reactive ER was associated with the trans Golgi saccules or GERL. G6Pase activity thus serves as a reliable marker for the perikaryal and dendritic ER and related structures. These observations support the theory that the ER is an integral component of the neuronal endomembrane system associated with the transfer of membrane or membrane molecules among intracellular compartments, the packaging and transport of exportable protein, and energy metabolism. G6Pase activity in the ER of axons and terminals is considered in detail in part two of this study.

Use of Thick Histochemical Preparations, Whole Mount Cultured Cells or Thick Sections from Embedded Tissues, for Three-Dimensional Observation by Ultrahigh Voltage Electron Microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 184-185
Author(s):  
Tetsuji Nagata ◽  
Nobuteru Usuda ◽  
Hongjun Ma
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Rat Hepatocytes ◽  
Wistar Rat ◽  
Specific Cell ◽  
Cell Organelles ◽  
Voltage Electron ◽  
Culture Cells ◽  
Whole Mount

We have observed very thick biological specimens, both whole mount cultured cells and thick sections from embedded tissues, which were stained with histochemical reactions for specific cell organelles, by means of high or ultrahigh voltage electron microscopy (UHVEM).Thick histochemical specimens, both whole mount cultured cells and semithin sections from embedded tissues were used. Two cell strains in culture, one established CHO-K1 cell line and the other primary culture cells of adult Wistar rat hepatocytes were used. Culture cells were seeded onto formval coated gold meshes and incubated in CO2 incubator. CHO-K1 cells were cultured in Ham’s F12 medium containing HRP (1 mg/ml), while rat hepatocytes were in L-15 medium containing Clofibrate (0.2 mM). The cultured cells were stained with DAB reaction, dried in a critical point dryer (Hitachi HCP-1). Thick sections (0.2-1.0 μm) from DEHP fed rat livers, fixed in 2.5% glutaraldehyde, stained with DAB reaction, postfixed in 1% osmium tetroxide and embedded in Epon, or semithin Epon sections (0.2μm) from the pancreases of 3H-thymidine or 3H-uridine injected mice, fixed doubly, radioautographed with Sakura NR-H2 emulsion.

scholarly journals Electron microscopic analysis of different cell types in human pancreatic cancer spheres

2017 ◽  
Author(s):  
Toshiyuki Ishiwata ◽  
Fumio Hasegawa ◽  
Masaki Michishita ◽  
Norihiko Sasaki ◽  
Naoshi Ishikawa ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Electron Microscopic Analysis ◽  
Human Pancreatic Cancer ◽  
Electron Microscopic ◽  
Different Cell Types

scholarly journals Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells.

1976 ◽  
Vol 143 (6) ◽  
pp. 1402-1420 ◽  
Author(s):  
N Nogueira ◽  
Z Cohn
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Cytoplasmic Matrix ◽  
Mode Of Entry ◽  
Intracellular Fate

The mode of entry and intracellular fate of epimastigotes and trypomastigotes of Trypanosoma cruzi in cultured cells was studied. Electron microscopic observations indicated the uptake by phagocytosis of both forms into mouse peritoneal macrophages and of trypomastigotes and transition forms into other cultured cell types. In each instance the organisms were initially surrounded by a plasma membrane-derived phagosome. Trypsin and chymotrypsin treatment of the macrophages completely abolished attachment and ingestion of both forms, indicating that protease-sensitive structures on the macrophage plasma membrane mediate ingestion. The macrophage Fc or C3b receptors were not essential for uptake of T. cruzi in the conditions used. Cytochalasin B inhibited ingestion but not the attachment of both forms by macrophages. Epimastigotes were not taken up by HeLa, L cells, and calf embryo fibroblasts. In macrophages, epimastigotes were killed and digested within phagolysosomes. In contrast, trypomastigotes and transition forms escaped from the phagocytic vacuole and then multiplied in the cytoplasmic matrix. Amastigotes released from infected cells exhibited properties similar to those of trypomastigotes and were able to enter all cell types studied and multiply intracellularly.

scholarly journals Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites.

1984 ◽  
Vol 98 (4) ◽  
pp. 1279-1290 ◽  
Author(s):  
S E Sasaki-Sherrington ◽  
J R Jacobs ◽  
J K Stevens
Keyword(s):  
Surface Area ◽  
Smooth Endoplasmic Reticulum ◽  
Optic Tract ◽  
Microscopic Analysis ◽  
Amacrine Cells ◽  
Cell Types ◽  
Sectional Area ◽  
Electron Microscopic ◽  
Direct Role ◽  
Cross Sectional

AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape.

Electron Microscopic Examination of DNA in an Alkaline Sucrose Gradient

1975 ◽  
Vol 33 ◽  
pp. 612-613
Author(s):  
W. D. Garner ◽  
R. E. Nordquist ◽  
R. H. Bottomley ◽  
J. M. Hawrylko
Keyword(s):  
Mammalian Cells ◽  
Sucrose Gradient ◽  
Electron Microscopic Examination ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Ultrastructural Characteristics ◽  
Rat Hepatoma ◽  
Hepatoma Tissue

The technique of lysing cells on top of an alkaline sucrose gradient prior to sedimentation was first designed to examine single stranded DNA from protoplasts. The lysis of mammalian cells and sedimentation of the released DNA has not been examined thoroughly by EM. The present work describes the ultrastructural characteristics of DNA species found after whole cell lysing and sedimentation of the DNA through an alkaline sucrose gradient.The DNA of H4-II-E rat hepatoma tissue culture cells was prelabelled with H3TdR (luci/ml) for 18 to 24 hours prior to lysis. Cells were layered into a lysing layer (0.1 M EDTA and 0.5 N NaOH, pH 13.25) on top of a 5-202or 5-25% alkaline sucrose gradient (0.01 M EDTA, 0.1 N NaOH and 0.9 M NaCl). After centrifugation the gradients were fractionated from the bottom. The fractions containing the majority of H3TdR were applied directly on grids at pH 8.5 or 12. Samples were stained with uranyl acetate.

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