Electroporation-mediated uptake of proteins into mammalian cells

1990 ◽  
Vol 68 (4) ◽  
pp. 729-734 ◽  
Author(s):  
Helene Lambert ◽  
Roumen Pankov ◽  
Johanne Gauthier ◽  
Ronald Hancock
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Histone H1 ◽  
Dna Transfection ◽  
Optimum Conditions ◽  
Gold Particles ◽  
Protein Uptake ◽  
Particle Uptake ◽  
20 Nm

Proteins of up to 230 kilodaltons are taken up by Chinese hamster ovary fibroblasts exposed to electroporation under conditions generally similar to those used to mediate DNA transfection. The uptake of catalase, ovalbumin, and histone H1 labelled with fluorescein was visualized by fluorescence microscopy. Under the same conditions, the uptake of colloidal gold particles (20 nm diameter) was visualized by electron microscopy. In optimum conditions, about 25% of the cells remained viable and grew normally and about 25% of these retained labelled proteins during two cycles of further growth. About 6 × 104 molecules of catalase were retained per cell. Proteins were taken up when presented to the cells up to 4 h after electroporation, suggesting that mechanisms other than classical electropore formation may operate in these conditions. The proteins were localized in the cytoplasm in a predominantly vesicular pattern and histone H1 entered the nucleus in some cells.Key words: electroporation, protein uptake, gold particle uptake, endocytosis, cell membrane.

scholarly journals Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

2019 ◽  
Author(s):  
Yongdeng Zhang ◽  
Lena K. Schroeder ◽  
Mark D. Lessard ◽  
Phylicia Kidd ◽  
Jeeyun Chung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Membrane Structures ◽  
Molecular Specificity ◽  
20 Nm

AbstractCombining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.One Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

The Lyme Disease Spirochete, Borrelia Burgdorferi, Forms Spheroplasts In the Presence of Spingolipid Analogue Ppmp

1999 ◽  
Vol 5 (S2) ◽  
pp. 1136-1137
Author(s):  
Lori L. Lubke ◽  
Claude F. Garon
Keyword(s):  
Electron Microscopy ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
The United States ◽  
Eukaryotic Cell ◽  
Dark Field ◽  
Multisystem Disorder

Lyme disease is the most common arthropod-borne infection in the United States. Over 100,000 cases have been reported to date. The disease is a multisystem disorder with dermatologic, neurologic and rheumatologic manifestations. The causative agent, Borrelia burgdorferi1has been isolated from ticks, animals and humans from around the world and can generally be cultured in the laboratory using BSK-II medium.While biologically significant concentrations of eukaryotic cell inhibitors such as aphidicolin, ara C, cis platinum, CPX, hydroxyurea, mimosine, nalidixic acid, trioxsalen and boromethyglycine showed little effect on the growth of Borrelia burgdorferiwhen added to BSK-II cultures, PPMP, a spingolipid analogue, showed immediate and profound inhibitory effects when monitored by dark-field microscopy, transmission electron microscopy (TEM), field emission scanning electron microscopy (SEM) and by optical density measurements at 600 mμ. PPMP (dl-thzreo-l-phenyl-2-palmitoylamino-3-morpholino-l-propanol) is known to inhibit the synthesis of spingomyelin in Chinese hamster ovary cells and to inhibit the synthesis of glucosylceramides in a large variety of mammalian cells.

scholarly journals THE NUCLEOLI IN MITOTIC DIVISIONS OF MAMMALIAN CELLS IN VITRO

1965 ◽  
Vol 26 (2) ◽  
pp. 539-553 ◽  
Author(s):  
T. C. Hsu ◽  
Frances E. Arrighi ◽  
Robert R. Klevecz ◽  
B. R. Brinkley
Keyword(s):  
Electron Microscopy ◽  
Protein Synthesis ◽  
Mammalian Cells ◽  
Fluorescent Microscopy ◽  
Chinese Hamster ◽  
Methyl Green ◽  
Azure B ◽  
Nucleolar Material ◽  
Cell Strains

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

Specificity of Lectins Labeled with Colloidal Gold to the Exopolymeric Matrix Carbohydrates of the Sulfate-Reducing Bacteria Biofilm Formed on Steel

2020 ◽  
Vol 82 (5) ◽  
pp. 11-20
Author(s):  
D.R. Abdulina ◽  
◽  
L.M. Purish ◽  
G.O. Iutynska ◽  
◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Colloidal Gold ◽  
Steel Surface ◽  
Lectin Binding ◽  
Sulfate Reducing Bacteria ◽  
Gold Particles ◽  
Sulfate Reducing ◽  
Transmission Electron ◽  
Reducing Bacteria

The studies of the carbohydrate composition of the sulfate-reducing bacteria (SRB) biofilms formed on the steel surface, which are a factor of microbial corrosion, are significant. Since exopolymers synthesized by bacteria could activate corrosive processes. The aim of the study was to investigate the specificity of commercial lectins, labeled with colloidal gold to carbohydrates in the biofilm exopolymeric matrix produced by the corrosive-relevant SRB strains from man-caused ecotopes. Methods. Microbiological methods (obtaining of the SRB biofilms during cultivation in liquid Postgate B media under microaerophilic conditions), biochemical methods (lectin-binding analysis of 10 commercial lectins, labeled with colloidal gold), transmission electron microscopy using JEM-1400 JEOL. Results. It was shown using transmission electron microscopy that the binding of lectins with carbohydrates in the biofilm of the studied SRB strains occurred directly in the exopolymerіс matrix, as well as on the surfaces of bacterial cells, as seen by the presence of colloidal gold particles. For detection of the neutral carbohydrates (D-glucose and D-mannose) in the biofilm of almost all studied bacterial strains PSA lectin was the most specific. This lectin binding in biofilms of Desulfotomaculum sp. К1/3 and Desulfovibrio sp. 10 strains was higher in 90.8% and 94.4%, respectively, then for ConA lectin. The presence of fucose in the SRB biofilms was detected using LABA lectin, that showed specificity to the biofilm EPS of all the studied strains. LBA lectin was the most specific to N-аcetyl-D-galactosamine for determination of amino sugars in the biofilm. The amount of this lectin binding in D. vulgaris DSM644 biofilm was 30.3, 10.1 and 9.3 times higher than SBA, SNA and PNA lectins, respectively. STA, LVA and WGA lectins were used to detect the N-acetyl-Dglucosamine and sialic acid in the biofilm. WGA lectin showed specificity to N-acetyl-D-glucosamine in the biofilm of all the studied SRB; maximum number of bounded colloidal gold particles (175 particles/μm2) was found in the Desulfotomaculum sp. TC3 biofilm. STA lectin was interacted most actively with N-acetyl-D-glucosamine in Desulfotomaculum sp. TC3 and Desulfomicrobium sp. TC4 biofilms. The number of bounded colloidal gold particles was in 9.2 and 7.4 times higher, respectively, than using LVA lectin. The lowest binding of colloidal gold particles was observed for LVA lectin. Conclusions. It was identified the individual specificity of the 10 commercial lectins to the carbohydrates of biofilm matrix on the steel surface, produced by SRB. It was estimated that lectins with identical carbohydrates specificity had variation in binding to the biofilm carbohydrates of different SRB strains. Establishing of the lectin range selected for each culture lead to the reduction of the scope of studies and labor time in the researching of the peculiarities of exopolymeric matrix composition of biofilms formed by corrosiverelevant SRB.

scholarly journals Screening for Effects of Inhaled Nanoparticles in Cell Culture Models for Prolonged Exposure

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 606
Author(s):  
Claudia Meindl ◽  
Kristin Öhlinger ◽  
Verena Zrim ◽  
Thomas Steinkogler ◽  
Eleonore Fröhlich
Keyword(s):  
Prolonged Exposure ◽  
A549 Cells ◽  
Cytokine Release ◽  
Transient Effects ◽  
Particle Uptake ◽  
Particle Accumulation ◽  
Low Concentrations ◽  
Culture Models ◽  
20 Nm ◽  
Inhaled Nanoparticles

Respiratory exposure of humans to environmental and therapeutic nanoparticles repeatedly occurs at relatively low concentrations. To identify adverse effects of particle accumulation under realistic conditions, monocultures of Calu-3 and A549 cells and co-cultures of A549 and THP-1 macrophages in the air–liquid interphase culture were exposed repeatedly to 2 µg/cm2 20 nm and 200 nm polystyrene particles with different functionalization. Particle accumulation, transepithelial electrical resistance, dextran (3–70 kDa) uptake and proinflammatory cytokine secretion were determined over 28 days. Calu-3 cells showed constant particle uptake without any change in barrier function and cytokine release. A549 cells preferentially ingested amino- and not-functionalized particles combined with decreased endocytosis. Cytokine release was transiently increased upon exposure to all particles. Carboxyl-functionalized demonstrated higher uptake and higher cytokine release than the other particles in the A549/THP-1 co-cultures. The evaluated respiratory cells and co-cultures ingested different amounts and types of particles and caused small (partly transient) effects. The data suggest that the healthy cells can adapt to low doses of non-cytotoxic particles.

Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

1983 ◽  
Vol 61 (6) ◽  
pp. 421-427 ◽  
Author(s):  
James R. Lepock ◽  
Kwan-Hon Cheng ◽  
Hisham Al-Qysi ◽  
Jack Kruuv
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Water Soluble ◽  
Growth Data ◽  
Protein Fluorescence ◽  
Polar Groups ◽  
Spin Resonance ◽  
Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

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