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.

scholarly journals Tracking Lysosome Migration within Chinese Hamster Ovary (CHO) Cells Following Exposure to Nanosecond Pulsed Electric Fields

2018 ◽  
Vol 5 (4) ◽  
pp. 103
Author(s):  
Gary Thompson ◽  
Hope Beier ◽  
Bennett Ibey
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Lateral Diffusion ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Membrane Repair ◽  
Osmotic Swelling

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

scholarly journals Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

2003 ◽  
Vol 374 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Substrate Binding ◽  
Chinese Hamster ◽  
Water Soluble ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

scholarly journals THE INTERACTION OF MAMMALIAN CELLS WITH ANTIBODIES. I

1961 ◽  
Vol 113 (3) ◽  
pp. 599-610 ◽  
Author(s):  
M. Oda ◽  
T. T. Puck
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Antibody Activity ◽  
Specific Cell ◽  
Plating Technique ◽  
Killing Activity ◽  
Killing Action ◽  
Very High ◽  
Cell Plating

The single cell plating technique has been applied to quantitation of the reproductive killing of mammalian cells by specific antibodies. This method confirms previous demonstrations by other workers of localization of all the killing activity in the γ-globulin fraction of specific cell antisera but not of normal sera; the need for complement for the killing action in low doses of antibody and the leakage of cell constituents attending cell killing under these conditions. In concentrations of 4 per cent or higher of heated antiserum cell killing occurs without added complement. The cell plating technique permits highly reproducible quantitation of antibody action and demonstrates antibody activity in sera diluted 1:3000. It permits demonstration of very high degrees of species specificity as shown by virtually complete absence of cross-reaction between antisera to Chinese hamster and S3 HeLa cells, respectively. Somatic cells which have been sensitized by absorption of specific antibody lose their sensitization when incubated at 37° unless complement is added within 1 hour.

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.

BODIPY Fluorophores for Membrane Potential Imaging

2019 ◽  
Author(s):  
Jenna Franke ◽  
Benjamin Raliski ◽  
Steven Boggess ◽  
Divya Natesan ◽  
Evan Koretsky ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Water Solubility ◽  
Water Soluble ◽  
Voltage Sensitivity ◽  
Chemical Transformations ◽  
Direct Modification ◽  
Biological Compatibility ◽  
Meso Position ◽  
Ionizable Groups ◽  
Boron Center

Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based voltage-sensitive fluorophores. The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.

scholarly journals Scaling parameter of the lethal effect of mammalian cells based on radiation-induced OH radicals: effectiveness of direct action in radiation therapy

2020 ◽  
Vol 62 (1) ◽  
pp. 86-93
Author(s):  
Tamon Kusumoto ◽  
Ryo Ogawara ◽  
Kazuyo Igawa ◽  
Kentaro Baba ◽  
Teruaki Konishi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Direct Action ◽  
Absorbed Dose ◽  
Vertical Axis ◽  
Lethal Effect ◽  
Cell Killing ◽  
Oh Radicals ◽  
Survival Curves ◽  
Biological Effectiveness ◽  
Indirect Action

ABSTRACT We have been studying the effectiveness of direct action, which induces clustered DNA damage leading to cell killing, relative to indirect action. Here a new criterion Direct Ation-Based Biological Effectiveness (DABBLE) is proposed to understand the contribution of direct action for cell killing induced by C ions. DABBLE is defined as the ratio of direct action to indirect action. To derive this ratio, we describe survival curves of mammalian cells as a function of the number of OH radicals produced 1 ps and 100 ns after irradiation, instead of the absorbed dose. By comparing values on the vertical axis of the survival curves at a certain number of OH radicals produced, we successfully discriminate the contribution of direct action induced by C ions from that of indirect action. DABBLE increases monotonically with increasing linear energy transfer (LET) up to 140 keV/μm and then drops, when the survival curves are described by the number of OH radicals 1 ps after irradiation. The trend of DABBLE is in agreement with that of relative biological effectiveness (RBE) of indirect action. In comparison, the value of DABBLE increases monotonically with LET, when the survival curves are described by the number of OH radicals 100 ns after irradiation. This finding implies that the effectiveness of C ion therapy for cancer depends on the contribution of direct action and we can follow the contribution of direct action over time in the chemical phase.

scholarly journals LION/web: a web-based ontology enrichment tool for lipidomic data analysis

GigaScience ◽  
2019 ◽  
Vol 8 (6) ◽  
Author(s):  
Martijn R Molenaar ◽  
Aike Jeucken ◽  
Tsjerk A Wassenaar ◽  
Chris H A van de Lest ◽  
Jos F Brouwers ◽  
...  
Keyword(s):  
Membrane Fluidity ◽  
Plasma Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lateral Diffusion ◽  
Decanoic Acid ◽  
Web Based ◽  
Ovary Cells ◽  
Lipid Species ◽  
Significant Enrichment

Abstract Background A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems. Results We built a new lipid ontology (LION) that associates >50,000 lipid species to biophysical, chemical, and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including “plasma membrane", “headgroup with negative charge", "glycerophosphoserines", “above average bilayer thickness", and “below average lateral diffusion". A second validation was performed by analyzing the membrane fluidity of Chinese hamster ovary cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ("above average", "very high", and "high lateral diffusion" and "below average transition temperature"). Conclusions The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.

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