Ultrastructural autoradiography of L6 skeletal-muscle cells exposed to a tritiated macrolide antibiotic (LY237216) in vitro

1992 ◽  
Vol 50 (1) ◽  
pp. 612-613
Author(s):  
S.L. White ◽  
C.B. Jensen ◽  
D.D. Giera ◽  
D.A. Laska ◽  
M.N. Novilla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Quantitative Analysis ◽  
Macrolide Antibiotic ◽  
Circle Method ◽  
Apical Surface ◽  
Polycarbonate Membrane ◽  
L6 Cells ◽  
Low Viscosity ◽  
Erythromycin A

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

Ultrastructural evaluation of the effects induced by a macrolide antibiotic (LY281389) on L6 muscle cells in vitro

1991 ◽  
Vol 49 ◽  
pp. 80-81
Author(s):  
J. W. Horn ◽  
S. L. White ◽  
D. A. Laska ◽  
M. K. Buening ◽  
M. N. Novilla ◽  
...  
Keyword(s):  
Macrolide Antibiotic ◽  
Uniform Density ◽  
Solution Ph ◽  
L6 Cells ◽  
Vacuolar Change ◽  
Interference Range ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
L6 Muscle Cells

Toxic doses of LY281389 (9-N(n-propyl)erythromycylamine), a cationic amphophilic macrolide antibiotic, induce a generalized cytoplasmic vacuolar change in tissues of rats and dogs. The present in vitro study using L6 cells (a line derived from neoplastic skeletal muscle) was done to evaluate sequentially early cytologic changes.The L6 cells seeded on tissue culture plates were allowed to grow to a uniform density. The culture medium was then replaced with control medium or medium containing 0.25 mg/ml LY281389 for exposures of 0.5, 2, 6, 12, 24, or 48 hours. The L6 cells were then fixed for 1-hour in modified Karnovsky's solution (pH 7.2), rinsed with 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed for an additional hour in 2% osmium tetroxide (pH 7.2). Following a second buffer rinse, the L6 cells were dehydrated in graded ethanol solutions. The cells on each plate were embedded with epoxy resin (EPON 812), selected areas were scribed out and re-embedded for orientation. Ultrathin sections (silver to gold color interference range) were mounted on copper 200 mesh grids, counterstained, and examined using a Philips EM410LS electron microscope at 60kV.

scholarly journals Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles

2020 ◽  
Vol 21 (20) ◽  
pp. 7545
Author(s):  
Matej Skočaj ◽  
Maruša Bizjak ◽  
Klemen Strojan ◽  
Jasna Lojk ◽  
Mateja Erdani Kreft ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Polyacrylic Acid ◽  
In Vitro Toxicity ◽  
Continuous Exposure ◽  
Short Term ◽  
Tio2 Nps ◽  
L6 Cells ◽  
Novel Approach

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

scholarly journals Targeting of skeletal muscle in vitro using biotinylated immunoliposomes

2004 ◽  
Vol 377 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Anita SCHNYDER ◽  
Stefan KRÄHENBÜHL ◽  
Michael TÖRÖK ◽  
Jürgen DREWE ◽  
Jörg HUWYLER
Keyword(s):  
Skeletal Muscle ◽  
Cellular Uptake ◽  
Transferrin Receptor ◽  
Drug Transport ◽  
Binding Capacity ◽  
L6 Cells ◽  
Coupling Procedure ◽  
Immunofluorescence Labelling ◽  
Biotin Binding

In the present study, a non-covalent (biotin–streptavidin) coupling procedure for the preparation of pegylated immunoliposomes is presented, which simplifies the attachment of targeting vectors to sterically stabilized liposomes. A biotinylated poly(ethylene glycol) (PEG)-phospholipid [bio-PEG-distearoylphosphatidylethanolamine (DSPE)] was used as a linker between a streptavidin-conjugated monoclonal antibody (mAb) (i.e. the OX26 mAb raised against the rat transferrin receptor) and 150 nm liposomes. OX26–streptavidin had a biotin binding capacity of two to three biotin molecules per OX26–streptavidin conjugate. Immunostaining experiments with the OX26 mAb followed by fluorescent confocal microscopy revealed immunofluorescence labelling of the transferrin receptor on skeletal muscle, as well as in L6 cells, a continuous cell line derived from rat skeletal muscle. Uptake experiments with L6 cells using the OX26 mAb, fluorescence-labelled OX26–streptavidin or fluorescent OX26-immunoliposomes demonstrated cellular uptake and accumulation within an intracellular compartment of the OX26 mAb and its conjugates. Cellular uptake of OX26 conjugates was sensitive to competition with free OX26 antibody. In summary, these studies describe the design of biotinylated immunoliposomes as a universal drug transport vector and their potential for targeting of the transferrin receptor of skeletal muscle.

scholarly journals Influence of an Intermediary Base on the Microleakage of Simulated Class II Composite Resin Restorations

2014 ◽  
Vol 39 (3) ◽  
pp. 301-307 ◽  
Author(s):  
MCC Giorgi ◽  
NMAP Hernandes ◽  
MM Sugii ◽  
GMB Ambrosano ◽  
GM Marchi ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Composite Resin ◽  
Glass Ionomer Cement ◽  
In Vitro Study ◽  
Qualitative Evaluation ◽  
Class Ii ◽  
Low Viscosity ◽  
Significant Difference ◽  
Composite Resin Restorations

SUMMARY The aim of this in vitro study was to qualitatively and quantitatively evaluate the microleakage of Class II cavities restored with a methacrylate-based composite (Filtek Z250, 3M ESPE) or silorane-based composite (Filtek LS, 3M ESPE), varying the application of an intermediary base, using a low-viscosity composite resin (Filtek Z350 Flow, 3M ESPE) or resin-modified glass ionomer cement (RMGIC) (Vitrebond, 3M ESPE) and no intermediary base (control groups). Sixty cavities were prepared on the proximal surfaces of bovine teeth and were randomly divided according to the experimental groups (n=10). Following the restorative procedures and thermocycling, the samples were immersed in methylene blue for two hours. The qualitative evaluation was made using a stereomicroscope, whereby two observers analyzed the infiltration level of the dye within the tooth/filling. Microleakage scores among the groups were compared using the Kruskal-Wallis test followed by the Mann-Whitney test (p≤0.05). The samples were then ground and the powder was prepared for quantitative analysis in an absorbance spectrophotometer. The results were statistically analyzed by analysis of variance and the Tukey test (p≤0.05). Results from the quantitative analysis showed that LS presented higher values of microleakage than did Z250. There was a significant difference between both composites concerning the intermediary materials, with the lowest values obtained using RMGIC as an intermediary base. Results from the qualitative analysis showed that there were no statistically significant differences between composites; however, there were significant differences for both composites concerning the intermediary materials, with the lowest values obtained using RMGIC as an intermediary. It is possible to conclude that using RMGIC as an intermediary base provided lower microleakage, indicating better sealing of the tooth-restoration interface.

Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

1997 ◽  
Vol 78 (02) ◽  
pp. 934-938 ◽  
Author(s):  
Hsiun-ing Chen ◽  
Yueh-I Wu ◽  
Yu-Lun Hsieh ◽  
Guey-Yueh Shi ◽  
Meei-Jyh Jiang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Platelet Adhesion ◽  
Treatment Time ◽  
Shape Change ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Apical Surface ◽  
Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

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