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1.
J Cell Physiol ; 232(11): 3139-3145, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28059438

ABSTRACT

Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.


Subject(s)
Cell Separation/methods , Endothelial Cells/physiology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/pathology , Animals , Aorta/cytology , Biological Transport , Biomarkers/blood , Calcium/metabolism , Cell Proliferation , Collagenases/metabolism , Endopeptidases/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelin-1/pharmacology , Flow Cytometry , Lipoproteins, LDL/metabolism , Microscopy, Fluorescence , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Neovascularization, Physiologic , Phenotype , Sus scrofa , Time Factors
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 73(4): 730-7, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19419902

ABSTRACT

The vibrational frequencies, normal mode assignments and the internal-rotation and inversion potential energy surfaces of methylarsine and methylstibine are reported. The potential energy distribution (PED) for each frequency is found using Kim's Correspondence Rules of unified group theory and the program MOLVIB. The predominant motion of each PED is used to name the corresponding vibrational frequency. We also report the infrared spectra prediction of deuterated isotopomers. Our predicted infrared spectra show good agreement with experiment. In the potential energy surfaces of both molecules, there are three minimum energy states; and three first-order internal-rotation, six first-order inversion, and six second-order inversion transition-states. Calculations are performed at the HF, DFT/B3LYP, and MP2 levels of theory using GAUSSIAN 03 quantum chemistry code. The 6-311G** basis set is used for methylarsine and the CEP-121G basis set is used for methylstibine.


Subject(s)
Arsenicals/chemistry , Organometallic Compounds/chemistry , Spectrophotometry, Infrared/methods , Computer Simulation , Models, Molecular , Molecular Structure , Quantum Theory , Thermodynamics
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