Abstract 432: Endothelial Cells Express miR-33a-5p and Release Extracellular Vesicles Containing miR-33a-5p

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Alexis Stamatikos ◽  
Lucia Vojtech ◽  
Nagadhara Dronadula ◽  
David Dichek
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Cholesterol Efflux ◽  
Lipid Membrane ◽  
Cell Types ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Background: The microRNA miR-33a-5p increases cellular cholesterol by posttranscriptional silencing of genes involved in cholesterol efflux. Previous studies showed that miR-33a-5p inhibition protects against atherosclerosis. Atheroprotective effects of miR-33a-5p inhibition are attributed to actions in hepatocytes that raise plasma HDL and actions in macrophages that enhance cholesterol efflux. We hypothesized that miR-33a-5p is also expressed in primary arterial endothelial cells (EC) and that miR-33a-5p might contribute to atherosclerosis by promoting cholesterol accumulation in EC. We also hypothesized that EC could release miR-33a-5p in extracellular vesicles (EV; including exosomes and microvesicles) that transferred miR-33a-5p to neighboring vascular cells such as SMC and macrophages, inhibiting cholesterol efflux, promoting lipid accumulation in these cells, and accelerating atherosclerosis. Methods: We cultured bovine aortic endothelial cells (BAEC) in serum-free medium, collected the medium, and isolated EV by ultracentrifugation. To test whether we had isolated EV, we compared EV and BAEC lysates by SDS-PAGE, Coomassie blue staining, and immunoblotting. We measured EV size with nanoparticle tracking analysis and imaged the particles with transmission electron microscopy. We extracted total RNA from BAEC and EV, measured RNA size using BioAnalyzer, and detected miR-33a-5p expression using RT-PCR and restriction digestion as well as qRT-PCR. Results: Coomassie blue staining revealed large differences between BAEC and EV lysates. The exosome marker CD9 was present at higher levels in EV lysate compared to BAEC lysate. Most of the EV were in the size range of exosomes, and appeared as typical lipid membrane-bound spheres by electron microscopy. EV contained primarily small RNA (~25-200 nucleotides). miR-33a-5p was detected both in BAEC lysates and in detergent-soluble EV. Conclusions: Primary arterial EC express miR-33a-5p and release EV rich in small RNA, including miR-33a-5p. Manipulating miR-33a-5p expression in EC, for example with a miR-33a-5p antagomiR, may be an effective therapeutic approach for increasing cholesterol efflux from multiple vessel wall cell types and preventing/reversing atherosclerosis.

Coomassie blue staining for high sensitivity gel-based proteomics

2013 ◽  
Vol 90 ◽  
pp. 96-106 ◽  
Author(s):  
Victoria J. Gauci ◽  
Matthew P. Padula ◽  
Jens R. Coorssen
Keyword(s):  
High Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

scholarly journals Heating Greatly Speeds Coomassie Blue Staining and Destaining

BioTechniques ◽  
2000 ◽  
Vol 28 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Connie Wong ◽  
Srinavas Sridhara ◽  
James C.A. Bardwell ◽  
Ursula Jakob
Keyword(s):  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase

1977 ◽  
Vol 55 (9) ◽  
pp. 988-994 ◽  
Author(s):  
A. McGeer ◽  
B. Lavers ◽  
G. R. Williams
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Blue Staining ◽  
Beef Heart ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS–polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with the activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.

PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III

1987 ◽  
Author(s):  
Deborah A Rathjen ◽  
Carolyn L Geczy
Keyword(s):  
Cultured Cells ◽  
Factor Xa ◽  
Lymphocyte Activation ◽  
Blue Staining ◽  
Aortic Endothelial Cells ◽  
Tissue Sections ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
At Iii

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

scholarly journals Expression of Human Salivary Histatin and Cystatin/ Histatin Chimeric cDNAs in Escherichia coli

1993 ◽  
Vol 4 (3) ◽  
pp. 581-590 ◽  
Author(s):  
L.A. Bobek ◽  
H. Tsai ◽  
M.J. Levine
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Expression System ◽  
Full Length ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Overlap Extension ◽  
Cystatin Sn ◽  
The Individual

We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.

scholarly journals Extracellular Vesicles in Tumors: A Potential Mediator of Bone Metastasis

2021 ◽  
Vol 9 ◽  
Author(s):  
Shenglong Li ◽  
Wei Wang
Keyword(s):  
Bone Metastasis ◽  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Bone Cells ◽  
Membrane Vesicles ◽  
Metastatic Tumor ◽  
Cell Types ◽  
Biological Phenomena ◽  
Breast And Prostate Cancer ◽  
Metastatic Sites

As one of the most common metastatic sites, bone has a unique microenvironment for the growth and prosperity of metastatic tumor cells. Bone metastasis is a common complication for tumor patients and accounts for 15–20% of systemic metastasis, which is only secondary to lung and liver metastasis. Cancers prone to bone metastasis include lung, breast, and prostate cancer. Extracellular vesicles (EVs) are lipid membrane vesicles released from different cell types. It is clear that EVs are associated with multiple biological phenomena and are crucial for intracellular communication by transporting intracellular substances. Recent studies have implicated EVs in the development of cancer. However, the potential roles of EVs in the pathological exchange of bone cells between tumors and the bone microenvironment remain an emerging area. This review is focused on the role of tumor-derived EVs in bone metastasis and possible regulatory mechanisms.

Sign in / Sign up

Export Citation Format

Share Document