scholarly journals Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration

Membranes ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 172 ◽  
Author(s):  
Karolina Sapoń ◽  
Dominika Maziarz ◽  
Teresa Janas ◽  
Aleksander F. Sikorski ◽  
Tadeusz Janas
Keyword(s):  
Cholera Toxin ◽  
Fluorescence Probe ◽  
Rapid Determination ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Gm1 Ganglioside ◽  
Cholera Toxin Subunit B ◽  
Order Of Magnitude ◽  
Subunit B

We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 108 isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 108 to 6 × 109 exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r2 = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.

scholarly journals Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL.

1979 ◽  
Vol 81 (3) ◽  
pp. 543-554 ◽  
Author(s):  
K C Joseph ◽  
A Stieber ◽  
N K Gonatas
Keyword(s):  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Cholera Toxin ◽  
Plasma Membranes ◽  
Neuroblastoma Cells ◽  
Gm1 Ganglioside ◽  
Murine Neuroblastoma ◽  
Positive Elements ◽  
Subunit B ◽  
Cytochemical Marker

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.

Encapsulation of Nisin and Lysozyme in Liposomes Enhances Efficacy against Listeria monocytogenes

2004 ◽  
Vol 67 (5) ◽  
pp. 922-927 ◽  
Author(s):  
LILIAN M. WERE ◽  
BARRY BRUCE ◽  
P. MICHAEL DAVIDSON ◽  
JOCHEN WEISS
Keyword(s):  
Antimicrobial Activity ◽  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Fluorescence Probe ◽  
Size Exclusion ◽  
Phospholipid Liposomes ◽  
Exclusion Chromatography ◽  
Delivery Vehicles ◽  
Strain Dependent

The efficacy and stability against Listeria monocytogenes of nisin and lysozyme encapsulated in phospholipid liposomes was evaluated. Antimicrobial-containing liposomes were prepared by hydrating dried lipids with buffer containing nisin, nisin plus the fluorescence probe calcein, or calcein and lysozyme. Mixtures were then centrifuged and sonicated, and encapsulated liposomes were collected using size-exclusion chromatography. Antimicrobial concentration in liposomes was determined by bicinchoninic acid assay prior to determination of antimicrobial activity against strains of L. monocytogenes. When nisin was encapsulated in liposomes, protein concentrations of 0.39, 0.27, and 0.23 mg/ml for phosphatidylcholine (PC), PC-cholesterol (7:3), and PC–phosphatidylglycerol (PG)–cholesterol (5:2:3), respectively, were obtained. Encapsulation of nisin with calcein yielded protein concentrations of 0.35, 0.39, and 0.28 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulation of calcein with lysozyme resulted in protein concentrationsof 0.43, 0.26, and 0.19 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulated nisin in 100% PC and PC-cholesterol liposomes inhibited bacterial growth by >2 log CFU/ml compared with free nisin. Growth inhibition with liposomal lysozyme was strain dependent, with greater inhibition observed for strains 310 and Scott A with PC-cholesterol and PC-PG-cholesterol liposomes. Inhibition of L. monocytogenes indicated the potential of liposomes to serve as delivery vehicles for antimicrobials in foods while improving stability of antimicrobials.

Use of cholera toxin subunit B to label neural projections to lower urinary tract organs v1

2021 ◽  
Author(s):  
Janet R Keast ◽  
Peregrine B Osborne ◽  
John-Paul Fuller-Jackson
Keyword(s):  
Urinary Tract ◽  
Cholera Toxin ◽  
Lower Urinary Tract ◽  
Animal Experimentation ◽  
Female Rat ◽  
Cholera Toxin Subunit B ◽  
Autonomic Neurons ◽  
Subunit B ◽  
Operative Monitoring ◽  
Lower Urinary

This protocol is used to visualise sensory and autonomic neurons innervating organs of the lower urinary tract in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

Sign in / Sign up

Export Citation Format

Share Document