Specificity of carbohydrate binding by AAV1 capsids on a glycan array. Interestingly, an older version of the array, containing glycans immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate, was used to screen the sugar binding specificity of the parvovirus minute virus of mice (MVM) capsids (M. This array, containing sialylated and nonsialylated sugars with […]
ΔOD595 for triplicate experiments is shown: ΔnanT (A) and ΔyjhB (B). ΔOD595 for triplicate experiments is shown: BW25113 (B), ΔyjhC (C), and complemented yjhC (D). Deletion of yjhC resulted in loss of growth on 2,7-anhydro-Neu5Ac but not on Neu5Ac (Fig. 5C), which could be complemented in trans with yjhC (Fig. 5D), suggesting that the gene […]
HepG2 (A) and Pro-5 (B) cells were transduced with a constant amount of AAV1-luc or AAV6-luc vectors in the presence of a 200-fold-excess amount of competing AAV1, AAV6, or AAV2 encapsidated λ phage DNA-containing vector at 37°C for 1 h. However, more and more studies suggest that AAV2-based vectors are rate limiting in certain tissues […]
For experiments, PC-12 cells were collected via 0.25% trypsin (Gibco) and seeded for culturing in collagen IV (Sigma-Aldrich) coated 4-chamber slides (Nunc Lab-Tek, Merck). PC-12 cells grew as small clusters in suspension and were maintained in a density 5 × 105 cells/ml in culture flasks coated by 0.1% collagen type IV (Sigma-Aldrich). For testing bioactivity […]
The resulting weight gain during the last 2 min of raising the venous pressure was divided by the concomitant increment of capillary pressure, and values were normalized to predicted lung weight. The amounts of activated Rac1 or Cdc42 (arbitrary units) are based on the optical density values obtained after subtracting negative control values. A “recombinant […]
Finally, is there a threshold of sialic acid loss that must be attained before barrier disruption occurs? More specifically, treatment of endothelial monolayers with neuraminidases leads to endothelial barrier disruption. However, the fact that there was an actual increase in resistance suggests that there is something more going on, something we do not yet understand. […]
This observation was further supported by the staining with fluorescently tagged lectin from Arachis hypogaea following neuraminidase treatment. TRITC-tagged lectin from Arachis hypogaea did not bind to treated cells (B), and the monolayer was not disrupted (C). Treated cells exhibited severely disrupted monolayers. B: Confluent monolayers of PAECs and PMVECs were treated with Texas Red-tagged […]
This was followed by the addition of neuraminidase solution or buffer control to each well and continued resistance measurement 4 (or more) h. More specifically, following treatment with neuraminidase from Clostridium perfringens, the PAECs appeared to lose cell-cell contacts, resulting in rather evenly dispersed individual cells. In addition, among all other cell lines tested, AAV6 […]
Whole cell lysate (20 μl) was combined with 80 μl of 0.05 N H2SO4 (hydrolysis reagent) and incubated at 80°C for 60 min. Samples were briefly centrifuged at 14,000 revolution/min (16,000 g), after which 20 μl of 1 M NaOH (neutralization reagent) was added and the mixture centrifuged again at 14,000 revolution/min. Afterwards, wells were […]
Previous work using spectrophotometric assays reported that E. coli YjhC showed a weak interaction with Neu5Ac, with an apparent Km of 68.8 mm (20). Here, we used ESI-MS to assess the activity of YjhC against 2,7-anhydro-Neu5Ac or Neu5Ac. The glycan array binding data provide independent support of AAV1 interaction with α2,3 and α2,6 trisaccharides. Our […]
