The substrates Galβ1,3GalNAcα1 pNP (CAS 59837-14-8), GlCAScβ1,3GalNAcα1 pNP (CAS 125455-64-3), Galβ1,3GlcNAcα1 pNP (CAS 57467-13-7), GalNACAS pNP (CAS 23646-68-6), calf κ-casein, human glycophorin A, porcine mucin, calf fetuin, and calf asialofetuin were purchased from Sigma.
Hundred per cent of PNP removal was achieved in the whole operating period with a maximum specific PNP loading rate of 0.26 g PNP g−1 VSS d−1.
The kinetic parameters obtained through optimization, validated with an additional respirometric test, were kmax = 1.02 mg PNP mg−1 COD d−1, Ks = 1.6 mg PNP L−1 and Ki = 54 mg PNP L−1.
An adenovirus with Tyrex2 regulating PNP (Ad2Tyr2-PNP) was tested for cytotoxicity.
pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC50=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH3+ 1b without 2-NHAc showed little activity (IC50 >50 mM).
Based on the results presented above, we propose orf pnp E1 and pnp E2 as genes encoding for functional cognate subunits of HqD that catalyze transformation reaction involved in HQ branch.
For 2C4NP and PNP transformation, the reaction mixture contained 20 mM phosphate buffer (pH 7.2), crude cell extracts, substrate (2C4NP or PNP, 20 μM), NADPH (100 μM), FAD (10 μM) and MgSO4 (1 mM) in a final volume of 500 μl.
In addition to 2C4NP, strain CNP-8 was also able to utilize 2C5NP and MNP; but it unable to degrade PNP, 4C2NP and 5C2NP (Additional file 1: Table S1).
Sera were obtained from 20 patients with clinically, histologically and immunopathologically typical PNP [2], [3].
These parameters were a slightly conservative estimate of those estimated from past elephant demographic patterns in PNP [10].
In the first experiment 5 µg of pNP, pM2 or pCAGGS (control) in 100 µl of PBS was injected intramuscularly per mouse per vaccination.
