Treatment effect of auranofin on <i>S. japonicum</i> in mice.

*<p>Auranofin treatment versus control, <i>P</i><0.01 by t-test.</p

Protein profile of purified recombinant SjTGR.

<p>Lane 1: Purified recombinant SjTGR protein on SDS-PAGE gel stained with Commassie Blue. MW: Protein molecular weight marker.</p

Electrophoretic patterns of expression products of recombinant SjTGR.

<p>(A) Expression products from <i>E. coli</i> BL21 transformed with recombinant SjTGR-pET41a and induced with 1 mmole/L of IPTG at 37°C for 4 h. Lane 1: Supernatant of bacterial lysate. Lane 2: Precipitate of bacteria lysate resuspended with PBS. The black arrow indicates the expressed SjTGR protein band. MW: Protein molecular weight marker. (B) Expression products from transformed or non-transformed <i>E. coli</i> BL21 induced with 0.5 mmole/L of IPTG at 24°C for 24 h. Products in precipitates (lane 1) or supernatant (lane 2) of induced bacteria containing plasmid SjTGR-pET41a show expression of the recombinant SjTGR (black arrow). Expression products of induced bacteria containing plasmid pET41a (lane 3) or of induced non-transformed bacteria (lane 4) are also shown. MW: Protein molecular weight marker.</p

Comparison of recombinant TGR enzyme activities of <i>S. japonicum</i> and <i>S. mansoni</i>.

<p>Comparison of recombinant TGR enzyme activities of <i>S. japonicum</i> and <i>S. mansoni</i>.</p

Kinetics of recombinant SjTGR with different substrates.

<p>Kinetic constants for recombinant SjTGR (48 nM) were determined in assays performed at 25°C in 0.1 M potassium phosphate (pH 7.4) with 10 mM EDTA and 100 µM NADPH. All assays were conducted in triplicate.</p

Comparison of kinetics of recombinant TGR from <i>S. mansoni</i> and <i>S. japonicum</i> with different substrates.

<p>Comparison of kinetics of recombinant TGR from <i>S. mansoni</i> and <i>S. japonicum</i> with different substrates.</p

Analysis of recombinant SjTGR-pET41a by enzyme restriction.

<p>(A) Agarose gel (1%) electrophoresis of recombinant SjTGR-pET41a or pET41a vectors. Lane 1: Purified PCR amplified SjTGR gene fragment of about 1,800 bp. Lane 2: Products of recombinant plasmid SjTGR-pET41a digested by <i>Nde</i> I/<i>Sal</i> I. The sizes of the pET41a backbone and the SjTGR gene were about 5,000 bp and 1,800 bp, respectively. Lane 3: Products of plasmid pET 41a digested by <i>Nde</i> I/<i>Sal</i> I. The sizes of the fragments from <i>Nde</i> I/<i>Sal</i> I digestion of pET41a were about 5,000 bp and 900 bp. Lane 4: Undigested pET 41a plasmid of 5933 bp. Lane 5: Undigested recombinant plasmid SjTGR-pET41a of about 6,800 bp. Note that both undigested plasmids migrated lower in the gel due to the super-helical structures. MW: DNA molecular weight marker. (B) Schematic diagram of the SjTGR-pET41a plasmid showing relevant enzyme sites and the SjTGR insert size.</p

Western blotting analysis of <i>S. japonicum</i> worm homogenate.

<p>Adult worm supernatant (lane 1) and purified recombinant SjTGR protein (lane 2) detected by mice polyclonal serum antibodies against recombinant SjTGR. MW: Protein molecular weight marker.</p

Analysis of isotope ⁷⁵Se-cysteine incorporation.

<p>(A) Profile of expressed protein products on a 12% SDS-PAGE gel. (B) Autoradiogram profile of expressed products corresponding to the proteins on SDS-PAGE. Lane 1: Expression products of <i>E.coli</i> BL21 containing plasmid SjTGR-pET41a. Lane 2: Expression products of <i>E.coli</i> BL21 containing plasmids SjTGR-pET41a and pSUABC. MW: Protein molecular weight marker. The black dots on the SDS-PAGE gel are the expression products of <i>E.coli</i> BL21 containing plasmids SjTGR-pET41a and pSUABC. The expression products containing isotope ⁷⁵Se-cysteine (SeCys) were dotted on the SDS-PAGE gel after electrophoresis as a positive control for autoradiography. The black arrow indicates the SjTGR selenoprotein developed by autoradiography.</p

Inhibition of <i>S. japonicum</i> adult worms by auranofin in vitro.

<p>(A) Effects of auranofin (AUF 1, 5, 10, 20, 30 µg/ml) on TrxR and GR activities of adult worms after 3 h of treatment. Worms were mock treated in the control groups (RPMI 1640 and 1.2% DMSO). Worms were treated with 30 µg/ml of praziquantel in the PZQ 30 µg/ml group. (B) Inhibition of auranofin on TrxR activity of adult worms over time. Worms were treated with 5 and 10 µg/ml of auranofin, and the TrxR activities of worm homogenates were tested after 0, 1, 3 and 6 h. (C) Inhibition of auranofin on GR activity of adult worms over time. Worms were treated with 5 and 10 µg/ml of auranofin, and the GR activities of worm homogenates were tested after 0, 1, 3 and 6 h.</p