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

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

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

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

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

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

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

Inhibition of auranofin on enzymatic activities of recombinant SjTGR.

<p>(A) Percentages of TrxR, GR and Grx activities inhibited by different doses of auranofin with DTNB, GSSG or HED as substrate. (B) Inhibition constant () values of auranofin on recombinant SjTGR in the TrxR assay. The straight lines were fitted on the basis of the reciprocal of concentration of substrate and initial velocity. The concentrations of auranofin were 0 nM, 5 nM, 10 nM and 15 nM, and DTNB ranged from 60 to 1,000 µM. (C) Inhibition constant () values of auranofin on SjTGR in the GR activity assay. The straight lines were fitted on the basis of the reciprocal of concentration of substrate and initial velocity. The concentrations of auranofin were 0 nM, 5 nM, 10 nM and 20 nM, and GSSG ranged from 10 to 100 µM.</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