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Articles by K. F Chan
Total Records ( 2 ) for K. F Chan
  K. F Chan , P Zhang and Z. Song
 

The Golgi CMP-sialic acid transporter (CST) is a type III transmembrane protein with 10 transmembrane domains that are linked by eight hydrophilic loops. To investigate the function of these hydrophilic loops, the green fluorescent protein (GFP) was inserted into each loop of the transporter. Expression and localization of the resulting CST-GFP fusion proteins were confirmed by analyzing the fluorescence of GFP. The transport activity of the CST-GFP proteins was analyzed by a previously described erythropoietin/isoelectric focusing assay in CST-deficient MAR-11 cells. Interruption of the second and fourth lumenal loops and the fourth cytosolic loop of CST with GFP resulted in complete or partial loss of transport activity. Regions in these loops that play crucial roles in CST’s activity were identified by Gly substitutions. Single amino acid substitution experiments revealed that Lys272 of the fourth loop on the cytosolic side of CST is essential for transport activity. Mutation of the conserved Lys residue (Lys297) in the UDP-galactose transporter (UGT) also resulted in a complete loss of its activity. Point mutations of highly conserved amino acid residues in the loop regions identified Leu136 of CST as essential for its activity. However, mutation of the conserved Leu residue in UGT (Leu160) did not affect the transport activity of UGT. Finally, mutation of Leu224 in UGT completely inactivated the activity of UGT, although mutation of its conserved counterpart in CST, Leu199, did not have any effect on CST. This study provides a structure–function analysis of the loop regions in CST and UGT.

  I. L. K Wong , K. F Chan , Y Zhao , T. H Chan and L. M. C. Chow
  Objectives

The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites.

Methods

Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effects of different combinations of quinacrine and an apigenin dimer on modulating the pentamidine resistance levels of LePentR50 and LdAG83PentR50 using an MTS proliferation assay. We then measured the accumulation level of pentamidine using HPLC. The fractional inhibitory concentration index (FICI) method was used to evaluate the interaction between quinacrine and the apigenin dimer on reversing pentamidine resistance in Leishmania.

Results

LePentR50 and LdAG83PentR50 promastigotes were ~8.6- and 4.6-fold more resistant to pentamidine than their wild-type parents. Amastigotes derived from LePentR50 and LdAG83PentR50 were also pentamidine-resistant. We found that quinacrine can increase the susceptibility of Leishmania to pentamidine. Quinacrine, when used at 6 µM, can increase the IC50 of pentamidine by 3.8-, 3.4-, 3.5- and 6.3-fold in wild-type Leishmania enriettii Le, LePentR50, wild-type Leishmania donovani LdAG83 and LdAG83PentR50, respectively. Quinine, quinidine and verapamil did not show any sensitizing effect. The sensitizing effect of quinacrine was: (i) dose-dependent; (ii) not associated with an increase in pentamidine accumulation; and (iii) only observed in pentamidine-resistant but not sodium stibogluconate-resistant or vinblastine-resistant parasites. Other than quinacrine, we also found that an apigenin dimer (compound 9d), previously shown to be able to inhibit ABCB1-mediated cancer drug resistance in mammalian cells, can also increase the pentamidine susceptibility of Leishmania. 9d, when used at 6 µM, can increase the IC50 of pentamidine by 2.5-, 4.2-, 1.6- and 1.9-fold in Le, LePentR50, LdAG83 and LdAG83PentR50, respectively. Unlike quinacrine, sensitization by 9d was accompanied by an increase in pentamidine accumulation, presumably due to the inhibition of an ABC transporter. Using the FICI method, we found that quinacrine and 9d can act synergistically. When they are used in a 1:1 ratio, they sensitize LePentR50 to pentamidine by 19-fold, with an FICI of 0.48 (P < 0.005), indicating that they might act synergistically.

Conclusions

Our findings support the notion that the pentamidine susceptibility of Leishmania is mediated by multiple targets. Quinacrine and apigenin dimer 9d, each inhibiting its own target, can have a synergistic effect when used together to sensitize Leishmania to pentamidine.

 
 
 
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