Sanjay Shahi - Academia.edu (original) (raw)

Papers by Sanjay Shahi

Trends in Pharmacological Sciences, 2006

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic micro... more ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in threedimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

European Journal of Pharmacology, 2006

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barri... more Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamicpituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 μM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 μM the same three increased sensitivity of pglycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

A functional steroid-binding element in an ATP-binding cassette multidrug transporter

Mol Biochem Parasitol, 2001

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete seque... more Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

The Journal of Biological Chemistry, Jun 6, 2003

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the... more The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the antiestrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [ 3 H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

Molecular Microbiology, 2002

Molecular and Biochemical Parasitology, 2001

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete seque... more Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

Journal of Endocrinology, 1998

A prominent functional change during differentiation of lutein cells from follicular thecal and g... more A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPAR , a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPAR in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPAR in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1·5-fold of the basal level) to an endogenous ligand of PPAR , 15-deoxy-12,14 prostaglandin J 2 (15-dPGJ 2 ) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPAR level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPAR proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPAR was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPAR in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ 2 , suggesting that 15-dPGJ 2 exerts its effect on steroidogenic activity via PPAR and that the 15-dPGJ 2 -PPAR system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Journal of Biological Chemistry, 2003

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a struc... more LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [ 3 H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures

Journal of Biological Chemistry, 2003

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the... more The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the antiestrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [ 3 H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug r... more We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione-spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Biochemical and Biophysical Research Communications, 2006

We have previously shown ginsenosides derived from Panax ginseng exert opposing effects on angiog... more We have previously shown ginsenosides derived from Panax ginseng exert opposing effects on angiogenesis. Here, we examined protopanaxadiol-containing ginsenosides (Rg3, Rh2, and PPD) and protopanaxatriol-containing ginsenosides (Rg1, Rh1, and PPT) as potential inhibitors of breast cancer resistance protein (BCRP). Among these ginsenosides, metabolites Rh2, PPD, and PPT significantly enhanced the cytotoxicity of mitoxantrone (MX) to human breast carcinoma MCF-7/MX cells

Molecular Pharmacology, 2008

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter... more The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid

Molecular and Biochemical Parasitology, 2006

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug r... more We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione-spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Journal of Bacteriology, 2005

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gra... more MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gramnegative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K i values of 16 and 11 M, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K i of 57 M, in a similar range as the K i for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbAmediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G.

International Journal of Antimicrobial Agents, 2003

The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport ... more The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli , provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 Å . The E. coli crystal structure of MsbA reveals a dimer. Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface. We considered the interface in a two-armed MsbA dimer, named spiral . Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E. coli MsbA represents a crystallization artefact. A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described. #

International Journal of Antimicrobial Agents, 2003

Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals... more Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATPbinding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium , Leishmania , Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included. #

European Journal of Pharmacology, 2006

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barri... more Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamicpituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 μM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 μM the same three increased sensitivity of pglycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

BMC Research Notes, 2011

Background: The present study represents a genome-wide transcriptomic analysis of the response of... more Background: The present study represents a genome-wide transcriptomic analysis of the response of the model streptomycete Streptomyces coelicolor A3(2) M145 to fermentor culture in Modified Evans Media limited, respectively, for nitrogen, phosphate and carbon undertaken as part of the ActinoGEN consortium to provide a publicly available reference microarray dataset. Findings: A microarray dataset using samples from two replicate cultures for each nutrient limitation was generated. In this report our analysis has focused on the genes which are significantly differentially expressed, as determined by Rank Products Analysis, between samples from matched time points correlated by growth phase for the three pairs of differently limited culture datasets. With a few exceptions, genes are only significantly differentially expressed between the N6/N7 time points and their corresponding time points in the C and P-limited cultures, with the vast majority of the differentially expressed genes being more highly expressed in the N-limited cultures. Our analysis of these genes indicated expression of several members of the GlnR regulon are induced upon nitrogen limitation, as assayed for by [NH 4 + ] measurements, and we are able to identify several additional genes not present in the GlnR regulon whose expression is induced in response to nitrogen limitation. We also note SCO3327 which encodes a small protein (32 amino acid residues) unusually rich in the basic amino acids lysine (31.25%) and arginine (25%) is significantly differentially expressed in the nitrogen limited cultures. Additionally, we investigate the expression of known members of the GlnR regulon and the relationship between gene organization and expression for the SCO2486-SCO2487 and SCO5583-SCO5585 operons.

Biochemical Society Transactions, 2005

The movement of drugs across biological membranes is mediated by two major classes of membrane tr... more The movement of drugs across biological membranes is mediated by two major classes of membrane transporters. Primary-active, ABC (ATP-binding cassette) multidrug transporters are dependent on ATP-binding/hydrolysis, whereas secondary-active multidrug transporters are coupled to the proton (or sodium)-motive force that exists across the plasma membrane. Recent work on LmrA, an ABC multidrug transporter in Lactococcus lactis, suggests that primary- and secondary-active multidrug transporters share functional and structural features. Some of these similarities and their implications for the mechanism of transport by ABC multidrug transporters will be discussed.

Trends in Pharmacological Sciences, 2006

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic micro... more ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in threedimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

European Journal of Pharmacology, 2006

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barri... more Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamicpituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 μM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 μM the same three increased sensitivity of pglycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

A functional steroid-binding element in an ATP-binding cassette multidrug transporter

Mol Biochem Parasitol, 2001

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete seque... more Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

The Journal of Biological Chemistry, Jun 6, 2003

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the... more The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the antiestrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [ 3 H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

Molecular Microbiology, 2002

Molecular and Biochemical Parasitology, 2001

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete seque... more Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

Journal of Endocrinology, 1998

A prominent functional change during differentiation of lutein cells from follicular thecal and g... more A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPAR , a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPAR in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPAR in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1·5-fold of the basal level) to an endogenous ligand of PPAR , 15-deoxy-12,14 prostaglandin J 2 (15-dPGJ 2 ) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPAR level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPAR proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPAR was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPAR in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ 2 , suggesting that 15-dPGJ 2 exerts its effect on steroidogenic activity via PPAR and that the 15-dPGJ 2 -PPAR system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Journal of Biological Chemistry, 2003

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a struc... more LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [ 3 H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures

Journal of Biological Chemistry, 2003

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the... more The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the antiestrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [ 3 H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug r... more We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione-spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Biochemical and Biophysical Research Communications, 2006

We have previously shown ginsenosides derived from Panax ginseng exert opposing effects on angiog... more We have previously shown ginsenosides derived from Panax ginseng exert opposing effects on angiogenesis. Here, we examined protopanaxadiol-containing ginsenosides (Rg3, Rh2, and PPD) and protopanaxatriol-containing ginsenosides (Rg1, Rh1, and PPT) as potential inhibitors of breast cancer resistance protein (BCRP). Among these ginsenosides, metabolites Rh2, PPD, and PPT significantly enhanced the cytotoxicity of mitoxantrone (MX) to human breast carcinoma MCF-7/MX cells

Molecular Pharmacology, 2008

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter... more The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid

Molecular and Biochemical Parasitology, 2006

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug r... more We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione-spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Journal of Bacteriology, 2005

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gra... more MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gramnegative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K i values of 16 and 11 M, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K i of 57 M, in a similar range as the K i for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbAmediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G.

International Journal of Antimicrobial Agents, 2003

The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport ... more The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli , provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 Å . The E. coli crystal structure of MsbA reveals a dimer. Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface. We considered the interface in a two-armed MsbA dimer, named spiral . Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E. coli MsbA represents a crystallization artefact. A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described. #

International Journal of Antimicrobial Agents, 2003

Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals... more Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATPbinding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium , Leishmania , Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included. #

European Journal of Pharmacology, 2006

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barri... more Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamicpituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 μM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 μM the same three increased sensitivity of pglycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

BMC Research Notes, 2011

Background: The present study represents a genome-wide transcriptomic analysis of the response of... more Background: The present study represents a genome-wide transcriptomic analysis of the response of the model streptomycete Streptomyces coelicolor A3(2) M145 to fermentor culture in Modified Evans Media limited, respectively, for nitrogen, phosphate and carbon undertaken as part of the ActinoGEN consortium to provide a publicly available reference microarray dataset. Findings: A microarray dataset using samples from two replicate cultures for each nutrient limitation was generated. In this report our analysis has focused on the genes which are significantly differentially expressed, as determined by Rank Products Analysis, between samples from matched time points correlated by growth phase for the three pairs of differently limited culture datasets. With a few exceptions, genes are only significantly differentially expressed between the N6/N7 time points and their corresponding time points in the C and P-limited cultures, with the vast majority of the differentially expressed genes being more highly expressed in the N-limited cultures. Our analysis of these genes indicated expression of several members of the GlnR regulon are induced upon nitrogen limitation, as assayed for by [NH 4 + ] measurements, and we are able to identify several additional genes not present in the GlnR regulon whose expression is induced in response to nitrogen limitation. We also note SCO3327 which encodes a small protein (32 amino acid residues) unusually rich in the basic amino acids lysine (31.25%) and arginine (25%) is significantly differentially expressed in the nitrogen limited cultures. Additionally, we investigate the expression of known members of the GlnR regulon and the relationship between gene organization and expression for the SCO2486-SCO2487 and SCO5583-SCO5585 operons.

Biochemical Society Transactions, 2005

The movement of drugs across biological membranes is mediated by two major classes of membrane tr... more The movement of drugs across biological membranes is mediated by two major classes of membrane transporters. Primary-active, ABC (ATP-binding cassette) multidrug transporters are dependent on ATP-binding/hydrolysis, whereas secondary-active multidrug transporters are coupled to the proton (or sodium)-motive force that exists across the plasma membrane. Recent work on LmrA, an ABC multidrug transporter in Lactococcus lactis, suggests that primary- and secondary-active multidrug transporters share functional and structural features. Some of these similarities and their implications for the mechanism of transport by ABC multidrug transporters will be discussed.