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P Borst

Publications and source records attributed to P Borst.

At least 109 records · Page 6Linked to original sources
PubMed1995

New mechanisms of drug resistance in parasitic protozoa.

The main line of defense now available against parasitic protozoa--which are responsible for major diseases of humans and domestic animals--is chemotherapy. This defense is being eroded by drug resistance and, with few new drugs in the pipeline, prevention and circumvention of resistance are medical and veterinary priorities. Although studies of resistance mechanisms in parasites have lagged behind similar studies in bacteria and cancer cells, the tools to tackle this problem are rapidly improving. Transformation with exogenous DNA is now possible with all major parasitic protozoa of humans. Hence, putative resistance genes can be tested in sensitive protozoa, allowing an unambiguous reconstruction of resistance mechanisms. Gene cloning, the polymerase chain reaction, and monoclonal antibodies against resistance-related proteins have made it possible to analyze potential resistance mechanisms in the few parasites that can be obtained from infected people. Hence, the prospect of applying new knowledge about resistance mechanisms to parasites in patients is good, even though today virtually all knowledge pertains to parasites selected for resistance in the laboratory. Resistance mechanisms highlighted in this review include: 1. Decrease of drug uptake because of the loss of a transporter required for uptake. This decrease contributes to resistance to arsenicals and diamidines in African trypanosomes. 2. The export of drugs from the parasite by P-glycoproteins and other traffic ATPases. This export could potentially be an important mechanism of resistance, as these proteins are richly represented in the few protozoa analyzed. There are indications that such transmembrane transporters can be involved in resistance to emetine in Entamoeba spp., to mefloquine in Plasmodium spp., and to antimonials in Leishmania spp. 3. The possible involvement of the P-glycoprotein encoded by the Plasmodium falciparum pfmdr1 gene in chloroquine resistance. We present the available data that lead to the conclusion that overproduction of the wild-type version of this protein results in chloroquine hypersensitivity rather than resistance. 4. The involvement of the PgpA P-glycoprotein of Leishmania spp. in low-level resistance to arsenite and antimonials. We raise the possibility that this protein transports glutathione conjugates of arsenite and antimonials rather than the compounds themselves. 5. Loss of drug activation as the main mechanism of metronidazole resistance in Trichomonas and Giardia spp. Recent evidence indicates that a decrease of the proximal cellular electron donor for metronidazole activation, ferredoxin, is the main cause of resistance in Trichomonas. 6. Resistance arising through alteration of drug targets. The amino acid substitutions in the dihydrofolate reductase-thymidylate synthase of Plasmodium spp. are good examples of this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A.

We have previously shown that absence of the mouse mdr1a (also called mdr3) P-glycoprotein in mdr1a (-/-) "knockout" mice has a profound effect on the tissue distribution and elimination of vinblastine and ivermectin, and hence on the toxicity of these compounds. We show here that the mouse mdr1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. Injection of these radio-labeled drugs in mdr1a (-/-) and wild-type mice resulted in markedly (20- to 50-fold) higher levels of radioactivity in mdr1a (-/-) brain for digoxin and cyclosporin A, with more moderate effects for dexamethasone (2- to 3-fold) and morphine (1.7-fold). Digoxin and cyclosporin A were also more slowly eliminated from mdr1a (-/-) mice. Our findings show that P-glycoprotein can be a major determinant for the pharmacology of several medically important drugs other than anti-cancer agents, especially in the blood-brain barrier. These results may explain a range of pharmacological interactions observed between various drugs in patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.

The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

VSG gene expression site control in insect form Trypanosoma brucei.

When the African trypanosome Trypanosoma brucei is taken up from mammals by a tse-tse fly, it replaces its variant surface glycoprotein (VSG) coat by a procyclin coat. Transcription of VSG genes stops in the fly, but transcription of sequences derived from the promoter area of the VSG expression site(s) remains high. Whether this is due to continuing high activity of one promoter or to low activity of many promoters was unclear. We have used the small differences between the sequences of different expression sites to show that multiple expression site promoters are active in insect form trypanosomes. This is confirmed by the low expression of single copy marker genes introduced into the transcribed area. However, if the expression site promoter is removed from the genomic location of the expression site and inserted in the non-transcribed spacer of the ribosomal DNA (rDNA), it is derepressed. Derepression of transcription can also be accomplished by replacing the promoter of an expression site by an rDNA promoter. We conclude that the down-regulation of VSG gene expression site promoters in insect form trypanosomes is affected by both the DNA sequence of the promoter and the genomic context in which it resides.

Animals↗
PubMed1994

The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.

The mouse mdr2 P-glycoprotein (P-gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P-gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P-glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.

Amino Acid Sequence↗
PubMed1994

Tissue distribution of the human MDR3 P-glycoprotein.

BACKGROUND: P-glycoproteins (Pgps) belong to a family of well conserved plasma membrane proteins with two members in humans: MDR1 and MDR3. The MDR1 Pgp can transport drugs; the murine homologue of MDR3, mdr2, was recently shown by us to be involved in transport of the phospholipid phosphatidylcholine (lecithin) into bile. EXPERIMENTAL DESIGN: We have determined the MDR3 mRNA levels in a panel of human tissues by RNase protection. We have also generated polyclonal antibodies specific for the MDR3 Pgp. Detection of the MDR3 Pgp in human tissues with these antibodies was by a streptavidin-ABC procedure. RESULTS: The RNase protection results show that expression of the MDR3 gene has a more restricted distribution than that of MDR1. A high level of MDR3 mRNA was detected in the liver and in low levels in the adrenal gland, heart, striated muscle, spleen, and tonsil. In all of these tissues, some of the previously described splice variants of MDR3 were abundantly expressed. No indications were found for a tissue-specific regulation of alternative splicing of the MDR3 pre-mRNA. Two MDR3 Pgp-specific antibodies stained the bile canalicular membrane of hepatocytes across the entire liver lobule. No staining was found in the epithelial cells of the bile ductules and gall bladder, indicating that the staining at these sites with C219, a monoclonal antibody that recognizes both MDR1 and MDR3 Pgp, (mainly) represents the MDR1 Pgp. No MDR3 was detected by specific antibodies in the adrenal gland, spleen, and muscle. Since no staining was reported with MDR1-specific antibodies in muscle either, our results indicate that the C219 staining in some fibers of striated muscle represents a cross-reaction with another protein. One of the human MDR3-specific antibodies cross-reacted with the highly homologous mouse mdr2 Pgp. Staining with this antibody showed that the distribution of this protein in mouse liver and striated muscle is very similar to that of MDR3 Pgp in human tissues. CONCLUSIONS: The highest expression of the MDR3 Pgp was found in liver in the canalicular membranes of hepatocytes. This is in agreement with a role for MDR3 in the transport of phospholipid into bile.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei.

We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.

Animals↗
PubMed1993

Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein.

Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance. Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4. The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs. In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected. None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein. The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

The identification of hydroxymethyluracil in DNA of Trypanosoma brucei.

We have previously reported the detection of two unusual nucleotides, pdJ and pdV, in the DNA of Trypanosoma brucei (Gommers-Ampt et al., 1991). pdJ was found to be a novel nucleotide and is possibly involved in the regulation of variant specific surface antigen gene expression in trypanosomes. Recent evidence suggests that V could be a precursor of J, making V a key compound in the study of the biosynthesis and function of J. We have therefore determined the structure of V and here we present proof that V is HOMeU. The identity is based on a detailed comparison of dV(p) with authentic HOMedU(p), showing: I) co-migration in three different liquid chromatography analyses II) identical UV absorbance characteristics III) identical behavior in acetyl-pentafluorobenzyl derivatization and subsequent Gas chromatography/Mass spectrometry (GC/MS). The GC/MS technique has not been used before to analyse HOMedU purified from biological material. Because of its high sensitivity, it may also be useful for the detection of the low amounts of HOMedU resulting from oxidative damage of DNA.

Animals↗
PubMed1993

The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.

P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constructs in which the promoter area of the pgp genes was fused to the coding region of lacZ. Expression of pgp-1 and pgp-3, as inferred from pgp-lacZ transgenic nematodes, was confined to the intestinal cells. The expression patterns of both genes were virtually indistinguishable. Quantitative analysis of pgp mRNA levels during development showed that pgp-1, -2, and -3 were expressed throughout the life cycle of C.elegans, albeit with some variation indicating developmental regulation. The expression of P-glycoprotein genes in intestinal cells is an evolutionarily conserved feature of these genes, consistent with the hypothesis that P-glycoproteins provide a mechanism of protection against environmental toxins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals.

In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Stable transformation of Trypanosoma brucei.

We have further analyzed parameters affecting stable transformation of Trypanosoma brucei. Linear DNA was much more efficient than circular DNA and in the vast majority of transformants analyzed the plasmid DNA had inserted into the chromosomes by homologous recombination. The presence of non-homologous (vector) DNA at one or both ends of linear constructs inhibited transformation efficiency. Less than 1 kb of homologous flanking sequence was sufficient for efficient targeting of a marker gene into the tubulin gene array. When transformants with a single neomycin phosphotransferase (neo(r)) gene replacing a beta-tubulin gene were selected for higher levels of G418 resistance, the neo(r) gene was amplified and spread through the tubulin gene cluster. The additional neo(r) gene copies were adjacent in the tubulin gene array and were added to the array rather than replacing beta-tubulin genes. These results are compatible with asymmetric post-replication recombination (unequal sister chromatid exchange) as the mechanism for neo(r) gene amplification. Starting with a circular construct containing the neo(r) gene between tubulin intergenic regions, we obtained a single transformant that maintained the neo(r) genes as an extrachromosomal plasmid. We show this plasmid to consist of a circular pentamer of the input construct. All other attempts to derive a shuttle vector that replicates extrachromosomally in T. brucei were unsuccessful. Our experiments extend previous observations suggesting that T. brucei has a strong preference for chromosomal insertion of exogenous DNA by homologous recombination.

Animals↗
PubMed1993

Insertion of the promoter for a variant surface glycoprotein gene expression site in an RNA polymerase II transcription unit of procyclic Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei are invariably expressed near the ends of chromosomes (telomeres). We have targeted a VSG gene expression site (ES) promoter driving a selectable marker gene (neomycin phosphotransferase) into a chromosome-internal transcription unit, the tubulin gene array of procyclic trypanosomes. To avoid read through transcription of the marker gene from the tubulin promoter, we targeted the ES promoter in inverse orientation relative to tubulin gene transcription. The only correctly targeted transformant obtained contained the marker gene close to the border of the tubulin gene array, and expression of this gene was relatively low. Possible reasons for the low targeting efficiency and expression level are discussed.

Animals↗
PubMed1992

The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences.

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗