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P Borst

Publications and source records attributed to P Borst.

At least 55 records · Page 3Linked to original sources
PubMed1998

Analysis of a variant surface glycoprotein gene expression site promoter of Trypanosoma brucei by remodelling the promoter region.

Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3' of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5' of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5' ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5' of an ES promoter is required for optimal functioning of an ES.

Animals↗
PubMed1998

Changes in expression site control and DNA modification in Trypanosoma brucei during differentiation of the bloodstream form to the procyclic form.

We have adapted a system for in vitro differentiation of a monomorphic trypanosome strain to monitor changes in transcription and DNA modification in expression sites during the transition of the bloodstream-form to the procyclic trypanosome. We have used trypanosomes that have a gene for drug resistance integrated in an expression site, just downstream of either an expression site promoter, or a ribosomal promoter replacing the endogenous promoter. During the transition from bloodstream-form to procyclic, the promoters in an active expression site behave as expected on the basis of previous work on these promoters in procyclics, i.e. the ribosomal replacement promoter remains fully active, whereas the expression site promoter is (incompletely) down-regulated. A silent bloodstream-form expression site promoter does not remain tightly silenced, however. There is a transient increase of transcription of the marker gene during the transition from bloodstream-form to procyclic, indicating that the control of silent expression sites differs between the bloodstream-form and the procyclic trypanosome, and that a short time is required to reset the silencing mechanisms. One of the differences between bloodstream-form and procyclic trypanosomes is the presence of the modified base beta-D-glucosyl-hydroxymethyluracil (J) in and around bloodstream-form expression sites. We have studied loss of this DNA modification and find that the change in expression site control from bloodstream-form to procyclic does not require active removal of J. Base J is lost by synthesis of new, unmodified DNA, which happens after the major changes in expression site transcription have occurred.

Animals↗
PubMed1998

The modified DNA base beta-D-glucosylhydroxymethyluracil confers resistance to micrococcal nuclease and is incompletely recovered by 32P-postlabeling.

The hypermodified DNA base beta-D-glucosylhydroxymethyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.

Animals↗
PubMed1998

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

ATP-Binding Cassette Transporters↗
PubMed1998

beta-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres.

The unusual DNA base beta-D-glucosyl-hydroxymethyluracil, called "J, " replaces approximately 0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA)n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine flagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinoflagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.

Animals↗
PubMed1998

Control of VSG gene expression sites in Trypanosoma brucei.

Antigenic variation in African trypanosomes continues to be one of the most elaborate and intriguing strategies ever devised by a protozoan parasite to avoid complete destruction by the immune defense of its mammalian host. Here we review some of the recent advances in our understanding of this strategy, concentrating on (unpublished) work from our laboratory.

Animals↗
PubMed1998

The role of transferrin-receptor variation in the host range of Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. brucei to cope with the large sequence diversity in the transferrins of its hosts.

Animals↗
PubMed1998

Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (-/-) mice.

Mice homozygous for a disruption in the Mdr2 gene (Mdr2 (-/-) mice) lack the Mdr2 P-glycoprotein (P-gp) in the canalicular membrane of the hepatocyte and are unable to excrete phosphatidylcholine into the bile. These mice develop a nonsuppurative cholestatic liver disease, presumably caused by the high concentrations of free cytotoxic bile acids in bile. We generated transgenic mice that express the human homolog of Mdr2, MDR3, specifically in the liver by the use of an albumin promoter. In these mice the MDR3 P-gp is exclusively located in the canalicular membrane of hepatocytes and phospholipid excretion into bile is restored. Mice that contain the same amount of MDR3 P-gp as that of Mdr2 P-gp in wild-type mice, also excrete the same amount of phospholipids. No histopathological abnormalities were observed in the livers of these mice. In mice that express MDR3 at a higher or lower level, the phospholipid excretion correlated with the amount of MDR3 P-gp. We conclude that the human MDR3 P-gp is functionally homologous to the murine Mdr2 P-gp and that it can fully replace this P-gp in Mdr2 (-/-) mice, restoring the excretion of phospholipids into the bile. The phospholipid excretion is limited by the amount of MDR3 or Mdr2 P-gp. The excretion of cholesterol is not tightly coupled to the excretion of phospholipids in these mice, because a very low phospholipid excretion level is sufficient to give almost wild-type cholesterol excretion into the bile.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Frequent loss of the active site during variant surface glycoprotein expression site switching in vitro in Trypanosoma brucei.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.

Animals↗
PubMed1998

Biosynthesis and function of the modified DNA base beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

Animals↗
PubMed1998

Surface receptors and transporters of Trypanosoma brucei.

African trypanosomes combine antigenic variation of their surface coat with the ability to take up nutrients from their mammalian hosts. Uptake of small molecules such as glucose or nucleosides is mediated by translocators hidden from host antibodies by the surface coat. The multiple glucose transporters and transporters for nucleobases and nucleosides have been characterized. Receptors for host macromolecules such as transferrin and lipoproteins are visible to antibodies but hidden from the cellular arm of the host immune system in an invagination of the trypanosome surface, the flagellar pocket. The trypanosomal transferrin receptor is a heterodimer that resembles the major component of the surface coat of Trypanosoma brucei. The ability to make several versions of this receptor allows T. brucei to bind transferrins from a range of mammals with high affinity. The proteins required for uptake of nutrients by trypanosomes provide a target for chemotherapy that remains to be fully exploited.

Animals↗
PubMed1997

Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1.

The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

beta-D-glucosyl-hydroxymethyluracil, a novel base in African trypanosomes and other Kinetoplastida.

A novel base, beta-D-glucosyl-hydroxymethyluracil or J for short, was recently discovered in DNA of bloodstream form Trypanosoma brucei. The base is predominantly found in the hexameric repeat arrays of chromosome telomeres and in adjacent repetitive sub-telomeric DNA, and it is made by modification of specific thymines in DNA. J is present in inactive telomeric variant surface glycoprotein (VSG) genes, but not in active ones, suggesting a link between the presence of J and repression of the telomeric expression sites for VSG genes. The presence of J in DNA is specific for bloodstream form trypanosomes, as J is absent in insect form (procyclic) T. brucei. In addition to African trypanosomes, J has been found in DNA from other Kinetoplastida that do not undergo antigenic variation, such as Leishmania and Crithidia. The biological function of J remains to be deciphered.

Animals↗
PubMed1997

Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei.

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.

Animals↗
PubMed1997

Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833.

Mice lacking mdr1-type P-glycoproteins (mdr1a/1b [-/-] mice) display large changes in the pharmacokinetics of digoxin and other drugs. Using the kinetics of digoxin in mdr1a/1b (-/-) mice as a model representing a complete block of P-glycoprotein activity, we investigated the activity and specificity of the reversal agent SDZ PSC833 in inhibiting mdr1-type P-glycoproteins in vivo. Oral PSC833 was coadministered with intravenous [3H]digoxin to wild-type and mdr1a/1b (-/-) mice. The direct excretion of [3H]digoxin mediated by P-glycoprotein in the intestinal mucosa of wild-type mice was abolished by administration of PSC833. Hepatobiliary excretion of [3H]digoxin was markedly decreased in both wild-type and mdr1a/1b (-/-) mice by PSC833, the latter effect indicating that in vivo, PSC833 inhibits not only mdr1-type P-glycoproteins, but also other drug transporters. Upon coadministration of PSC833, brain levels of [3H]digoxin in wild-type mice showed a large increase, approaching (but not equaling) the levels found in brains of PSC833-treated mdr1a/1b (-/-) mice. Thus, orally administered PSC833 can inhibit blood-brain barrier P-glycoprotein extensively, and intestinal P-glycoprotein completely. These profound pharmacokinetic effects of PSC833 treatment imply potential risks, but also promising pharmacological applications of the use of effective reversal agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗