http://repub.eur.nl/res/aut/6281/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/16136/ 2009-04-01T00:00:00Z The phytoestrogen resveratrol has putative health-promoting effects and is present in several dietary constituents. Resveratrol is metabolized extensively in the gut epithelium, resulting in the formation of hydrophilic glucuronic acid and sulfate conjugates. These polar resveratrol conjugates need specific transporters to cross the cell membrane. We show here that vectorial transport of some of these metabolites is mediated by multidrug resistance protein 3 (MRP3, ABCC3) and/or breast cancer resistance protein (BCRP, ABCG2) located in the basolateral and apical membranes of enterocytes, respectively. In vitro, MRP3 transports resveratrol-glucuronide (Res-3-G). The absence of Mrp3 in mice results in altered disposition of Res- 3-G and its parent compound resveratrol, leading to a reduced percentage of resveratrol being excreted via the urine in Mrp3(-/-) mice. Circumstantial evidence suggests that circulating resveratrol is formed by deglucuronidating Res-3-G in vivo, providing a possible explanation for the health beneficial effects of resveratrol in vivo, despite its rapid and extensive conjugation. BCRP transports Res-3-G and resveratrol sulfates in vitro, and its absence in mice results in high plasma levels of resveratrol-di-sulfate, a resveratrol metabolite hardly detectable in the plasma of wild-type mice and in an increased disposal of resveratrol via the urine. The profound effects of ATP-binding cassette transporters on the disposal of resveratrol may be representative for the handling of several other polyphenols of dietary origin. http://repub.eur.nl/res/pub/2972/ 1983-01-01T00:00:00Z The nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramphenicol to be predicted. The 9S RNA has little homology with other rRNAs, but a possible secondary structure is not unlike that of the 2.5-fold larger E.coli 16S rRNA. We conclude that the 12S RNA (about 1230 nucleotides) and the 9S RNA (about 640 nucleotides) are the smallest homologues of the E.coli 23S and 16S rRNAs yet observed http://repub.eur.nl/res/pub/2357/ 1982-01-01T00:00:00Z Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment. http://repub.eur.nl/res/pub/2961/ 1981-01-01T00:00:00Z http://repub.eur.nl/res/pub/2966/ 1981-01-01T00:00:00Z http://repub.eur.nl/res/pub/2950/ 1980-01-01T00:00:00Z We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown. http://repub.eur.nl/res/pub/2951/ 1980-01-01T00:00:00Z Maxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by electron microscopy. Long loops up to 10 micrometer were present, extending from the network rim; these are considered typical of maxi-circles.The presence of maxi-circles was proven by digestion of kinetoplast DNA with restriction endonucleases and S1 nuclease. This released a minor DNA component, detectable by agarose gel electrophoresis, which hybridized to maxi-circle DNA from Trypanosoma brucei. The molecular weight of the linearized maxi-circle of Trypanosoma cruzi is 26 . 10(6), as judged from its electrophoretic mobility in 0.6% agarose. Our restriction enzyme analysis of the mini-circles of Trypanosoma cruzi has confirmed their sequence heterogeneity and internally-repeated structure. We have found that more than 90% of the mini-circles are cut into 1/4 length molecules by endonuclease TaqI. Denaturation and renaturation of mini-circles, cut once with endonuclease MboI, mainly yields linear and circular molecules with single-stranded eyes and tails in electron micrographs. This shows that 1/4 repeats contain sub-segments in which sequence divergence is extensive. Our EcoRI and HapII digests differ in fragment size distribution from those previously reported. This suggests that this distribution may not be a stable characteristic of the Y strain. http://repub.eur.nl/res/pub/2953/ 1980-01-01T00:00:00Z We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs. http://repub.eur.nl/res/pub/2954/ 1980-01-01T00:00:00Z Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene. http://repub.eur.nl/res/pub/2956/ 1980-01-01T00:00:00Z We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei. Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli. Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins. The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another. http://repub.eur.nl/res/pub/2959/ 1980-01-01T00:00:00Z We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains. http://repub.eur.nl/res/pub/2960/ 1980-01-01T00:00:00Z Sequential expression of variant surface glycoproteins (VSGs) enables the parasitic protozoan Trypanosoma brucei to evade the immune response of its mammalian hosts. Studies of several VSGs, which have been isolated as soluble molecules following disruption of cells in the absence of detergent, have indicated extensive amino acid diversity and the absence of a hydrophobic segment which might serve to anchor the carboxy terminus to the membrane. The carboxy-terminal tryptic peptides of six VSGs have recently been characterized and shown to be glycosylated. Three of these VSGs terminated with a glycosylated aspartate or asparagine residue (Asx), suggesting that the VSG was cleaved following synthesis and glycosylation and before characterization. We present here nucleotide sequence data which suggest that the primary translation product of one VSG gene contains a hydrophobic tail at the carboxy terminus which is not found on the isolated, mature glycoprotein. The data also predict that the glycosylated residue is aspartic acid rather than the anticipated asparagine. http://repub.eur.nl/res/pub/2945/ 1978-01-01T00:00:00Z We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms. http://repub.eur.nl/res/pub/2946/ 1978-01-01T00:00:00Z We have hybridized total cellular RNA of Crithidia luciliae with the kinetoplast DNA of this organism. To allow the discrimination of DNA from mini-circles (2300 base pairs) and maxi-circles (33 000 base pairs), kinetoplast DNA was digested with restriction endonucleases and the fragments were separated by electrophoresis through an agarose gel and transferred to nitrocellulose filters by blotting. No mini-cricle transcripts were found under conditions where maxi-circle fragments showed extensive and specific hybridization. Since maxi-circle sequences are present at less than 1% of the concentration of mini-circle sequences, we conclude that mini-circles may not be transcribed at all. Predominant hybridization with the maxi-circle fragments is obtained with a segment of only 2300--2500 base pairs. The possibility that this segment codes for unusually small mitochondrial ribosomal RNAs is discussed. http://repub.eur.nl/res/pub/2944/ 1977-01-01T00:00:00Z http://repub.eur.nl/res/pub/2332/ 1974-01-01T00:00:00Z