A nucleoside transporter from Trypanosoma brucei involved in drug resistance.
(9/1525)
Drug resistance of pathogens is an increasing problem whose underlying mechanisms are not fully understood. Cellular uptake of the major drugs against Trypanosoma brucei spp., the causative agents of sleeping sickness, is thought to occur through an unusual, so far unidentified adenosine transporter. Saccharomyces cerevisiae was used in a functional screen to clone a gene (TbAT1) from Trypanosoma brucei brucei that encodes a nucleoside transporter. When expressed in yeast, TbAT1 enabled adenosine uptake and conferred susceptibility to melaminophenyl arsenicals. Drug-resistant trypanosomes harbor a defective TbAT1 variant. The molecular identification of the entry route of trypanocides opens the way to approaches for diagnosis and treatment of drug-resistant sleeping sickness. (+info)
Selective irreversible inactivation of replicating mengovirus by nucleoside analogues: a new form of viral interference.
(10/1525)
We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection. (+info)
A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum.
(11/1525)
Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome. (+info)
Synthesis of 4'-C-ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta- D-ribo-pentofuranosyl pyrimidines, and their biological evaluation.
(12/1525)
4'-C-Ethynyl-beta-D-arabino-pentofuranosyl thymine (14) and cytosine (16), and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosyl thymine (25) and cytosine (27) were synthesized by properly protected 4'-C-hydroxy-methyl-3,5-di-O-benzyl-alpha-D-ribo-pentofuranose (1) from D-glucose. Among them, 2'-deoxy derivatives 25 and 27 exhibited antiviral activity, while cytidine derivatives 16 and 27 inhibited the growth of neoplastic cells. (+info)
The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1.
(13/1525)
Nephrotoxicity is the dose-limiting clinical adverse effect of cidofovir and adefovir, two potent antiviral therapeutics. Because renal uptake likely plays a role in the etiology of cidofovir- and adefovir-associated nephrotoxicity, we attempted to identify a renal transporter capable of interacting with these therapeutics. A cDNA clone was isolated from a human renal library and designated human organic anion transporter 1 (hOAT1). Northern analysis detected a specific 2.5-kilobase pair hOAT1 transcript only in human kidney. However, reverse transcription-polymerase chain reaction revealed hOAT1 expression in human brain and skeletal muscle, as well. Immunoblot analysis of human kidney cortex demonstrated that hOAT1 is an 80- to 90-kilodalton heterogeneous protein modified by abundant N-glycosylation. Xenopus laevis oocytes expressing hOAT1 supported probenecid-sensitive uptake of [(3)H]p-aminohippurate (K(m) = 4 microM), which was trans-stimulated in oocytes preloaded with glutarate. Importantly, both hOAT1 and rat renal organic anion transporter 1 (rROAT1) mediated saturable, probenecid-sensitive uptake of cidofovir, adefovir, and other nucleoside phosphonate antivirals. The affinity of hOAT1 toward cidofovir and adefovir (K(m) = 46 and 30 microM, respectively) was 5- to 9-fold higher compared with rROAT1 (K(m) = 238 and 270 microM, respectively). These data indicate that hOAT1 may significantly contribute to the accumulation of cidofovir and adefovir in renal proximal tubules and, thus, play an active role in the mechanism of nephrotoxicity associated with these antiviral therapeutics. (+info)
Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells.
(14/1525)
One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 - 20 microM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive c-Myc expression. (+info)
Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.
(15/1525)
The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase. (+info)
Early steps of replication of moloney murine leukemia virus in resting lymphocytes.
(16/1525)
We explored the role of cell type in the early steps of replication of Moloney murine leukemia virus (Mo-MLV) by comparing viral entry and reverse transcription in physiologically quiescent peripheral blood B and T lymphocytes. Virus entry was identical in both cell types. In contrast to previous results, full-length viral DNA was synthesized in resting B lymphocytes, but in agreement with earlier reports, reverse transcription was abortive in resting T lymphocytes. The addition of exogenous nucleosides in the culture medium of resting T lymphocytes allowed reverse transcription to proceed in these cells, without inducing cell cycling. These data suggest that the difference in the ability of quiescent T and B lymphocytes to sustain reverse transcription of Mo-MLV can be explained by a difference in the dNTP pool sizes of these two populations of quiescent cells. (+info)