Oligodendrocytes (OLs) are the glial cells of the central nervous system and are classically known to form myelin sheaths around most axons of higher vertebrates. Whether these cells might have other roles, in particular during development, has not been studied. Taking advantage of a transgenic mouse model in which OLs can be selectively killed in a desired time-frame, we have investigated the impact of OL ablation on cerebellar development. OL ablation was induced during the first 3 postnatal weeks, a time at which cerebellum development is ongoing. Strikingly, OL ablation triggers a profound perturbation of the known cerebellum developmental program, characterized by the disorganization of the cortical layers, abnormal foliation and a complete alteration of Purkinje cell dendritic arborization and axonal fasciculation. This phenotype is accompanied by decreased granule cell density, a disorganized Bergmann glia network and impaired migration of interneurons in the molecular layer. These results demonstrate a previously ignored role of OLs in the formation of the cerebellar cytoarchitecture. (+info)
In vitro drug combination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil with anti-human immunodeficiency virus or anticancer nucleosides.
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1-beta-D-Arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) and E-5-(2-bromovinyl)uracil, a metabolite of BV-araU, did not affect either the anti-human immunodeficiency virus activity or the cytotoxicity of azidothymidine in MT-4 and MOLT-4 cells. Similarly, the bromovinyl compounds did not affect the in vitro antitumor activities of arabinosylcytosine, 5-fluorouracil, and 5-fluoro-2'-deoxyuridine. The anti-varicella-zoster virus activity of BV-araU was not influenced by azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, or arabinosylcytosine, whereas relatively high concentrations of fluorinated antitumor agents enhanced the anti-varicella-zoster virus activity. (+info)
Synthesis and properties of oligonucleotides containing C-2 and/or C-5 substituted arabinofuranosyluracils.
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The oligodeoxyribonucleotides (ODN) containing C-2 and/or C-5 substituted arabinofuranosyluracils were synthesized by a post-synthetic modification method. The duplex stability of the ODN bearing isocytosine derivative with DNA was lower than that of the normal ODN/DNA duplex but the ODN bearing C5-substituted uracil derivative was similar to the normal ODN/DNA duplex. (+info)
Noninvasive imaging of transgene expression by use of positron emission tomography in a pig model of myocardial gene transfer.
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BACKGROUND: Radionuclide imaging of reporter gene expression may be useful for noninvasive monitoring of clinical cardiac gene therapy. Experience until now, however, has been limited to small animals. METHODS AND RESULTS: To evaluate feasibility in a clinically applicable setting, pigs were studied by conventional positron emission tomography (PET) 2 days after regional intramyocardial injection of control adenovirus or adenovirus carrying herpesviral thymidine kinase reporter gene (HSV1-tk). Myocardial blood flow was quantified by use of [13N]ammonia. Subsequently, kinetics of the reporter substrate [124I]-2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabino-furanosyluracil (FIAU) were assessed over a period of 2 hours. Areas infected with adenovirus expressing HSV1-tk showed significantly elevated FIAU retention during the first 30 minutes after injection. At later times, washout was observed, and retention was not different from that in areas infected with control virus or remote myocardium. Early in vivo FIAU uptake correlated with ex vivo images, autoradiography, and immunohistochemistry for reporter gene product after euthanasia. After intramyocardial injection of both adenoviruses, myocardial blood flow was mildly elevated compared with that in remote areas, consistent with histological signs of regional inflammation. CONCLUSIONS: In vivo quantification of regional myocardial transgene expression is feasible with clinical PET methodology, the radioiodinated reporter probe FIAU, and the HSV1-tk reporter gene. Radioactivity efflux after specific initial uptake was not observed previously in tumor studies, suggesting that tissue-specific differences in nucleoside metabolism influence reporter probe kinetics. By coregistering reporter gene expression with additional biological parameters such as myocardial blood flow, PET allows for noninvasive characterization of the success of cardiac gene transfer along with its functional correlates. (+info)
Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs.
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Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [14C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nm nitrobenzylthioinosine. Moreover, FIAU (50 microm) produced significant mitochondrial toxicity ( approximately 70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine. (+info)
Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.
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The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. METHODS: TK(1) activity; S-phase fraction; and uptake of TdR, 3'-deoxy-3'-fluorothymidine (FLT), and 2'-fluoro-5-methyl-1-(beta-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. RESULTS: TK(1) activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK(1) activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK(1) activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK(1) activity. The slope of the relationship between TdR uptake and TK(1) activity was nearly twice that for FLT and more than 40-fold that for FMAU. CONCLUSION: Although not all tumors show a strong TK(1) dependence of proliferation, in all cell lines for which proliferation is highly TK(1) dependent, phosphorylation of TdR or FLT accurately reflects changes in TK(1) enzyme activity. (+info)
Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy.
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We determined the frequency of integrated viral DNA in the livers of three woodchucks chronically infected with the woodchuck hepatitis virus before and during 30 weeks of therapy with the nucleoside analog L-FMAU [1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl)uracil, clevudine]. We found that although viral covalently closed circular DNA declined 20- to 100-fold, integrated viral DNA showed no discernable decrease over the course of treatment. Thus, chemotherapeutic clearance of covalently closed circular DNA did not involve the replacement of the infected hepatocyte population with uninfected progenitors, but rather, uninfected hepatocytes in the treated liver were derived from the infected hepatocyte population. The frequency of integrated DNA in chronically infected woodchucks was found to be 1 or 2 orders of magnitude higher than that in transiently infected woodchucks, implying that integration and other genomic damage accumulate over the duration of infection. Our results indicate that genetic changes from this damage remain in the liver even while virus infection is cleared and argue for early antiviral intervention in chronic hepatitis. (+info)
Preclinical pharmacology and pharmacokinetics of the anti-hepatitis virus agent 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil in mice and rats.
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The preclinical pharmacology and pharmacokinetics of 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (FEAU), a selective inhibitor of herpesvirus and hepatitis virus replication, were investigated in the mouse and rat. Following intravenous (i.v.) or oral (p.o.) administration, FEAU was cleared from the plasma primarily unchanged, with a terminal half-life of 58 to 80 min in the mouse and 63 to 78 min in the rat. The steady-state volumes of distribution times bioavailabilities of FEAU were approximately 2.1 and 3.4 times the total body water volumes after p.o. administration of 10 mg of drug per kg of body weight in mice and rats, respectively. A comparison of the area under the concentration-time curve after i.v. and p.o. FEAU administration indicated that the p.o. dose was completely absorbed in both species. When tritiated FEAU was used in mice, 35.0% of the i.v. dose and 33.5% of the p.o. dose were excreted in urine as unchanged FEAU, 8.1% (i.v. dose) and 9.2% (p.o. dose) were excreted as tritiated water, and 15.6% (i.v. dose) and 18.1% (p.o. dose) were excreted as unknown metabolite(s) in urine within 24 h of dosing. Only 1.24% (i.v. dose) and 2.6% (p.o. dose) of the total doses were found in urine as 3H2O when the FEAU dose was increased to 50 mg/kg. However, a higher percentage of the total dose (59.6% for the i.v. dose and 61.3% for the p.o. dose) was recovered within 24 h as intact FEAU in rat urine, less than 1.4% (i.v. dose) and 2.7% (p.o. dose) of the total dose were found to be 3H2O, and 5.6% (i.v. dose) and 6.7% (p.o. dose) of the total dose were excreted as known metabolite(s). The distribution ratios for total radioactivity in tissue relative to those in plasma were 0.5 to 1.3 in spleen, testes, muscle, and liver during the first hour after a 10-mg/kg dose in rats. Of the total FEAU radioactivity administered, only 1.38% was excreted in bile as unchanged FEAU. No FEAU glucuronide metabolite was detected. Tissue concentrations of 0.15 to 0.6 microM at 6 h after dosing are in the range of the effective antiviral concentration for FEAU. In conclusion, FEAU administered p.o. to mice and rats was well absorbed; FEAU was rapidly distributed into tissues and remained above in vitro antiviral concentrations for more than 6 h; in mice, [3H]FEAU showed metabolism-mediated tritium exchange with water; and in rats, FEAU was less extensively metabolized than in mice and clearance was primarily via renal processes, mainly in the form of unchanged FEAU. (+info)