Studies of Nucleoside Transporters Using Novel Autofluorescent Nucleoside Probes
Bibliographic record
Abstract
To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".