ALL -acute lymphoblastic leukemia DMEM -Dulbecco's modified eagle medium DMSO -dimethyl sulfoxide D-PBS -Dulbecco's phosphate buffered saline DY -dipyridamole ENBT1 -equilibrative nucleobase transporter 1 ENT1 -equilibrative nucleoside transporter 1 ENT2 -equilibrative nucleoside transporter 2 ENT4 -equilibrative nucleoside transporter 4 FBS -fetal bovine serum G418 -geneticin GAPDH -glyceraldehyde 3-phosphate dehydrogenase GFP -green fluorescent protein HEK293 -human embryonic kidney 293 HPRT -hypoxanthine-guanine phosphoribosyltransferase HRP -horseradish perdoxidase IMDM -Iscove's modified Dulbecco medium MRP -multidrug resistance protein This article has not been copyedited and formatted. The final version may differ from this version.
Film boiling heat transfer coefficients for a downwardfacing hemispherical surface are measured from the quenching tests in DELTA (Downward-boiling Experimental Laminar Transition Apparatus). Two test sections are made of copper to maintain low Biot numbers. The outer diameters of the hemispheres are 120 mm and 294 mm, respectively. The thickness of all the test sections is 30 mm. The effect of diameter on film boiling heat transfer is quantified utilizing results obtained from the test sections. The measured data are compared with the numerical predictions from laminar film boiling analysis. The measured heat transfer coefficients are found to be greater than those predicted by the conventional laminar flow theory on account of the interfacial wavy motion incurred by the Helmholtz instability. Incorporation of the wavy motion model considerably improves the agreement between the experimental and numerical results in terms of heat transfer coefficient. In addition, the interfacial wavy motion and the quenching process are visualized through a digital camera.
The effect of inclination angle of the downward facing flat plate on the interfacial wavy motion is investigated utilizing the water test apparatus DELTA FS (Downward Ebullient Laminar Transition Apparatus Flat Surface) in a quasi-steady state. Film boiling heat transfer coefficients are obtained on the relatively long surface in the flow direction. The measured heat transfer coefficients are compared with those predicted by the laminar film boiling and interfacial wavy film boiling correlations at the same experimental condition. Visualization of the vapor film revealed that the interfacial wavelength decreases as the flat plate moves from the vertical to downward facing locations.
Background Thiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity. Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature. To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature. Hypothesis We hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity. Methods The ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay. Results Oil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 & 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 & 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5). Conclusion Our results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessa...
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