Effect of AMP on the p50 of intact erythrocytes.

<p>(A) Shift of WT erythrocytes’ p50 in response to extracellular AMP concentration; (B) Average AMPD3^-/-/CD73^-/- erythrocyte p50 values in PBS and 2 mM AMP (N = 3). (C) Shift of erythrocytes’ p50 after 2 mM AMP incubation in the four genotypes (N = 3).</p

New insights on the regulation of the adenine nucleotide pool of erythrocytes in mouse models

<div><p>The observation that induced torpor in non-hibernating mammals could result from an increased AMP concentration in circulation led our investigation to reveal that the added AMP altered oxygen transport of erythrocytes. To further study the effect of AMP in regulation of erythrocyte function and systemic metabolism, we generated mouse models deficient in key erythrocyte enzymes in AMP metabolism. We have previously reported altered erythrocyte adenine nucleotide levels corresponding to altered oxygen saturation in mice deficient in both CD73 and AMPD3. Here we further investigate how these <i>Ampd3</i>^<i>-/-</i><i>/Cd73</i>^<i>-/-</i> mice respond to the administered dose of AMP in comparison with the control models of single enzyme deficiency and wild type. We found that <i>Ampd3</i>^<i>-/-</i><i>/Cd73</i>^<i>-/-</i> mice are more sensitive to AMP-induced hypometabolism than mice with a single enzyme deficiency, which are more sensitive than wild type. A dose-dependent rightward shift of erythrocyte p50 values in response to increasing amounts of extracellular AMP was observed. We provide further evidence for the direct uptake of AMP by erythrocytes that is insensitive to dipyridamole, a blocker for ENT1. The uptake of AMP by the erythrocytes remained linear at the highest concentration tested, 10mM. We also observed competitive inhibition of AMP uptake by ATP and ADP but not by the other nucleotides and metabolites tested. Importantly, our studies suggest that AMP uptake is associated with an erythrocyte ATP release that is partially sensitive to inhibition by TRO19622 and Ca⁺⁺ ion. Taken together, our study suggests a novel mechanism by which erythrocytes recycle and maintain their adenine nucleotide pool through AMP uptake and ATP release.</p></div

Concentration dependent uptake of AMP by erythrocytes.

<p>(A) A typical assessment of AMP uptake by erythrocytes. [¹⁴C]-AMP signal as a percentage of total added radioactivity in the supernatant, cells, and wash fractions over a time course. Uptake of AMP by wild type (B) and <i>Cd73</i>^<i>-/-</i> (C) erythrocytes up to 10 mM extracellular AMP were measured. The difference in AMP uptake by the two genotypes at low extracellular AMP concentrations is exhibited by plots of uptake in the range of 0–1 mM extracellular AMP concentrations in wild type (D) and <i>Cd73</i>^<i>-/-</i> (E) erythrocytes.</p

Nucleotides’ induction of ATP release by erythrocytes.

<p>(A) ATP release time course after addition of various nucleotides and adenosine measured by luciferase assays. (B) Quantification of multiple samples for ATP release stimulated by various nucleotides (N = 3). (C) Quantification of multiple samples of AMP and adenosine stimulated ATP release (N = 3). (D) Effect of CBX on ATP release stimulated by AMP. (E) Effect of TRO19622 on ATP release stimulated by AMP. (F) Effect of Ca²⁺ on ATP release stimulated by AMP. P values = *** < .0001.</p

AIHM responses among four genotypes of mice.

<p>(A). AIHM duration varies among four genotypes of mice. Mice from each genotype (N = 3) were injected with a sub-optimal dose of AMP (0.2 mg/gbw) and placed at an ambient temperature of 15°C. The amount of time in AIHM was calculated from the time of injection to when they increased their VO₂ to 1200 mL/kg/h or, in the case of the wild type (WT) mice, until their VO₂ started to increase. (B). Graphical representation of the percentage of mice remaining in AIHM relative to total injected animals as a function of time. Mice from each genotype (wild type (N = 16), <i>Ampd3</i>^<i>-/-</i> (N = 15), <i>Cd73</i>^<i>-/-</i> (N = 15), and <i>Ampd3</i>^<i>-/-</i><i>/Cd73</i>^<i>-/-</i> (N = 16)) were injected with the optimal dose of AMP (0.5 mg/gbw) and placed at an ambient temperature of 15°C. The length of time each mouse stayed in AIHM was calculated from injection to when their VO₂ rose above 1200 mL/kg/h. Sampling of animals in AIHM were taken every 2 h. P values = * < .05, ** < .005, and *** < .0001.</p

Evaluation of potential inhibitors of AMP uptake by erythrocytes.

<p>(A) CAT effect on erythrocyte AMP uptake. (B) Dipyridamole (Dip) effect on erythrocyte AMP uptake. (C) The inhibition of erythrocyte AMP uptake by Tannic Acid (TAN). (D) Titration of TAN concentration in percentages against erythrocyte AMP uptake. (E). AMP uptake inhibition assay to evaluate possible competitive nucleotide/metabolite inhibitors for AMP uptake. (F) Examining GMP inhibition of AMP uptake as a function of increasing concentrations of extracellular AMP.</p

AMPD activity is not detectable in erythrocytes (rbc) and is reduced in the heart of <i>Ampd3^−/−</i> mice.

<p>A. AMP deminase assays were carried out with cell lysates from the indicated organs. For all samples, except serum (WT n = 10; <i>Ampd3^−/−</i> n = 5), heart (n = 8) and erythrocytes (WT n = 15; <i>Ampd3^−/−</i> n = 7), the data represent the average of tissue samples (n = 3). B. The level of AMPD3 activities in erythrocytes from wild type, <i>Ampd3^+/−</i> and <i>Ampd3^−/−</i> mice. Error bars, mean ± SEM. T-test: *p<0.05.</p

Erythrocytes of <i>Ampd3^−/−</i> mice have elevated levels of ATP and ADP but normal levels of AMP.

<p>A. Nucleotides from methanol extracts of erythrocyte lysates from wild type (n = 4) and <i>Ampd3^−/−</i> (n = 4) were separated by HPLC and quantified by calibration with standards. B. Representative HPLC chromatograms from wild type and <i>Ampd3^−/−</i> erythrocyte nucleotide extracts. C. Fasting glucose levels in <i>Ampd3^−/−</i> (n = 16) and <i>Ampd3^+/−</i> mice (n = 9) and WT (n = 14) mice. Error bars, mean ± SEM. T-test: *p<0.05.</p

The “knockout-first” conditional construct for use at the <i>Ampd3</i> genomic loci.

<p>A. Recombination at the 5′ and 3′ homologous arms of the vector to the target sequences of the <i>Ampd3</i> gene allows the insertion of an en2-LacZ-neo cassette into a region spanning the intron between exon 5 and exon 6 of the <i>Ampd3</i> locus. With the “knockout-first” design, the 3′ end of exon 5, the first coding exon, is spliced into the splice acceptor (SA) site of the en2-LacZ-neo cassette in order to disrupt and destabilize the <i>Ampd3</i> transcript. B. RT-PCR showing that <i>Ampd3</i> mRNA from the heart of wild type (WT) mice is absent in <i>Ampd3^−/−</i> heart tissue. “-RT” indicates the negative control where reverse transcriptase was lacking in parallel reactions.</p

<i>In Situ</i> Imaging of Two-Dimensional Crystal Growth Using a Heat-Resistant Optical Microscope

Revealing low-dimensional material growth dynamics is critical for crystal growth engineering. However, in a practical high-temperature growth system, the crystal growth process is a black box because of the lack of heat-resistant imaging tools. Here, we develop a heat-resistant optical microscope and embed it in a chemical vapor deposition (CVD) system to investigate two-dimensional (2D) crystal growth dynamics. This in situ optical imaging CVD system can tolerate temperatures of ≤900 °C with a spatial resolution of ∼1 μm. The growth of monolayer MoS2 crystals was studied as a model for 2D crystal growth. The nucleation and growth process have been imaged. Model analysis and simulation have revealed the growth rate, diffusion coefficient, and spatial distribution of the precursor. More importantly, a new vertex–kink–ledge model has been suggested for monolayer crystal growth. This work provides a new technique for in situ microscopic imaging at high temperatures and fundamental insight into 2D crystal growth