HeLa cell growth is not affected by doxycycline supplement.

<p>Values are the mean of three independent cultures; error bars indicate ±1 S.E.</p

Comparative transcriptional chase analyses of a long-lived test mRNA.

<p>(A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t₀, and sacrificed at defined intervals. Levels of β^WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β^WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β^WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t_1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t_1/2 values calculated following correction for interval cell expansion.</p

Effects of cell doubling time on the apparent t_1/2 value of a study mRNA.

<p>(A) Hypothetical mRNA decay. Cell aliquots, each originally containing 1.0×10⁶ cells, are incubated for defined intervals between 0 and 24 hours. Cells in each aliquot double every 6 hours, in parallel with the total amount of β-actin mRNA. At T = 0, each aliquot contains an equal quantity [1 arbitrary unit (au)] of an infinitely stable β-globin mRNA, encoded by a gene that has been transcriptionally silenced. The decline in the ratio of the globin:actin mRNAs, which erroneously indicates a half-life value of 6 hours for the globin mRNA, fails to account for interval expansion in cell number. These same principles apply to mRNAs with finite stabilities (panel B). (B) True and uncorrected t_1/2 values in cells with different doubling times. Curves illustrate the uncorrected half-life for a test mRNA, following transcriptional silencing of its encoding gene, if the interval expansion in cell number is not considered. The examples utilize an mRNA with a true t_1/2 = 6 h, expressed in cells that are growth arrested (curve <i>a</i>) or display doubling times of 24, 8, 6, or 4 hours (curves <i>b</i> through <i>e</i>, respectively).</p

Values used in the current report.

<p>Values used in the current report.</p

Experimental schemata for transcriptional chase analyses of mRNA stability.

<p>(A) Conventional chase method. Identical aliquots cultured in doxycycline (dox)-supplemented media (thick line) are sacrificed at defined intervals (arrowheads). A hypothetical 80-hr chase experiment is illustrated. (B) Reverse-chase method. Identical aliquots cultured in dox-free media (thin line) are amended with dox at defined intervals, and sacrificed simultaneously at the conclusion of the experiment.</p

HeLa cell expansion under transcriptional chase conditions.

<p>Data from dox-supplementation experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g002" target="_blank">Fig. 2</a> (1 µg/mL) was regressed to an exponential function, and expansion factors defined [j = 0.0224 (h) or 0.0004 (min)].</p

Increasing the stability of <i>Lumbricus terrestris</i> erythrocruorin <i>via</i> poly(acrylic acid) conjugation

<p>Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm <i>Lumbricus terrestris</i> erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (<i>T</i>_m = 72 ± 0.84 °C) relative to native LtEc (<i>T</i>_m = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P₅₀ = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P₅₀ = 26.67 ± 0.4 mm Hg), but the cooperativity (<i>n</i> = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.</p

Design, Synthesis, and Biological Evaluation of Ester and Ether Derivatives of Antisickling Agent 5‑HMF for the Treatment of Sickle Cell Disease

Candidate drugs to counter intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) continue to represent a promising approach to mitigating the primary cause of the pathophysiology associated with sickle cell disease (SCD). One such compound is the naturally occurring antisickling agent, 5-hydroxymethyl-2-furfural (5-HMF), which has been studied in the clinic for the treatment of SCD. As part of our efforts to develop novel efficacious drugs with improved pharmacologic properties, we structurally modified 5-HMF into 12 ether and ester derivatives. The choice of 5-HMF as a pharmacophore was influenced by a combination of its demonstrated attractive hemoglobin modifying and antisickling properties, well-known safety profiles, and its reported nontoxic major metabolites. The derivatives were investigated for their time- and/or dose-dependent effects on important antisickling parameters, such as modification of hemoglobin, corresponding changes in oxygen affinity, and inhibition of red blood cell sickling. The novel test compounds bound and modified Hb and concomitantly increased the protein affinity for oxygen. Five of the derivatives exhibited 1.5- to 4.0-fold higher antisickling effects than 5-HMF. The binding mode of the compounds with Hb was confirmed by X-ray crystallography and, in part, helps explain their observed biochemical properties. Our findings, in addition to the potential therapeutic application, provide valuable insights and potential guidance for further modifications of these (and similar) compounds to enhance their pharmacologic properties

A Triazole Disulfide Compound Increases the Affinity of Hemoglobin for Oxygen and Reduces the Sickling of Human Sickle Cells

Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt “sickled” shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4′-di­(1,2,3-triazolyl)­disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with β-Cys93, which inhibits the salt-bridge formation between β-Asp94 and β-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the <i>P</i>₅₀ of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb β-Cys93, may provide new treatment options for patients with sickle cell disease