Folate deficiency causes massive incorporation of uracil into human DNA (4 million per cell) and chromosome breaks. The likely mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into DNA by DNA polymerase. During repair of uracil in DNA, transient nicks are formed; two opposing nicks could lead to chromosome breaks. Both high DNA uracil levels and elevated micronucleus frequency (a measure of chromosome breaks) are reversed by folate administration. A significant proportion of the U.S. population has low folate levels, in the range associated with elevated uracil misincorporation and chromosome breaks. Such breaks could contribute to the increased risk of cancer and cognitive defects associated with folate deficiency in humans.

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To survive, respiring organisms must sense and respond to changes in environmental oxygen levels. Complex III of the mitochondrial electron transport chain (ETC) has been implicated in the O2 sensing pathway in mammals through its ability to increase production of reactive oxygen species (ROS) during hypoxia. The present study tested whether Complex III in yeast also contributes to O2 sensing during hypoxia. Strains deficient in mitochondrial DNA (rho0), the Rieske iron-sulfur protein (DeltaRip1) in Complex III, or an enzyme responsible for coenzyme Q biosynthesis (DeltaCoq2) were studied to determine the importance of Complex III activity in the transcriptional response to hypoxia. Loss of Complex III function abrogated the hypoxia-induced increase in ROS in each strain. Northern analysis identified a set of genes that are activated by hypoxia in wild-type but not in rho0, DeltaRip1, or DeltaCoq2 strains. Yeast lacking the transcription factors Yap1p, Mga2p, and Msn2p were also deficient in hypoxic activation of gene transcription, suggesting the importance of redox regulation in hypoxic gene expression. The authors conclude that Complex III of the ETC is required for ROS production and for expression of a group of hypoxia-inducible genes in yeast. These findings indicate that the mitochondrial O2 sensing mechanism is highly conserved throughout evolution.

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

We studied the effect of maturation on contractile properties of tracheal smooth muscle from seventeen 2-wk-old swine (2ws) and fifteen 10-wk-old swine (10ws) in situ and in vitro. The response to parasympathetic stimulation was studied in situ in isometrically fixed segments. Contraction was elicited at lower frequencies [half-maximal response to electrical stimulation (ES50) = 6.7 +/- 0.05 Hz] in 2ws than in 10ws (ES50 = 9.1 +/- 0.4 Hz; P less than 0.01). Despite substantial differences in morphometrically normalized cross-sectional area in 2ws (0.012 +/- 0.003 cm2) and 10ws (0.028 +/- 0.001 cm2; P less than 0.01), maximal active tension elicited by parasympathetic stimulation was similar (12.4 +/- 3.2 g/cm in 2ws vs. 13.3 +/- 2.3 g/cm in 10ws; P = NS). In separate in vitro studies in 25 tracheal smooth muscle strips from 10 swine, concentration-response curves generated with potassium-substituted Krebs solution (KCl) were similar in 2ws and 10ws. In 58 other strips (10 swine), maximal active force elicited with acetylcholine (ACh) in 2ws was significantly greater than for 10ws (P less than 0.001). Removal of the epithelium had no effect. However, cholinesterase inhibition with 10(-7) M physostigmine augmented the response to ACh in 10ws (P less than 0.02) but not 2ws. We demonstrate increased force generation and sensitivity to vagal stimulation in 2ws vs. 10ws, which corresponds to increased reactivity to ACh in vitro. The relative hyperresponsiveness in 2ws is specific for cholinergic response and is attenuated at least in part by maturation of the activity of acetylcholinesterase enzyme.

Response to Reviewers: Manuscript FCSS-2016-0094 Note: We would like to thank the reviewer for very thoughtful feedback. We hope that our response is adequate, but should there be lingering issues, please let us know. Changes in the doc are indicated in blue highlighting. Reviewer 1 Authors' Response 1 Introduction • You discuss the negative perceptions of officiating-does any data exist regards the number of official who leave the sport due to these reasons? Anecdotally, retention is a significant issue in officiating however is there any data to support this perception?

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.

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