Signaling through reactive oxygen and nitrogen species is differentially modulated in sunflower seedling root and cotyledon in response to various nitric oxide donors and scavengers^*

<p>Sodium nitroprusside (SNP), diethylenetriamine NONOate (DETA), S-nitroso-n-acetyl-D,L- penicillamine (SNAP), and 4-(<i>p-</i>methoxyphenyl)-1,3,2- Oxathiazolylium-5-olate (CAY) exhibit differential NO releasing ability in aqueous solution and hemoglobin is a more efficient NO quencher than cPTIO in solution. DETA releases 16% more NO compared with SNP in solution. Various NO donors (SNP, DETA, SNAP, and CAY) also bring about a differential but concentration-dependent increase in endogenous NO in seedling cotyledons and roots. Two-day old, dark-grown seedling roots exhibit 95%, 77%, 59% and 45% increase in NO content in presence of each of 500 µM of DETA, SNAP, CAY and SNP, respectively, relative to control. NO accumulation in the tissue system as a response to NO donors is reflected in terms of corresponding peroxynitrite accumulation. Release of cyanide and free iron as byproducts of SNP dissociation in solution limits its usefulness as an NO donor. SNP leads to profuse ROS generation in sunflower seedling roots. Light is not a pre-requisite for NO generation from SNP. Present work also demonstrates the usefulness of hemoglobin over cPTIO as NO scavenger. Hemoglobin brings about increasing NO quenching with its increasing concentration from 2.5 to 10 µM. Greater sensitivity of the root system to the NO donor/scavenger treatments is evident, it being in direct contact with the molecules in the incubation/ growth medium. This differential effect does not seem to be significantly transmitted to the cotyledons (long-distance signaling).</p

Additional file 1 of The role of stigma in cannabis use disclosure: an exploratory study

Additional file 1. Complete Survey

(A) Relative protein expression levels of CBS in the liver of C57BL/6 <i>Ldlr^−/− Cbs^+/+</i> mice (grey bar) and of C57BL/6 <i>Ldlr^−/− Cbs^+/−</i> mice 2 weeks after gene transfer with 10¹¹ particles of Adnull (black bar) or 10¹¹ particles of AdCBS (black crosshatched bar).

<p>Data in Adnull- and AdCBS-treated mice were calculated using values in C57BL/6 <i>Ldlr^−/− Cbs^+/+</i> mice as the denominator. All data are shown as means ± SEM (n = 7 to 9 for each condition). (B) Representative Western blot of CBS expression in the liver of C57BL/6 <i>Ldlr^−/− Cbs^+/+</i> mice (lanes 1 and 2) and of C57BL/6 <i>Ldlr^−/− Cbs^+/−</i> mice injected with 10¹¹ particles of Adnull (lanes 3–5) or the same dose of AdCBS (lanes 6–8).</p

Poly(lactic-co-glycolic) acid nanoparticles localize in vesicles after diffusing into cells and are retained by intracellular traffic modulators supplementary figures

Aim: We investigated our previous finding of increased retention of poly(lactic-coglycolic) acid nanoparticles (PLGA-NPs) with metabolic inhibitors (MI) and studied the effect of some small molecule inhibitors on PLGA-NP assimilation. Materials & methods: Intracellular PLGA-NP colocalization in the presence of MI was investigated by confocal microscopy. Intracellular retention of PLGA-NPs by some small molecules was estimated by fluorescence microscopy and flow cytometry after Pulse/Chase experiments. Results: MI caused PLGA-NP colocalization in intracellular membranous structures, mainly endosomes and lysosomes. Some small molecule inhibitors demonstrated increased intracellular PLGA-NP accumulation. Conclusion: This study elucidates the movement of PLGA-NP in cells and suggests that clinically used small molecules can reduce their extrusion by enhancing their stay within intracellular vesicles, with possible clinically beneficial consequences.Plain language summary: Nanoparticles are increasingly being used to carry drugs for treatment of cancer. We wish to decrease their movement out of the cells. This may give time for them to unload their drugs. Cells were treated with nanoparticles for 30 min and observed. Then the nanoparticles were washed off. Cells were again observed after 30 min. Various intracellular trafficking inhibitors were also added. Nanoparticle retention and subcellular localization were measured. We found that nanoparticles are trapped in some membranous compartments within the cells after energy depletion. We also discovered some commonly used clinical molecules that can decrease the excretion of nanoparticles from the cells. These inhibitors can be utilized for increasing the intracellular stay of the drug-loaded nanoparticles.</p

Hemodynamic parameters in the left ventricle and in the aorta of Sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice and <i>at</i> day 28 after myocardial infarction in control and AdCBS treated C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice.

<p>Data are expressed as means ± S.E.M.</p>§<p>p<0.05;</p>§§§<p>p<0.001 versus Sham.</p>*<p>p<0.05 versus Control MI.</p

Representative Sirius red stained cross-sections of sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice, control MI mice, and AdCBS MI mice at day 28 after ligation of the LAD.

<p>Morphometric analysis was performed on tissue sections of 4 separate regions using Axiovision 4.6 software (Zeiss). Scale bar represents 1 mm.</p

Hemodynamic parameters in the left ventricle and in the aorta of sham C57BL/6 mice and of sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− and sham AdCBS treated C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice.

<p>Sham C57BL/6 mice (n = 10) were fed normal chow. Sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− (n = 11) and AdCBS treated (n = 11) C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice were fed with folate-depleted, methionine-enriched diet supplemented with 0.2% cholesterol and 10% coconut oil. Data are expressed as means ± S.E.M.</p

Morphometric analysis of the left ventricle of sham C57BL/6 mice and of sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− and sham AdCBS treated C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice.

<p>Sham C57BL/6 mice (n = 10) were fed normal chow. Sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− (n = 11) and sham AdCBS treated (n = 11) C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice were fed with folate-depleted, methionine-enriched diet supplemented with 0.2% cholesterol and 10% coconut oil. Data are expressed as means ± S.E.M.</p><p>LV: left ventricular.</p

Heart and lung weights of sham C57BL/6 mice and of sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− and sham AdCBS treated C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice.

<p>Sham C57BL/6 mice (n = 10) were fed normal chow. Sham C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− (n = 11) and sham AdCBS treated (n = 11) C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice were fed with folate-depleted, methionine-enriched diet supplemented with 0.2% cholesterol and 10% coconut oil. Data are expressed as means ± S.E.M.</p

Time course of plasma homocysteine levels (A) and plasma cholesterol (closed symbols) and HDL cholesterol levels (open symbols) (B) in female C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− control mice or in female C57BL/6 <i>Ldlr</i>^−/−<i>Cbs</i>^+/− mice injected with 5×10¹⁰ particles of AdCBS.

<p>A hyperhomocysteinemic and high saturated fat/high cholesterol diet (0.2 mg/kg folic acid, 4.1 g/kg L-methionine, 1.25% cholesterol (w/w), and 10% coconut oil (v/w)) was initiated 3 weeks before adenoviral gene transfer or saline injection and maintained throughout the experiment. The 0 week time point corresponds to the time point of gene transfer in the intervention group. Data are expressed as means ± S.E.M. (n = 8).</p