Mitochondria-targeted H2S donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing H2S that decreases oxidative damage. However, the rate of H2S release by current ...donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases H2S within mitochondria. MitoPerSulf is quickly taken up by mitochondria, where it reacts with endogenous thiols to generate a persulfide intermediate that releases H2S. MitoPerSulf is acutely protective against cardiac IR injury in mice, due to the acute generation of H2S that inhibits respiration at cytochrome c oxidase thereby preventing mitochondrial superoxide production by lowering the membrane potential. Mitochondria-targeted agents that rapidly generate H2S are a new class of therapy for the acute treatment of IR injury.
We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active ...molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified,
expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.
Unpreserved phenylephrine is often used as an off-licence intracameral surgical adjunct during cataract surgery to assist with pupil dilation and/or stabilise the iris in floppy iris syndrome. It can ...be delivered as a neat 0.2 ml bolus of either 2.5 or 10% strength, or in a range of ad-hoc dilutions. We wished to assess the accuracy of intracameral phenylephrine preparation in clinical practice.
Phenylephrine 0.2 ml was analysed both neat (2.5 and 10%) and in diluted form (ratio of 1:1 and 1:3). Samples were analysed using the validated spectrophotometric method.
A total of 36 samples were analysed. The standard curve showed linearity for phenylephrine (R
= 0.99). Wide variability was observed across all dilution groups. There was evidence of significant differences in the percentage deviations from intended results between dilutions (p < 0.001). Mean percentage deviation for 1:3 dilution was significantly greater than neat (p = 0.003) and 1:1 dilution (p = 0.001). There was no evidence of a significant difference between 1:1 and neat (p = 0.827).
Current ad-hoc dilution methods used to prepare intracameral phenylephrine are inaccurate and highly variable. Small volume 1 ml syringes should not be used for mixing or dilution of drug. Commercial intracameral phenylephrine products would address dosage concerns and could improve surgical outcomes in cases of poor pupil dilation and/or floppy iris syndrome.
There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive ...oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H2O2) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H2O2 content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H2O2 into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H2O2 in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H2O2 content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H2O2 content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H2O2 content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H2O2. The mechanism underlying this observed relationship between SMR and in vivo H2O2 content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H2O2.
Summary
In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change ...in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.
2-14CQuercetin-4′-glucoside (4 mg/kg body weight) was fed by gavage to rats housed in metabolic cages, and over an ensuing 72 h period, radiolabeled products in body tissues, plasma, feces, and urine ...were monitored by high-performance liquid chromatography with online radioactivity and MS2 detection. One and 6 h after ingestion, while in the small intestine, the flavonol glucoside was converted to glucuronide and methylated and sulfated derivatives of quercetin, but only trace amounts of these metabolites were excreted in urine. On entering the cecum and the colon, the flavonol metabolites declined as they were converted to phenolic acids, principally 3-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid, by the colonic microflora. Feces contained mainly 3-hydroxyphenylacetic acid. Urine collected 0−12 and 0−24 h after ingestion contained radiolabeled hippuric acid and 3-hydroxyphenylacetic acid. 14C-Hippuric acid declined markedly in the 24−48 and 48−72 h urine samples, and there was a concomitant increase in labeled benzoic acid. There was minimal accumulation of radioactivity in plasma, despite a 69% recovery of label in urine over the 72 h period, and likewise, very little radioactivity was detected in body tissues out with the gastrointestinal tract. This is reflected in the fact that 72 h after ingestion 96% of the ingested radioactivity was recovered in feces, urine, and the cage washes, which comprise a mixture of urine and feces. The study reveals that as it passes through the gastrointestinal tract, almost all of the of 2-14Cquercetin-4′-glucoside is converted to phenolic acids, compounds not monitored in previous flavonol bioavailability studies with model animal systems, some of which have used exceedingly high doses of the aglycone quercetin (500 mg/kg body weight), which is not a normal dietary component.
Photouncaging delivers compounds with high spatial and temporal control to induce or inhibit biological processes but the released compounds may diffuse out. We here demonstrate that sulfonate anions ...can be photocaged so that a membrane impermeable compound can enter cells, be uncaged by photoirradiation and trapped within the cell.
The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred‐fold within ...mitochondria in response to the mitochondrial membrane potential (Δψm). Typically, a simple alkane links the TPP to its “cargo”, increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria‐targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix‐facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria‐targeted TPP compounds within mitochondria to be fine‐tuned.
Triphenylphosphonium (TPP) cation is widely used for selective delivery of compounds into mitochondria. This study demonstrated that the replacement of an alkyl chain with polyethylene glycol (PEG) chain in TPP‐conjugated compounds can reduce the membrane adsorption of the compounds, leading to fine‐tuning of mitochondria targeting strategies.
In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred ...indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H
O
) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H
O
levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H
O
levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.
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