Carbon Monoxide-Driven Reduction of Ferric Heme and Heme Proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The \u27autoreduction\u27 of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized α and β chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O → CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome a3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This \u27607 nm complex\u27, described in 1981 by Nicholls and Chanady, forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, autoreduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome a3 to cytochrome a and cytochrome c

Kinetics of Reduction of Cytochrome \u3ci\u3ec\u3c/i\u3e Oxidase by Dithionite and the Effect of Hydrogen Peroxide

The reduction of cytochrome c oxidase by dithionite was reinvestigated with a flow-flash technique and with varied enzyme preparations. Since cytochrome a3 may be defined as the heme in oxidase which can form a photolabile CO adduct in the reduced state, it is possible to follow the time course of cytochrome a3 reduction by monitoring the onset of photosensitivity. The onset of photosensitivity and the overall rate of heme reduction were compared for Yonetani and Hartzell-Beinert preparations of cytochrome c oxidase and for the enzyme isolated from blue marlin and hammer-head shark. For all of these preparations the faster phase of heme reduction, which is dithionite concentration-dependent, is almost completed when the fraction of photosensitive material is still small. We conclude that cytochrome a3 in the resting enzyme is consistently reduced by an intramolecular electron transfer mechanism. To determine if this is true also for the pulsed enzyme, we examined the time course of dithionite reduction of the peroxide complex of the pulsed enzyme. It has been previously shown that pulsed cytochrome c oxidase can interact with H2O2 and form a stable room temperature peroxide adduct (Bickar, D., Bonaventura, J., and Bonaventura, C. (1982) Biochemistry 21, 2661-2666). Rather complex kinetics of heme reduction are observed when dithionite is added to enzyme preparations that contain H2O2. The time courses observed provide unequivocal evidence that H2O2 can, under these conditions, be used by cytochrome c oxidase as an electron acceptor. Experiments carried out in the presence of CO show that a direct dithionite reduction of cytochrome a3 in the peroxide complex of the pulsed enzyme does not occur

Cannabinoids in the treatment of epilepsy: current status and future prospects

Cannabidiol (CBD) is one of the prominent phytocannabinoids found in Cannabis sativa, differentiating from Δ9-tetrahydrocannabinol (THC) for its non-intoxicating profile and its antianxiety/antipsychotic effects. CBD is a multi-target drug whose anti-convulsant properties are supposed to be independent of endocannabinoid receptor CB1 and might be related to several underlying mechanisms, such as antagonism on the orphan GPR55 receptor, regulation of adenosine tone, activation of 5HT1A receptors and modulation of calcium intracellular levels. CBD is a lipophilic compound with low oral bioavailability (6%) due to poor intestinal absorption and high first-pass metabolism. Its exposure parameters are greatly influenced by feeding status (ie, high fatcontaining meals). It is mainly metabolized by cytochrome P 450 (CYP) 3A4 and 2C19, which it strongly inhibits. A proprietary formulation of highly purified, plant-derived CBD has been recently licensed as an adjunctive treatment for Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS), while it is being currently investigated in tuberous sclerosis complex. The regulatory agencies’ approval was granted based on four pivotal double-blind, placebocontrolled, randomized clinical trials (RCTs) on overall 154 DS patients and 396 LGS ones, receiving CBD 10 or 20 mg/kg/day BID as active treatment. The primary endpoint (reduction in monthly seizure frequency) was met by both CBD doses. Most patients reported adverse events (AEs), generally from mild to moderate and transient, which mainly consisted of somnolence, sedation, decreased appetite, diarrhea and elevation in aminotransferase levels, the last being documented only in subjects on concomitant valproate therapy. The interaction between CBD and clobazam, likely due to CYP2C19 inhibition, might contribute to some AEs, especially somnolence, but also to CBD clinical effectiveness. Cannabidivarin (CBDV), the propyl analogue of CBD, showed anti-convulsant properties in pre-clinical studies, but a plant-derived, purified proprietary formulation of CBDV recently failed the Phase II RCT in patients with uncontrolled focal seizures

Reliability of a 2-Bout exercise test on a Wattbike cycle ergometer

Purpose: To determine the intraday and interday reliability of a 2 × 4-min performance test on a cycle ergometer (Wattbike) separated by 30 min of passive recovery (2 × 4MMP). Methods: Twelve highly trained cyclists (mean ± SD; age = 20 ± 2 y, predicted VO2max = 59.0 ± 3.6 mL · kg–1 · min–1) completed six 2 × 4MMP cycling tests on a Wattbike ergometer separated by 7 d. Mean power was measured to determine intraday (test 1 [T1] to test 2 [T2]) and interday reliability (weeks 1–6) over the repeated trials. Results: The mean intraday reliabilities of the 2 × 4MMP test, as expressed by the typical error of measurement (TEM, W) and coefficient of variation (CV, %) over the 6 wk, were 10.0 W (95% confidence limits [CL] 8.2–11.8), and 2.6% (95%CL 2.1–3.1), respectively. The mean interday reliability TEM and CV for T1 over the 6 wk were 10.4 W (95%CL 8.7–13.3) and 2.7% (95%CL 2.3–3.5), respectively, and 11.7 W (95%CL 9.8–15.1) and 3.0% (95%CL 2.5–3.9) for T2. Conclusion: The testing protocol performed on a Wattbike cycle ergometer in the current study is reproducible in highly trained cyclists. The high intraday and interday reliability make it a reliable method for monitoring cycling performance and for investigating factors that affect performance in cycling events

Effects of the satiety signal oleoylethanolamide on binge-like food consumption in female rats

Several lines of evidence document the association between eating disorders and modern lifestyle, encompassing calorie-rich diets and psychological stress. Binge-eating disorder (BED) is a eating disorder characterized by excessive consumption of food in a short period of time, along with loss of control and psychological distress. Among the networks that partake in the neurobiological bases of BED a large body of evidence supports the activation of the hypothalamic-pituitary-adrenal stress (HPA) axis. Pharmacological treatments for BED are limited thus highlighting the need to identify novel targets that could lead to the development of more effective therapies. A large body of evidence has accumulated on the role played by the lipid signal oleoylethanolamide (OEA) as a pharmacological target for controlling aberrant eating disorders. As a drug, OEA reduces food intake and body weight gain in laboratory rodents by inducing a state of satiety. Additionaly, OEA dampens the hyperactivity of the HPA axis and ameliorates the effects of stress. On the bases of these premises, in the present study we investigated the effects of OEA on high palatable food (HPF) intake in a rat model of BED. Moreover, we assessed the impact of OEA on the corticotropin-releasing factor (CRF) system which plays a critical role in stress and on the oxytocinergic system which is crucial in mediating the pro-satiety effect of OEA. We used female rats with a history of intermittent food restriction which show binge-like palatable food consumption after the exposure to a “frustration stress”. On the test day, we either exposed or did not expose the rats to the sight of the palatable food (frustration stress) before assessing food consumption. OEA was administered at three different doses (2.5, 5, 10 mg/kg i.p.) and HPF intake was monitored over 2h. After the behavioural experiment brains were collected and in-situ hybridization experiment was performed to analyse CRF and oxytocin mRNA expression in selected brain areas. Our results demonstrate that OEA (10 mg/kg) was able to selectively prevent binge eating; the antibinge effect was accompained by a reduction of CRF mRNA within the central-amygdala. Finally, in keeping with our previous observations we found that the antibinge effect of OEA was accompanied by a significant increase of oxytocin mRNA at hypothalamic level. In the current study, we provide for the first time evidence to support that the endogenous fatty-gut lipid OEA exerts a selective inhibitory effects on binge-like eating behavior in female rats, supporting the hypothesis that OEA might represent a novel potential pharmacological target for the treatment of aberrant eating patterns