Search

The molecular circadian timing system involves genes known as "clock genes," such as the PER3 gene. Studies have demonstrated associations among a repeat polymorphism (VNTR) of the PER3 gene with chronotypes, with the occurrence of circadian rhythm disorders and with sleep homeostasis phenotypes. The aim of this study was to investigate, by actigraphy, sleep and circadian rhythm profiles of people with different genotypes for the VNTR polymorphism of the PER3 gene. We genotyped 467 individuals (46,39% male) for the PER3 VNTR polymorphism. The mean age of the participants was 21.84 ± 2.64, ranging from 18 to 30 y old. Actigraphy data were collected from a subsample of 81 subjects with PER3 4-repeats homozygous ( PER3 4/4 ) or 5-repeats homozygous ( PER3 5/5 subjects wore a wrist actigraph between 12 and 19 d. The sleep onset (weekdays, PER3 4/4 and 33 PER3 5/5 subjects wore a wrist actigraph between 12 and 19 d. The sleep onset (weekdays, p  = 0.015; weekend, p  = 0.022) and offset (weekdays, p  = 0.004; weekend, p  = 0.041) of the PER3 5/5 group occurred later than the PER3 4/4 group. Similar results were observed for the mid-sleep phase of weekdays (MSW) ( p  = 0.008) and free days (MSF) ( p  = 0.019), and for the mid-sleep phase corrected for sleep debt accumulated over the week (MSFsc) ( p  = 0.024). Despite the phase differences found between the PER3 4/4 and PER3 5/5 groups, no differences were found in sleep duration and social jet lag. However, the PER3 4/4 group presented, on average, a longer sleep rebound on the days off when compared to the PER3 5/5 ( p  = 0.002). The PER3 5/5 group showed lower interdaily stability (IS) ( p  = 0.032) and higher daily activity rhythm variability (IV) ( p  = 0.035). The findings of the present study revealed associations between the PER3 gene, sleep, and circadian rhythms. In general, we found that the gene is associated with the expression of sleep timing and duration and to the phase of the activity rhythm. The experiments carried out here occurred in the COVID-19 pandemic scenario, which should be considered as an environmental element with potential effects on the results obtained.

Studying communities at different stages of urbanisation and industrialisation can teach us how timing and intensity of light affect the circadian clock under real-life conditions. We have previously described a strong tendency towards morningness in the Baependi Heart Study, located in a small rural town in Brazil. Here, we tested the hypothesis that this morningness tendency is associated with early circadian phase based on objective measurements (as determined by dim light melatonin onset, DLMO, and activity) and light exposure. We also analysed how well the previously collected chronotype questionnaire data were able to predict these DLMO values. The average DLMO observed in 73 participants (40 female) was 20:03 ± 01:21, SD, with an earlier average onset in men (19:38 ± 01:16) than in women (20:24 ± 01:21; P ≤ .01). However, men presented larger phase angle between DLMO and sleep onset time as measured by actigraphy (4.11 hours vs 3.16 hours; P ≤ .01). Correlational analysis indicated associations between light exposure, activity rhythms and DLMO, such that early DLMO was observed in participants with higher exposure to light, higher activity and earlier light exposure. The strongest significant predictor of DLMO was morningness-eveningness questionnaire (MEQ) (beta=-0.35, P ≤ .05), followed by age (beta = -0.47, P ≤ .01). Sex, light exposure and variables derived from the Munich chronotype questionnaire were not significant predictors. Our observations demonstrate that both early sleep patterns and earlier circadian phase have been retained in this small rural town in spite of availability of electrification, in contrast to metropolitan postindustrial areas., (© 2020 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

A method is described for the isolation of respiratory mutants of Azotobacter vinelandii with increased amounts of d-type cytochrome by selecting for the inability to reduce tetrazolium red. Five stable mutants were obtained that had six-fold higher levels of cytochrome d, increased amounts of b-type and lower amounts of o-type and c-type cytochromes than the wild-type strain. Spectral alterations in cytochrome alpha 1 were also observed in the mutants. NADH and succinate oxidase activities of membrane particles were about two fold higher in the mutants compared to the wild-type strain. Ascorbate-N,N,N',N'-tetramethyl-p-phenylene diamine oxidase activity was barely detectable in membrane particles of the mutants. These results are consistent with an increase in the amount of the cytochrome d oxidase branch and a decrease in the amount and activity of the cytochrome o, alpha 1 oxidase branch in the mutants. Growth rates under oxygen-excess conditions and respiratory-linked proton translocation ratios of the mutant and wild-type stains were similar as were the photochemical spectral and kinetic properties of cytochrome d.

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mössbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mössbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mössbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mössbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.