Sleep duration,nightshift work,and the timing of meals and urinary levels of 8-isoprostane and 6-sulfatoxymelatonin in Japanese women

It has been hypothesized that disruption of circadian rhythms affects human health. Shift work and sleep deprivation are thought to disrupt the normal light–dark cycle, although the disruption due to shiftwork may be dependent on sleep deprivation. Both conditions have been suggested to be associated with an increased risk of cardiometabolic disorders. Non-photic environmental factors, such as the timing of eating, are also thought to regulate circadian rhythm and thus, may have effects on health, but the evidence from human studies is scarce. Oxidative stress is a risk factor of cardiometabolic disorders. Some laboratory studies suggest an involvement of circadian clock genes in the regulation of the redox system. The present study aimed to examine the association of sleeping habits, nightshift work, and the timing of meals with urinary levels of 8-isoprostane, a marker of oxidative stress, and 6-sulfatoxymelatonin, the principal metabolite of melatonin. Study subjects were 542 women who had previously attended a breast cancer mass screening in a community in Japan. Information on bedtimes and wake-up times, history of nightshift work, and the timing of meals was obtained by a self-administered questionnaire. The 8-isoprostane and 6-sulfatoxymelatonin were measured using the first morning void of urine and expressed per mg of creatinine. The geometric mean of 8-isoprostane levels was 12.1% higher in women with ≤6 hours of sleep than that in those with >8 hours of sleep on weekdays, and longer sleep duration on weekdays was significantly associated with lower urinary levels of 8-isoprostane after controlling for covariates (p for trend = 0.04). Women who were currently working the nightshift had a 33.3% higher geometric mean of 8-isoprostane levels than those who were not working nightshift (p = 0.03). Urinary 6-sulfatoxymelatonin levels were unrelated to sleep habits or nightshift work. Women who ate breakfast at irregular times had a 19.8% higher geometric mean of 8-isoprostane levels than those who ate breakfast at a regular time or who did not eat (p = 0.02). Women who ate nighttime snacks at irregular times had a 16.2% higher geometric mean of 8-isoprostane levels than those who did not eat nighttime snacks or who ate nighttime snacks at a regular time (p = 0.003). Among women who ate dinner at a regular time, earlier times for dinner were associated with higher 8-isoprostane and 6-sulfatoxymelatonin levels (p values for trends were 0.01 and 0.02, respectively). However, the times of dinner and nighttime snack are overlapping, and the time of last meal of the day was not associated with 8-isoprostane and 6-sulfatoxymelatonin levels. The time of breakfast or lunch was not associated with these biomarkers among women who ate the meal at regular times. Disturbing the rhythmicity of daily life may be associated with oxidative stress.     

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.     

Transcriptomic characterization of gall tissue of Japanese elm tree (Ulmus davidiana var. japonica) induced by the aphid Tetraneura nigriabdominalis

Insect galls are abnormal plant tissues induced by parasitic insect(s) for use as their habitat. In previous work, we suggested that gall tissues induced by the aphid Tetraneura nigriabdominalis on Japanese elm trees are less responsive than leaf tissues to jasmonic acid (JA), which is involved in the production of volatile organic compounds as a typical defensive reaction of plants against attack by insect pests. A comprehensive analysis of gene expression by RNA sequencing indicated that the number of JA responsive genes was markedly lower in gall tissues than in leaf tissues. This suggests that gall tissues are mostly defective in JA signaling, although JA signaling is not entirely compromised in gall tissue. Gene ontology analysis sheds light on some stress-related unigenes with higher expression levels in gall tissues, suggesting that host plants sense aphids as a biotic stress but are defective in the JA-mediated defense response in gall tissues.     

Antidiabetic effect of enterolactone in cultured muscle cells and in type 2 diabetic model db/db mice

Enterolactone (ENL) is formed by the conversion of dietary precursors like strawberry lignans via the gut microbiota. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of Type 2 diabetes (T2D). In the present study, antidiabetic effect of ENL and its modes of action were studied in vitro and in vivo employing a rat skeletal muscle-derived cell line, L6 myocytes in culture, and T2D model db/db mice. ENL dose-dependently increased glucose uptake in L6 myotubes under insulin absent condition. This increase by ENL was canceled by compound C, an inhibitor of 5′-adenosine monophosphate-activated protein kinase (APMK). Activation (=phosphorylation) of AMPK and translocation of glucose transporter 4 (GLUT4) to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with glut4 cDNA-coding vector. T2D model db/db mice were fed the basal 20 % casein diet (20C) or 20C supplemented with ENL (0.001 or 0.01 %) for 6 weeks. Fasting blood glucose (FBG) levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01 % in 20C) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(α-d-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase α and β. However, since the α-configuration of SQDG with two stearic acids (α-SQDG-C₁₈) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (α-SQMG-C₁₈) from α-SQDG-C₁₈ by hydrolysis with a pancreatic lipase. α-SQMG-C₁₈ inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. α-SQMG-C₁₈ arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that α-SQMG-C₁₈ induced cell death through a p53-dependent apoptotic pathway.     

Effects of various forms of surfactant protein C on tidal volume in ventilated immature newborn rabbits.

Surfactant protein (SP)-C is characterized by alpha-helix structure and palmitoyl groups attached to two cysteine residues. We examined the function of palmitoylation and dimerization in promotion of tidal volume in immature newborn rabbits. Reconstituted surfactants were made from a mixture of synthetic phospholipids and porcine SP-B (basic mixture) by adding various forms of SP-Cs: normal SP-C isolated from porcine lungs and monomeric or dimeric forms of SP-C. These latter two were isolated from patients with pulmonary alveolar proteinosis and were less palmitoylated. Animals were ventilated at an inspiratory pressure of 25 cmH2O. Median tidal volumes were <2 ml/kg in nontreated controls, 7.7 ml/kg in animals receiving the basic mixture without SP-C, and >18 ml/kg in animals treated with reconstituted surfactants containing 3% normal or 2% dimeric SP-C (P < 0.05 vs. basic mixture). The physiological effect of basic mixture was not improved by monomeric SP-C. We conclude that palmitoyl groups are important for the physiological effects of SP-C and that the dimeric form also improves physiological effects.     

Activation mechanism of Gi and Go by reactive oxygen species.

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.     

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration

The dissociation constants (pK_ms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a ¹³C nuclear magnetic resonance (NMR) titration method employing their N-¹³CH₃ (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 μM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pK_m values by allowing that the phenothiazines in the aqueous phase could be neglected. The pK_m values were calculated from the chemical shift dependence of the N-dimethyl ¹³C NMR signal on the pH value of sample solutions. The pK_m values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pK_m values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pK_m decrease and provided their precise values.