). † These authors contributed equally to this work. SUMMARYLOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) constitute a family of Arabidopsis F-box proteins that regulate the circadian clock. Over-expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over-expressing LKP2, CO and FT expression was down-regulated under longday conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Overexpression of LKP2 Kelch repeats induced late flowering under long-day conditions. lkp2 ztl double mutant plants flowered earlier than wild-type plants under short-day (non-inductive) conditions, and both CO and FT expression levels were up-regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non-inductive conditions, and this is dependent on FKF1.

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C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.

The levels of endogenous brassinosteroids (BRs) and the expression of the biosynthesis/metabolism/perception genes involved have been investigated during the development and germination of pea (Pisum sativum) seeds. When seeds were rapidly growing, the level of biologically active BRs (brassinolide [BL] and castasterone [CS]) and the transcript levels of two BR C-6 oxidases (CYP85A1 and CYP85A6) reached a maximum, suggesting the significance of BL and CS in seed development. In the early stages of germination, CS, but not BL, appeared and its level increased in the growing tissues in which the transcript level of CYP85A1 and CYP85A6 was high, suggesting the significance of CS in seed germination and early seedling growth of pea. 6-Deoxocathasterone (6-deoxoCT) was the quantitatively major BR in mature seeds. At the early stage of germination, the level of 6-deoxoCT was specifically decreased, whereas the levels of downstream intermediates were increased. It seems that 6-deoxoCT is the major storage BR and is utilized during germination and early growth stages. The level of the mRNAs of BR biosynthesis and perception genes fluctuated during seed development. In mature seeds, most of mRNAs were present, but the level was generally lower compared with immature seeds. However, CYP90A9 mRNA rapidly increased during seed development and reached the maximum in mature seeds. The mRNAs stored in mature pea seeds seem to be utilized when seeds germinate. However, it was found that de novo transcription of mRNAs also starts as early as during seed imbibition.

The harmful effects of ultraviolet (UV) exposure on the skin are associated with the generation of reactive oxygen species (ROS) such as superoxide anion radical ( O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical ( OH), and singlet oxygen ((1)O(2)) as well as with lipid peroxides and their radicals (LOOH and LOO ). To give direct proof that such ROS are generated in UV-exposed skin, we proposed the in vivo detection and imaging method in which both a sensitive and specific chemiluminescence (CL) probe, such as CLA, and an ultralow-light imaging apparatus with a CCD camera were used. With this method we found that O(2)(-) is formed intrinsically and that (1)O(2) and O(2)(-) are generated in the UVA-exposed skin of mice. In addition, we indicated that antioxidative ability against ROS in the skin of hairless rats decreased as age increased. Using these findings, we demonstrated the protective abilities of sodium ascorbate, caffeic acid, essential aroma oils, and zinc(ii) ion and its complexes, which we administered to mice both topically and orally. We present a review for the current state of our research proposing the sensitive CL method as a useful in vivo tool in photobiological research for the detection of oxidative stress as well as for the evaluation of antioxidative agents to the skin.

We studied the effects of calcium, cyclic nucleotides, and protein kinase C on albumin transfer, electrical resistance, and cytoskeletal actin filaments in cultured human umbilical vein endotheiiai cells. The endotheiiai monoiayer grown on collagentreated filters markedly restricted the transfer of albumin relative to Its transfer across the filter alone. Both Ca ++ lonophore A23187 and ethyleneglycol tetraacetlc acid disrupted the Integrity of the endotheiiai monoiayer, thereby Increasing endotheiiai albumin transfer and decreasing electrical resistance In a concentration-dependent manner. Neither W-7, a calmodulln antagonist, nor TMB-8, an intracellular Ca suggest that the pathogenesis of atherosclerosis involves endotheiiai damage, which reduces the effectiveness of these cells as a barrier.The development of EC culture has allowed the study of permeability in EC monolayers in porous-bottom dishes.3 Such in vitro models have the advantage over in vivo studies of permitting direct measurement of EC permeability. Transendothelial movements of several macromolecules of different molecular weights and radii have been investigated. 45 We chose to study albumin because it is one of the most important physiologic macromolecules, determining the balance of osmotic pressure across the endothelium and acting as a carrier of various substances, including hormones and drugs.Although it is known that ECs can be targets of thrombogenic and inflammatory mediators, which disrupt the EC monoiayer and thereby increase its permeability, the initial transmembrane events that activate EC functions remain unclear. Calcium, cyclic nucleotides, and degradation products of phosphatidyl inositol are important intracellular second messengers and are involved in

No abstract

On 1 April 2017, six years have passed since the Fukushima Daiichi Nuclear Power Station (FDNPS) accident, and the Japanese government declared that some residents who lived in Tomioka Town, Fukushima Prefecture could return to their homes. We evaluated environmental contamination and radiation exposure dose rates due to artificial radionuclides in the livelihood zone of residents (living space such as housing sites), including a restricted area located within a 10-km radius from the FDNPS, immediately after residents had returned home in Tomioka town. In areas where the evacuation orders had been lifted, the median air dose rates were 0.20 μSv/h indoors and 0.26 μSv/h outdoors, and the radiation exposure dose rate was 1.6 mSv/y. By contrast, in the “difficult-to-return zone,” the median air dose rate was 2.3 μSv/h (20 mSv/y) outdoors. Moreover, the dose-forming artificial radionuclides (radiocesium) in the surface soil were 0.018 μSv/h (0.17 mSv/y) in the evacuation order-lifted areas and 0.73 μSv/h (6.4 mSv/y) in the difficult-to-return zone. These findings indicate that current concentrations of artificial radionuclides in soil samples have been decreasing in the evacuation order-lifted areas of Tomioka town; however, a significant external exposure risk still exists in the difficult-to-return zone. The case of Tomioka town is expected to be the first reconstruction model including the difficult-to-return zone.