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991.
Keisuke Sakurai Jeannie Chen Shahrokh C. Khani Vladimir J. Kefalov 《The Journal of biological chemistry》2015,290(14):9239-9250
Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light. 相似文献
992.
993.
Identification of two essential aspartates for polymerase activity in parainfluenza virus L protein by a minireplicon system expressing secretory luciferase 下载免费PDF全文
Yusuke Matsumoto Keisuke Ohta Natsuko Yumine Hideo Goto Machiko Nishio 《Microbiology and immunology》2015,59(11):676-683
Gene expression of nonsegmented negative‐strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase‐active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase‐complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions. 相似文献
994.
Naturally high heavy metal concentrations in feathers of the flightless Kagu Rhynochetos jubatus 下载免费PDF全文
Jörn Theuerkauf Tokushi Haneda Nozomu J. Sato Keisuke Ueda Ralph Kuehn Roman Gula Izumi Watanabe 《Ibis》2015,157(1):177-180
We assessed heavy metal concentrations in feathers of 38 Kagus Rhynochetos jubatus living on ultramafic soils in New Caledonia. Concentrations of heavy metals in down feathers were similar to concentrations in shafts of primary or secondary feathers, whereas the concentrations in vanes were much higher, indicating that concentrations in down feathers were not due to external contamination but rather to ingestion. Although there was no anthropogenic pollution in our study area, concentrations of iron (Fe), zinc (Zn), manganese (Mn), chromium (Cr), selenium (Se), strontium (Sr) and cobalt (Co) in feathers were 1.2–21 times higher in Kagu than the average in other bird species studied, the majority of those from polluted environments. Kagus may have specific adaptations that enable them to live in environments with naturally high heavy metal concentrations. 相似文献
995.
Keisuke Yoshida Satoshi Hara Toru Hisabori 《The Journal of biological chemistry》2015,290(23):14278-14288
Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. 相似文献
996.
Although it has been reported that levels of hyaluronan are decreased in the dermis of aged skin, little is known about the cellular mechanism(s) underlying that hyaluronan deficiency. Since hyaluronan is produced by dermal fibroblasts and is secreted into the surrounding dermal tissues, we examined the secretion of hyaluronan by dermal fibroblasts and characterized its cellular mechanism using real-time RT-PCR and western blotting for its synthesizing and degrading enzymes, hyaluronan synthase and hyaluronidase, respectively. The secretion of hyaluronan by dermal fibroblasts derived from differently aged human donors, was higher in the younger human fibroblasts tested (0 and 19 years old) compared to the older human fibroblasts tested (39, 56 and 77 years old). The relative secretion levels of hyaluronan by the different human fibroblasts tested were attributable to the relative expression of hyaluronan synthases 1, 2, 3 but not hyaluronidases 1, 2 enzymes at the gene and protein levels among those fibroblasts. These findings indicate that the deficiency of hyaluronan in the aged dermis might result from the down-regulation in the potential of older human fibroblasts to secrete hyaluronan and that decrease in secretory potential is mainly associated with the down-regulated expression of hyaluronan synthases, especially hyaluronan synthase 2, but not with the expression levels of hyaluronidases. 相似文献
997.
Tomoaki Kozaki Ayaka Kubokawa Ryunosuke Taketomi Keisuke Hatae 《Journal of physiological anthropology》2015,34(1)
Background
Bright nocturnal light has been known to suppress melatonin secretion. However, bright light exposure during the day-time might reduce light-induced melatonin suppression (LIMS) at night. The effective proportion of day-time light to night-time light is unclear; however, only a few studies on accurately controlling both day- and night-time conditions have been conducted. This study aims to evaluate the effect of different day-time light intensities on LIMS.Methods
Twelve male subjects between the ages of 19 and 23 years (mean ± S.D., 20.8 ± 1.1) gave informed consent to participate in this study. They were exposed to various light conditions (<10, 100, 300, 900 and 2700 lx) between the hours of 09:00 and 12:00 (day-time light conditions). They were then exposed to bright light (300 lx) again between 01:00 and 02:30 (night-time light exposure). They provided saliva samples before (00:55) and after night-time light exposure (02:30).Results
A one-tailed paired t test yielded significant decrements of melatonin concentration after night-time light exposure under day-time dim, 100- and 300-lx light conditions. No significant differences exist in melatonin concentration between pre- and post-night-time light exposure under day-time 900- and 2700-lx light conditions.Conclusions
Present findings suggest the amount of light exposure needed to prevent LIMS caused by ordinary nocturnal light in individuals who have a general life rhythm (sleep/wake schedule). These findings may be useful in implementing artificial light environments for humans in, for example, hospitals and underground shopping malls. 相似文献998.
Fumiaki Katagiri Dario Canelon-Suarez Kelsey Griffin John Petersen Rachel K. Meyer Megan Siegle Keisuke Mase 《PloS one》2015,10(5)
Plant growth chambers produce controlled environments, which are crucial in making reproducible observations in experimental plant biology research. Commercial plant growth chambers can provide precise controls of environmental parameters, such as temperature, humidity, and light cycle, and the capability via complex programming to regulate these environmental parameters. But they are expensive. The high cost of maintaining a controlled growth environment is often a limiting factor when determining experiment size and feasibility. To overcome the limitation of commercial growth chambers, we designed and constructed an inexpensive plant growth chamber with consumer products for a material cost of $2,300. For a comparable growth space, a commercial plant growth chamber could cost $40,000 or more. Our plant growth chamber had outside dimensions of 1.5 m (W) x 1.8 m (D) x 2 m (H), providing a total growth area of 4.5 m2 with 40-cm high clearance. The dimensions of the growth area and height can be flexibly changed. Fluorescent lights with large reflectors provided a relatively spatially uniform photosynthetically active radiation intensity of 140–250 μmoles/m2/sec. A portable air conditioner provided an ample cooling capacity, and a cooling water mister acted as a powerful humidifier. Temperature, relative humidity, and light cycle inside the chamber were controlled via a z-wave home automation system, which allowed the environmental parameters to be monitored and programmed through the internet. In our setting, the temperature was tightly controlled: 22.2°C±0.8°C. The one-hour average relative humidity was maintained at 75%±7% with short spikes up to ±15%. Using the interaction between Arabidopsis and one of its bacterial pathogens as a test experimental system, we demonstrate that experimental results produced in our chamber were highly comparable to those obtained in a commercial growth chamber. In summary, our design of an inexpensive plant growth chamber will tremendously increase research opportunities in experimental plant biology. 相似文献
999.
Timothy J. Mitchison Keisuke Ishihara Phuong Nguyen Martin Wühr 《Cold Spring Harbor perspectives in biology》2015,7(10)
The first 12 cleavage divisions in Xenopus embryos provide a natural experiment in size scaling, as cell radius decreases ∼16-fold with little change in biochemistry. Analyzing both natural cleavage and egg extract partitioned into droplets revealed that mitotic spindle size scales with cell size, with an upper limit in very large cells. We discuss spindle-size scaling in the small- and large-cell regimes with a focus on the “limiting-component” hypotheses. Zygotes and early blastomeres show a scaling mismatch between spindle and cell size. This problem is solved, we argue, by interphase asters that act to position the spindle and transport chromosomes to the center of daughter cells. These tasks are executed by the spindle in smaller cells. We end by discussing possible mechanisms that limit mitotic aster size and promote interphase aster growth to cell-spanning dimensions.How components and processes within cells scale in size and rate with the size of the cell has become a topic of considerable interest in recent years (reviewed in Chan and Marshall 2012; Goehring and Hyman 2012; Levy and Heald 2012). For molecular machines with precise architectures (e.g., ribosomes), size is invariant, but rates of assembly and function, which depend on regulation and energy, might scale. For assemblies whose dimensions are not hard wired (e.g., cytoskeleton assemblies and organelles), both size and rate might scale. For pathways involving distributed biochemical change (e.g., the cell-cycle oscillator), size is not well defined, but rate might scale in interesting ways. Here, we will address only size scaling, and refer the reader to interesting recent progress on cell-cycle timing in early Xenopus embryos (Chang and Ferrell 2013; Tsai et al. 2014).Size-scaling relationships, which are part of the science of allometry, have long informed on whole organism physiology. Explicitly seeking them at the subcellular level is a newer endeavor, which in our mind holds two kinds of promise. It can inform on mechanism at the level of integrated cell physiology (e.g., on establishment of cleavage plane geometry). It can also inform on molecular processes involved in assembly growth and dynamics, and perhaps help us discern logic in often frustratingly complex molecular architectures. It is not obvious, for example, why ∼100 protein complexes are required to build a mitotic spindle in higher eukaryotes (Hutchins et al. 2010), when bacteria can segregate plasmids with far fewer (Salje et al. 2010). Part of the answer is the need for higher fidelity in the eukaryotic process. Gerhart and Kirschner (1997) also emphasized the need for highly adaptable processes in the evolution of higher eukaryotes. At least part of the complexity of subcellular assemblies might reflect the need for adaptable scaling of size, shape, and timing.Vertebrate embryos derived from large eggs provide a natural experiment in size scaling (Fig. 1). A Xenopus laevis egg, for example, is ∼1.2 mm in diameter. Following fertilization, it cleaves completely ∼12 times at an approximately constant rate of ∼2 divisions/h (most rates in early development are temperature dependent, and can vary up to about eightfold over the tolerated range). These divisions generate a quasispherical array of quasispherical cells that are, on average, smaller by 212-fold in volume, or 24-fold in radius. The first 12 divisions occur with little gene expression and little change in cell physiology, and it may be reasonable to assume approximately constant biochemistry (discussed below), other than periodic cell-cycle regulation. After the 12th division, cell physiology changes dramatically as part of the midblastula transition (MBT) (discussed below), which provides a natural cut-off for size-scaling investigations. An interesting and potentially informative complication is that cleaving amphibian embryos develop a gradient in blastomere sizes, with larger cells at the vegetal pole where yolk is more abundant (evident in Fig. 1C,D). Larger blastomeres tend to divide more slowly, which gradually eliminates division synchrony (Gerhart 1980).Open in a separate windowFigure 1.Spindle-size scaling in Xenopus laevis. A–D show confocal images of eggs and early embryos fixed at different stages, stained for tubulin (red) and DNA (green), cleared and imaged by confocal microscopy. Embryos containing metaphase spindles were selected for analysis. (A) Unfertilized egg with meiosis-II spindle (blue arrow). (B) First mitosis. Note scaling mismatch between the spindle and egg. (C,D) Cleavage stages. (E) Spindle lengths and cell lengths derived from confocal images like A–D. Note spindle length is approximately constant in the large-cell regime and scales with cell size in the small-cell regime. (F) Spindle assembled in a droplet of unfertilized egg extract containing fluorescent probes suspended in oil and imaged live. aNuMA, anti-nuclear mitotic apparatus. (A–E from Wühr et al. 2008; adapted, with permission, from the author; F is an unpublished image provided by Jesse Gatlin, University of Wyoming, which is similar to images in Hazel et al. 2013.)Embryos from different species have pros and cons for experimental analysis of size scaling during early divisions. Amphibian eggs provide a large dynamic range in cell size, complete division, and quasispherical geometry of both cells and embryos. In the minus column, they are opaque unless fixed and cleared and difficult to manipulate using genetics. Undiluted, cell-free extracts from Xenopus eggs and early embryos provide access to live imaging and molecular analysis and recapitulate the biology of intact eggs, including scaling relationships (Wilbur and Heald 2013), but it is important to go back to the intact embryo to check validity of key findings where possible. Zebrafish eggs provide a transparent, genetically tractable vertebrate system with very large cells but incomplete cleavage at early stages. Caenorhabditis elegans and Drosophila embryos have excellent imaging and genetics, which are advantages for scaling analysis, especially rate scaling (e.g., Carvalho et al. 2009; Hara and Kimura 2013), but these embryos start smaller, so they provide a lower dynamic range for analyzing size-scaling behavior. 相似文献
1000.
Yusuke Katsuda Yoshimi Niwano Takuji Nakashima Takayuki Mokudai Keisuke Nakamura Satomi Oizumi Taro Kanno Hiroyasu Kanetaka Hiroshi Egusa 《PloS one》2015,10(8)
Cytoprotective effects of short-term treatment with grape seed extract (GSE) upon human gingival fibroblasts (hGFs) were evaluated in relation to its antioxidant properties and compared with those of a water-soluble analog of vitamin E: trolox (Tx). GSE and Tx showed comparable antioxidant potential in vitro against di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH; a stable radical), hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Pretreatment or concomitant treatment with GSE for 1 min protected hGFs from oxidative stressors, including H2O2, acid-electrolyzed water (AEW), and 1O2, and attenuated the intracellular formation of reactive oxygen species induced by H2O2 and AEW. Tx also reduced the H2O2- and AEW-induced intracellular formation of reactive oxygen species, but showed no cytoprotective effects on hGFs exposed to H2O2, AEW, or 1O2. These results suggest that the cytoprotective effects of GSE are likely exerted independently of its antioxidant potential. 相似文献