E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.     

Change in sleep state of the elderly before and after cataract surgery

It seems likely that the influences of light upon circadian rhythms will decrease with aging, particularly those rhythms that are more influenced by light with a higher color temperature and richer in short wavelengths. More specifically, cataract patients' optical systems transmit light poorly, especially the shorter wavelengths that affect the circadian system more. The present study investigated melatonin secretion profiles and sleep patterns before and after cataract surgery. Fifteen subjects were studied for 3 consecutive weekdays before, and one month after, their cataract surgery. UV-cutting intra-ocular lenses were used for patients after surgery. No statistically significant differences between before and after surgery were observed in the amount of light received and the amount of activity. This means that there were no significant changes in their lifestyle during the experimental period. Considering the group as a whole, no significant differences were present in melatonin secretion, sleep parameters, or sleepiness before and after the surgery. However, individual subjects responded differently. The subjects showed a negative correlation between the wake-up (p=0.067) or retiring times (p=0.017) and sleep efficiency after surgery. The amount of light received during the nighttime influenced sleep more significantly than during the daytime.     

Radical formation in cytochrome c oxidase

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50μs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO(2)(-) ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O(2) with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H(2)O(2)) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O(2) and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a(3). This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".     

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.     

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By 1H NMR Spectroscopy

Recently, we discovered novel silver(I)-mediated cytosine–cytosine base pair (C–Ag^I–C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C–Ag^I–C base pair flanked by 14 A–T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.     

Life cycle inventory analysis of co<Subscript>2</Subscript> emissions manufacturing commodity plastics in japan

The goal of this study was to calculate the average CO₂ emissions for manufacturing three commodity plastics, polyethylene (PE), polypropylene (PP), and polyvinyl chloride (PVC) in Japan. The CO₂ emissions were calculated from cradle to gate, excluding the calcination processes after use. As the results, the followings were observed: 1) The gross CO₂ emissions for the manufacture of plastics in Japan were 1.3, 1.4, and 1.7 kg-CO₂/kg-PE, PP, and PVC, respectively. These mainly reflected the difference of CO₂ emissions for the in-house electricity generation. 2) The CO₂ emissions for the electricity used for manufacturing PVC were higher than that used for PE and PP, because additional electricity was required for the electrolysis to produce chlorine. The gross electricity consumption for manufacturing PVC was 1.3 kWh/kg-PVC, and the other plastics consumed 0.5 kWh/kg-Products. In addition, the effects of energy saving were studied using a projected gas-diffusion electrode for the electrolysis of salt on the reduction of CO₂ emissions. It was estimated that the reduction in CO₂ emissions was 7% compared with the present PVC manufacturing processes.     

Identification of the lantibiotic nisin Q,a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.     

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.