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991.
992.
Elnaz Mehdizadeh Aghdam Lena Mahmoudi Azar Abolfazl Barzegari Farrokh Karimi Majid Mesbahfar Naser Samadi Mohammad Saeid Hejazi 《Gene》2012
Oxidative stress occurs as a result of imbalance between generation and detoxification of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Relation between cytoplasmic recombinant protein expression with H2O2 concentration and catalase activity variation was already reported. The periplasmic space of E. coli has different oxidative environment in relative to cytoplasm and there are some benefits in periplasmic expression of recombinant proteins. In this study, hydrogen peroxide concentration and catalase activity following periplasmic expression of mouse IL-4 were measured in E. coli. After construction of pET2mIL4 plasmid, the expression of recombinant mouse interleukin-4 (mIL-4) was confirmed. Then, the H2O2 concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H2O2 concentration of the cells. However, the H2O2 concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H2O2 elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H2O2 concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase. 相似文献
993.
Sugiura M Ogami S Kusumi M Un S Rappaport F Boussac A 《The Journal of biological chemistry》2012,287(16):13336-13347
The main cofactors that determine the photosystem II (PSII) oxygen evolution
activity are borne by the D1 and D2 subunits. In the cyanobacterium
Thermosynechococcus elongatus, there are three
psbA genes coding for D1. Among the 344 residues
constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between
PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first
study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption
spectroscopy, we show that: (i) the time-resolved EPR spectrum of TyrZ• in the
(S3TyrZ•)′ is slightly modified; (ii) the split EPR signal
arising from TyrZ• in the (S2TyrZ•)′ state induced by near-infrared
illumination at 4.2 K of the S3TyrZ state is significantly
modified; and (iii) the slow phases of P680+⋅ reduction by TyrZ are
slowed down from the hundreds of μs time range to the ms time range,
whereas both the S1TyrZ• → S2TyrZ and
the S3TyrZ• → S0TyrZ + O2
transition kinetics remained similar to those in PsbA(1/3)-PSII. These results
show that the geometry of the TyrZ phenol and its environment, likely
the Tyr-O···H···Nϵ-His bonding,
are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to
the dynamics of the proton-coupled electron transfer processes associated with
the oxidation of TyrZ being affected. From sequence comparison, we
propose that the C144P and P173M substitutions in PsbA2-PSII
versus PsbA(1/3)-PSII, respectively located upstream of the
α-helix bearing TyrZ and between the two α-helices
bearing TyrZ and its hydrogen-bonded partner, His-190, are
responsible for these changes. 相似文献
994.
M Szekeres GL Nádasy G Turu E Soltész-Katona ZE Tóth A Balla KJ Catt L Hunyady 《The Journal of biological chemistry》2012,287(37):31540-31550
In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II. 相似文献
995.
Ferrante P Ballottari M Bonente G Giuliano G Bassi R 《The Journal of biological chemistry》2012,287(20):16276-16288
The photosystem II antenna of Chlamydomonas reinhardtii is composed of monomeric and trimeric complexes, the latter encoded by LHCBM genes. We employed artificial microRNA technology to specifically silence the LHCBM2 and LHCBM7 genes, encoding identical mature polypeptides, and the LHCBM1 gene. As a control, we studied the npq5 mutant, deficient in the LHCBM1 protein. The organization of LHCII complexes, functional antenna size, capacity for photoprotection, thermal energy dissipation and state transitions, and resistance to reactive oxygen species was studied in the various genotypes. Silencing of the LHCBM2/7 genes resulted in a decrease of an LHCII protein with an apparent molecular mass of 22 kDa, whereas silencing/lack of LHCBM1 caused the decrease/disappearance of a 23-kDa protein. A decrease in the abundance of trimeric LHCII complexes and in functional antenna size was observed in both LHCBM2/7 and LHCBM1 knockouts. In agreement with previous data, depletion of LHCBM1 decreased the capacity for excess energy dissipation but not the ability to perform state transitions. The opposite was true for LHCBM2/7, implying that this polypeptide has a different functional role from LHCBM1. The abundance of LHCBM1 and LHCBM2/7 is in both cases correlated with resistance to superoxide anion, whereas only LHCBM1 is also involved in singlet oxygen scavenging. These results suggest that different LHCBM components have well defined, non-redundant functions despite their high homology, implying that engineering of LHCBM proteins can be an effective strategy for manipulating the light harvesting system of Chlamydomonas reinhardtii. 相似文献
996.
TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)m(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed. 相似文献
997.
998.
García-Hoz C Sánchez-Fernández G García-Escudero R Fernández-Velasco M Palacios-García J Ruiz-Meana M Díaz-Meco MT Leitges M Moscat J García-Dorado D Boscá L Mayor F Ribas C 《The Journal of biological chemistry》2012,287(10):7792-7802
Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis. 相似文献
999.
Arnaud Germain David B. Stern 《The Plant journal : for cell and molecular biology》2012,72(6):960-971
Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3′→5′ exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non‐viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two‐step maturation of mRNA 3′ ends, with RNR1 polishing 3′ termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non‐polysomal fractions, and half‐life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3′→5′ exoribonucleases in chloroplast mRNA 3′ end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes. 相似文献
1000.
Simone Barera Cristina Pagliano Tillmann Pape Guido Saracco James Barber 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2012,367(1608):3389-3399
It was the work of Jan Anderson, together with Keith Boardman, that showed it was possible to physically separate photosystem I (PSI) from photosystem II (PSII), and it was Jan Anderson who realized the importance of this work in terms of the fluid-mosaic model as applied to the thylakoid membrane. Since then, there has been a steady progress in the development of biochemical procedures to isolate PSII and PSI both for physical and structural studies. Dodecylmaltoside (DM) has emerged as an effective mild detergent for this purpose. DM is a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric centre of the carbohydrate head group, axial in α-DM and equatorial in β-DM. We have compared the use of α-DM and β-DM for the isolation of supramolecular complexes of PSII by a single-step solubilization of stacked thylakoid membranes isolated from peas. As a result, we have optimized conditions to obtain homogeneous preparations of the C2S2M2 and C2S2 supercomplexes following the nomenclature of Dekker & Boekema (2005 Biochim. Biophys. Acta
1706, 12–39). These PSII–LHCII supercomplexes were subjected to biochemical and structural analyses. 相似文献