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991.
Isabel A. Abreu Diane E. Cabelli 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(2):263-274
Superoxide dismutases are enzymes that function to catalytically convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis at near diffusion controlled rate constants via a general mechanism that involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals. That the catalytically active metal can be copper, iron, manganese or, recently, nickel is one of the fascinating features of this class of enzymes. In this review, we describe these enzymes in terms of the details of their catalytic properties, with an emphasis on the mechanistic differences between the enzymes. The focus here will be concentrated mainly on two of these enzymes, copper, zinc superoxide dismutase and manganese superoxide dismutase, and some relatively subtle variations in the mechanisms by which they function. 相似文献
992.
M.J. Cardador M. Gallego 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(21):1824-1830
Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na2SO4 (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water. 相似文献
993.
Jihye Jung Hyun Joo Park Ki-Nam Uhm Dooil Kim Hyung-Kwoun Kim 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(9):1841-1849
Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively. 相似文献
994.
Hyun-Hee Jang Arvind P. Jamakhandi Shane Z. Sullivan Chul-Ho Yun Paul F. Hollenberg Grover P. Miller 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(6):1285-1293
As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)2•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners. 相似文献
995.
Kuniko Akama Tomoe Horikoshi Atsushi Sugiyama Satoko Nakahata Aoi Akitsu Nobuyoshi Niwa Atsushi Intoh Yasutaka Kakui Michiko Sugaya Kazuo Takei Noriaki Imaizumi Takaya Sato Rena Matsumoto Hitoshi Iwahashi Shin-ichi Kashiwabara Tadashi Baba Megumi Nakamura Tosifusa Toda 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(6):1272-1284
In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization. 相似文献
996.
Khakpay R Polster D Köles L Skorinkin A Szabo B Wirkner K Illes P 《Purinergic signalling》2010,6(3):349-359
Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg2+-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic
currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio
ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude
of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene
ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated
the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic
acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded
in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency
of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors
appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate
onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise
separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons. 相似文献
997.
In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X7 receptor activity. We show here that in the absence of extracellular Ca2+, BzATP-generated increase in [Ca2+]i
via Ca2+ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca2+ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X7 receptor, did not reduce any of the two components of the Ca2+ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca2+ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and
BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y2 but not the P2X7 receptors. 相似文献
998.
Hassenklöver T Schulz P Peters A Schwartz P Schild D Manzini I 《Purinergic signalling》2010,6(4):429-445
Like in other vertebrates, the anterior part of the telencephalon of amphibians mainly consists of the olfactory bulb (OB),
but different from higher vertebrates, the lateral telencephalic ventricles of larval Xenopus laevis expand deep into the anterior telencephalon. The neurogenic periventricular zone (PVZ) of the lateral ventricles generates
new OB neurons throughout the animal’s lifetime. We investigated the ultrastructural organization of the PVZ and found that
within a time period of 24 h, 42.54 ± 6.65% of all PVZ cells were actively proliferating. Functional purinergic receptors
are widespread in the central nervous system and their activation has been associated with many critical physiological processes,
including the regulation of cell proliferation. In the present study we identified and characterized the purinergic system
of the OB and the PVZ. ATP and 2MeSATP induced strong [Ca2+]i increases in cells of both regions, which could be attenuated by purinergic antagonists. However, a more thorough pharmacological
investigation revealed clear differences between the two brain regions. Cells of the OB almost exclusively express ionotropic
P2X purinergic receptor subtypes, whereas PVZ cells express both ionotropic P2X and metabotropic P1 and P2Y receptor subtypes.
The P2X receptors expressed in the OB are evidently not involved in the immediate processing of olfactory information. 相似文献
999.
The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro
Agrawal A Buckley KA Bowers K Furber M Gallagher JA Gartland A 《Purinergic signalling》2010,6(3):307-315
The P2X7 receptor (P2X7R) has been implicated in the process of multinucleation and cell fusion. We have previously demonstrated
that blockade of P2X7Rs on osteoclast precursors using a blocking antibody inhibited multinucleated osteoclast formation in
vitro, but that P2X7R KO mice maintain the ability to form multinucleated osteoclasts. This apparent contradiction of the
role the P2X7R plays in multinucleation has prompted us to examine the effect of the most commonly used and recently available
P2X7R antagonists on osteoclast formation and function. When added to recombinant RANKL and M-CSF human blood monocytes cultures,
all but one compound, decreased the formation and function of multinucleated TRAP-positive osteoclasts in a concentration-dependent
manner. These data provide further evidence for the role of the P2X7R in the formation of functional human multinucleated
osteoclasts and highlight the importance of selection of antagonists for use in long-term experiments. 相似文献
1000.
Ol’ga Vančová Ladislav Bačiak Svatava Kašparová Jarmila Kucharská Hector H. Palacios Jaromír Horecký Gjumrakch Aliev 《Journal of cellular and molecular medicine》2010,14(11):2667-2674
Brain energy disorders can be present in aged men and animals. To this respect, the mitochondrial and free radical theory of aging postulates that age‐associated brain energy disorders are caused by an imbalance between pro‐ and anti‐oxidants that can result in oxidative stress. Our study was designed to investigate brain energy metabolism and the activity of endogenous antioxidants during their lifespan in male Wistar rats. In vivo brain bioenergetics were measured using 31P nuclear magnetic resonance (NMR) spectroscopy and in vitro by polarographic analysis of mitochondrial oxidative phosphorylation. When compared to the young controls, a significant decrease of age‐dependent mitochondrial respiration and adenosine‐3‐phosphate (ATP) production measured in vitro correlated with significant reduction of forward creatine kinase reaction (kfor) and with an increase in phosphocreatine (PCr)/ATP, PCr/Pi and PME/ATP ratio measured in vivo. The levels of enzymatic antioxidants catalase, GPx and GST significantly decreased in the brain tissue as well as in the peripheral blood of aged rats. We suppose that mitochondrial dysfunction and oxidative inactivation of endogenous enzymes may participate in age‐related disorders of brain energy metabolism. 相似文献