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991.
Spin adducts of superoxide,alkoxyl, and lipid-derived radicals with EMPO and its derivatives 总被引:1,自引:0,他引:1
The compound 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) is a hydrophilic cyclic nitrone spin trap, which, in contrast to DMPO, forms a relatively stable superoxide adduct (t(1/2)=8.6 min) with an EPR spectrum similar to the respective DMPO adduct. In order to find the optimal degree of lipophilicity of this novel type of spin trap with respect to the detection of radicals formed during lipid peroxidation, the ethoxy group of EMPO was replaced by alkoxy substituents of increasing chain length, leading to the methoxy- (MeMPO), 1-propoxy- (PrMPO), 1-butoxy- (BuMPO), and 1-octyloxy- (OcMPO) derivatives of EMPO. The stability of their superoxide adducts was found to be strongly dependent on the size of the alkoxycarbonyl group. Increasing chain length of the alkoxyl substituent decreased the stability of alkoxyl radical adducts of MeMPO, EMPO, and PrMPO, but increased the stability of OcMPO adducts. The stability of alkoxyl radical adducts of BuMPO, on the other hand, were practically independent of the size of the alkoxyl group. Detection of lipid alkoxyl radicals formed by peroxidizing linoleic acid in a stationary system was therefore only possible with the most lipophilic spin trap, OcMPO. However, with the more hydrophilic spin traps MeMPO, EMPO, PrMPO, and BuMPO optimal EPR signal intensity could be obtained when a slow-flow system was used. Thus, within this series EMPO is the best spin trap for the detection of superoxide; OcMPO, on the other hand, is most suitable for the detection of lipid alkoxyl radicals. 相似文献
992.
The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different. 相似文献
993.
Kikuchi K Ekwall H Tienthai P Kawai Y Noguchi J Kaneko H Rodriguez-Martinez H 《Zygote (Cambridge, England)》2002,10(4):355-366
Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development. 相似文献
994.
Can MMTV exploit TLR4? 总被引:3,自引:0,他引:3
The recognition of microbial pathogens based on their molecular patterns is essential for host defense. Recently, Toll-like receptors have been shown not only to recognize viruses as well as bacteria and fungi, but also to trigger an efficient immune response. A recent publication proposed that the retrovirus mouse mammary tumor virus exploits the pattern-recognition receptor Toll-like receptor 4 to achieve more efficient infection. 相似文献
995.
McCammon MG Scott DJ Keetch CA Greene LH Purkey HE Petrassi HM Kelly JW Robinson CV 《Structure (London, England : 1993)》2002,10(6):851-863
Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein. 相似文献
996.
Raaijmakers H Macieira S Dias JM Teixeira S Bursakov S Huber R Moura JJ Moura I Romão MJ 《Structure (London, England : 1993)》2002,10(9):1261-1272
Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel. 相似文献
997.
Folkesson HG Matthay MA Chapin CJ Porta NF Kitterman JA 《American journal of physiology. Lung cellular and molecular physiology》2002,282(3):L508-L515
Knowledge about the conversion of the epithelium in the distal air spaces of the lung from secretion to absorption is imperative to the understanding of postnatal lung development; little such information is available in rats. Distal air space fluid clearance was therefore measured in 21- to 22-day gestation rat fetuses and newborn (40 h) rats. Distal air space fluid clearance was measured from the increase in (131)I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. There was no net fluid movement across the distal air space epithelium in the lungs of 21-day gestation fetuses. Twenty-four hours later, distal air space fluid was cleared at a rapid rate in the 22-day gestation fetuses. Within the first 40 h after birth, the rate rapidly declined to adult levels. The high distal air space fluid clearance at 22 days gestation and at 40 h after birth was mediated by beta-adrenergic receptors as demonstrated by elevated plasma epinephrine levels and inhibition by propranolol. Interestingly, the elevated distal air space fluid clearance in the 22-day gestation fetuses was only minimally amiloride sensitive; however, amiloride sensitivity increased over the first 40 h after birth. In conclusion, these studies demonstrate that 1) rapid rates of net alveolar fluid clearance occur late in gestation in the rat and 2) this clearance is driven by elevations of endogenous epinephrine. 相似文献
998.
Biessen EA Sliedregt-Bol K 'T Hoen PA Prince P Van der Bilt E Valentijn AR Meeuwenoord NJ Princen H Bijsterbosch MK Van der Marel GA Van Boom JH Van Berkel TJ 《Bioconjugate chemistry》2002,13(2):295-302
In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP. 相似文献
999.
Serum deprivation stimulates endothelial apoptosis while albumin inhibits this and has been proposed as important in confining apoptotic remodelling to poorly perfused vessels. Tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta are also reported to induce endothelial apoptosis. To investigate the comparative roles of these stimuli, the effect of TNF-alpha and TGF-beta, alone or in combination, in the presence or absence of serum or albumin was studied. There was strong synergy between the cytokines in inducing human umbilical vein endothelial cell apoptosis, but only in the absence of serum. Synergy was destroyed by boiling cytokines and was not affected by polymyxin B. Dose response experiments revealed greater activity of TGF-beta(1) than TGF-beta(2). The synergy was protein synthesis dependent and apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and FACS analysis. Data suggests a role for synergistic activation of endothelial cell apoptosis by TNF-alpha and TGF-beta(1) but perhaps only in poorly perfused vessels deprived of serum factors. 相似文献
1000.
Dournon C Membre H Brohm PE Coince A Cornu N Dreyer L Florentin J Jeanneau L Henniquin C Houbre M Guerard M Lecomte N Maxant L Schluraff M Venandet AS Jusyte A Simmet D Bocking D Flaig D Santak L Bolek S Goppel V Rossignon JP Trossat MA Raux M Forster S Staudenmaier G Boser S Horn E 《Journal of gravitational physiology : a journal of the International Society for Gravitational Physiology》2002,9(1):P375-P376
The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools. 相似文献