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991.
A choline-containing phospholipid (PL-4) in Methanopyrus kandleri cells was identified as archaetidylcholine, which has been described by Sprott et al. (1997). The PL-4 consisted of a variety of molecular species differing in hydrocarbon composition. Most of the PL-4 was acid-labile because of its allyl ether bond. The identity of PL-4 was confirmed by thin-layer chromatography followed by positive staining with Dragendorff reagent and fast-atom bombardment-mass spectrometry. A new method of LiAlH4 hydrogenolysis was developed to cleave allyl ether bonds and recover the corresponding hydrocarbons. We confirmed the validity of the LiAlH4 method in a study of the model compound synthetic unsaturated archaetidic acid (2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate). Saturated ether bonds were not cleaved by the LiAlH4 method. The hydrocarbons formed following LiAlH4 hydrogenolysis of PL-4 were identified by gas-liquid chromatography and mass spectrometry. Four kinds of hydrocarbons with one to four double bonds were detected: 47% of the hydrocarbons had four double bonds; 11% had three double bonds; 14% had two double bonds; 7% had one double bond; and 6% were saturated species. The molecular species composition of PL-4 was also estimated based on acid lability: 77% of the molecular species had two acid-labile hydrocarbons; 11% had one acid-labile and one acid-stable hydrocarbon; and 11% had two acid-stable hydrocarbons. To our knowledge, this is the first report of a specific chemical degradation method for the structural analysis of allyl ether phospholipid in archaea. 相似文献
992.
Proteomic analysis of human brain identifies alpha-enolase as a novel autoantigen in Hashimoto's encephalopathy 总被引:4,自引:0,他引:4
Ochi H Horiuchi I Araki N Toda T Araki T Sato K Murai H Osoegawa M Yamada T Okamura K Ogino T Mizumoto K Yamashita H Saya H Kira J 《FEBS letters》2002,528(1-3):197-202
Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies. 相似文献
993.
Tanimura A Nezu A Morita T Hashimoto N Tojyo Y 《The Journal of biological chemistry》2002,277(32):29054-29062
The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha. 相似文献
994.
Tanaka T Ito T Furuta M Eguchi C Toda H Wakabayashi-Takai E Kaneko K 《The Journal of biological chemistry》2002,277(33):30382-30387
We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts. 相似文献
995.
Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes 总被引:10,自引:0,他引:10
Amano S Yamagishi Si Kato N Inagaki Y Okamoto T Makino M Taniko K Hirooka H Jomori T Takeuchi M 《Biochemical and biophysical research communications》2002,299(2):183-188
The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy. 相似文献
996.
Ikebe C Kominami K Toda T Nakayama K 《Biochemical and biophysical research communications》2002,290(5):1399-1407
The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast. 相似文献
997.
Akuta T Eguchi A Okuyama H Senda T Inokuchi H Suzuki Y Nagoshi E Mizuguchi H Hayakawa T Takeda K Hasegawa M Nakanishi M 《Biochemical and biophysical research communications》2002,297(4):779-786
The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity. 相似文献
998.
999.
Maruyama T Asada M Shiraishi T Yoshida H Maruyama T Ohuchida S Nakai H Kondo K Toda M 《Bioorganic & medicinal chemistry》2002,10(6):1743-1759
To identify a new selective EP4-agonist with improved chemical stability, further chemical modification of those reported previously was continued. We focused our attention on chemical modification of the alpha chain of 3,7-dithiaPGE(1) and selected 5-thiaPGE(1) as a new chemical lead. Introduction of an optimized omega chain to the 5-thiaPG skeleton afforded m-methoxymethyl derivative 33a, which showed the most potent EP4-receptor agonist activity and good subtype-selectivity both in vitro and in vivo. 9beta-HaloPGF derivatives were also synthesized and biologically evaluated in an attempt to block self-degradation of the beta-hydroxyketone moiety. Among these series, and 39b showed potent agonist activity and good subtype-selectivity. Structure-activity relationships (SARs) are also discussed. 相似文献
1000.
New orally active PDE4 inhibitors with therapeutic potential 总被引:1,自引:0,他引:1
Ochiai H Ishida A Ohtani T Kusumi K Kishikawa K Yamamoto S Takeda H Obata T Nakai H Toda M 《Bioorganic & medicinal chemistry》2004,12(15):4089-4100
The design, synthesis, and biological evaluation of a series of pyrazolopyridines was carried out. Structural optimization of the aniline moiety of 4-anilinopyrazolopyridine derivative 3a, which is one of the newly discovered chemical leads for PDE4 inhibitors from our in-house library, was performed successfully. The details of the discovery of new orally active PDE4 inhibitors, which are expected to show therapeutic potential, are presented and their structure-activity relationships are discussed. Pharmacological evaluation and pharmacokinetic data for representative compounds are also presented. 相似文献