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101.
Ogawa K Hiraku Y Oikawa S Murata M Sugimura Y Kawamura J Kawanishi S 《Mutation research》2003,539(1-2):145-155
Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide], a hydrazine derivative, which has been shown to have effective antineoplastic activity, induces cancer in some experimental animals and humans. To clarify a new mechanism for its carcinogenic effect, we examined DNA damage induced by procarbazine in the presence of metal ion, using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Procarbazine plus Cu(II) induced piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at the 5'-ACG-3' sequence, complementary to a hotspot of the p53 gene, and the 5'-TG-3' sequence. Catalase partially inhibited DNA damage, suggesting that not only H(2)O(2) but also other reactive species are involved. Procarbazine plus Cu(II) significantly increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was completely inhibited by calatase. Electron spin resonance spin-trapping experiments revealed that methyl radicals were generated from procarbazine and Cu(II). On the basis of these findings, it is considered that procarbazine causes DNA damage through non-enzymatic formation of the Cu(I)-hydroperoxo complex and methyl radicals. In conclusion, in addition to alkylation, oxidative DNA damage may play important roles in not only antitumor effects but also mutagenesis and carcinogenesis induced by procarbazine. 相似文献
102.
Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells 总被引:11,自引:0,他引:11
Sakai K Matsumoto K Nishikawa T Suefuji M Nakamaru K Hirashima Y Kawashima J Shirotani T Ichinose K Brownlee M Araki E 《Biochemical and biophysical research communications》2003,300(1):216-222
Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus. 相似文献
103.
Miyashita T Kawakami A Tamai M Izumi Y Mingguo H Tanaka F Abiru S Nakashima K Iwanaga N Aratake K Kamachi M Arima K Ida H Migita K Origuchi T Tagashira S Nishikaku F Eguchi K 《Biochemical and biophysical research communications》2003,312(2):397-404
Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression. 相似文献
104.
Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals. 相似文献
105.
Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1 下载免费PDF全文
An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol. 相似文献
106.
Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells 总被引:4,自引:0,他引:4
107.
Oda A Wada I Miura K Okawa K Kadoya T Kato T Nishihara H Maeda M Tanaka S Nagashima K Nishitani C Matsuno K Ishino M Machesky LM Fujita H Randazzo P 《The Journal of biological chemistry》2003,278(8):6456-6460
Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions. 相似文献
108.
To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site. 相似文献
109.
A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P 总被引:7,自引:0,他引:7
Takebe G Yarimizu J Saito Y Hayashi T Nakamura H Yodoi J Nagasawa S Takahashi K 《The Journal of biological chemistry》2002,277(43):41254-41258
Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells. 相似文献
110.
Honma M Namikawa K Mansur K Iwata T Mori N Iizuka H Kiyama H 《Journal of neurochemistry》2002,82(4):961-975
Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate. 相似文献