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951.
Kasai K Banba N Hishinuma A Matsumura M Kakishita H Matsumura M Motohashi S Sato N Hattori Y 《American journal of physiology. Cell physiology》2000,279(6):C1859-C1869
A cyclopentenone-type prostaglandin,15-deoxy-12,14-prostaglandin J2(15-d-PGJ2), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-. We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ2-treatedcells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production wasdecreased in untreated cells according to duration of culture. PGD2 and PGJ2, which are precursors to15-d-PGJ2, exhibited an effect similar to15-d-PGJ2. However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-, and WY-14643, a specific PPAR- ligand, lacked this effect. 15-d-PGJ2 and itsprecursors, but not the thiazolidinediones, induced gene expression forheme oxygenase-1 (HO-1), a stress-related protein, and stronglyinhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production.Cyclopentenone-type PGs have been recently shown to inhibit nuclearfactor-B (NF-B) activation via a direct and PPAR-independentinhibition of inhibitor-B kinase, suggesting that, in humanthyrocytes, such PGs may inhibit IL-1-induced NO production, possiblyvia an inhibition of NF-B activation. On the other hand, sodiumarsenite, a known activator of the stress response pathway, inducedHO-1 mRNA expression but lacked a promoting effect on TG production.Thus 15-d-PGJ2 and its precursors appear to facilitate TGproduction via a PPAR-independent mechanism and through a differentpathway from the cellular stress response that is available tocyclopentenone-type PGs. Our findings reveal a novel role of these PGsassociated with thyrocyte differentiation. 相似文献
952.
To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress. 相似文献
953.
Detection of mutations in genes responsible for hereditary diseases or tumors is important clinically. It is necessary to establish a simple technique for screening mutations in large numbers of samples. The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has proved to be a useful technique for analyzing mutations or DNA polymorphisms. Non-radioisotopic versions using fluorescent dye and an automated DNA sequencer have also been exploited to extend this technique into the clinical field. We have examined mutations of exons 5-9 of the p53 gene in 112 colorectal, 28 esophageal and 33 hepatocellular carcinomas by fluorescence-based PCR-SSCP (F-SSCP) under various conditions. We found 64 types of mutations in 63, 17 and 12 cases of colon, esophageal and hepatocellular carcinomas by F-SSCP. We determined the sequence of all samples, and confirmed that all mutations were successfully detected by F-SSCP. With the low-pH buffer system, 61 types of mutants were detected, while 51 types were detected by TBE and 57 types were detected by TBE with glycerol gel. The polyacrylamide gel in TME or TBE without glycerol was tough and could be used repeatedly, but the glycerol containing gel was fragile and could not stand repeated use. Thus, use of a low-pH buffer in the electrophoresis of F-SSCP is simpler and better at detecting mutations than the conventional TBE buffer system. We believe that low-pH F-SSCP analysis is an efficient and powerful technique for examination of a large number of samples, in particular clinical specimens obtained by biopsy or surgery. 相似文献
954.
Recently, we have found a transient intermediate on the folding pathway of src SH3. Intending to investigate the structure of the transient intermediate, we tested a mutant of src SH3, named A45G, using circular dichroism, fluorescence and X-ray solution scattering, and incidentally found that it forms a stable alpha-helix-rich intermediate (I(eq)) (different from the native beta-sheet-based secondary structure) at pH 3.0, but contains only beta-sheets at pH 6.0, whereas wild-type SH3 forms only beta-sheets at both pH 3.0 and pH 6.0. The intermediate I(eq) shows a circular dichroism measured at theta(222)=-10,300 deg.cm(2) dmol(-1), indicating a 31% alpha-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I(eq) as 19.1 A at pH 3.0 and 15.4 A at pH 6.0, and Kratky plots showed a clear peak at pH 3.0, 4.0 and 6.0, indicating that I(eq) too is compact. In these parameters, I(eq) closely resembles the kinetically-obtained intermediate I(kin) which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 A and theta(222)=-8700 deg.cm(2)dmol(-1)), indicating a 26% alpha-helix proportion in our previous paper. Refolding experiments with A45G were done at pH 6.0 by stopped-flow apparatus monitored by circular dichroism, and compared to kinetic experiments with wild-type SH3 at pH 6.0. The result showed an alpha-helix-rich intermediate at the same dichroism amplitude, but nine times slower in formation-rate. A pH-jump experiment from pH 3.0 to pH 5.9 on A45G was also performed. This showed no bursts, and the rate of conformation-change was almost as fast as the refolding rate of A45G at pH 6.0. These kinetic experiment data would be consistent with I(eq) being nearly identical to the I(kin), which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3. 相似文献
955.
Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol. 相似文献
956.
High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A 总被引:3,自引:0,他引:3
Yao D Taguchi T Matsumura T Pestell R Edelstein D Giardino I Suske G Rabbani N Thornalley PJ Sarthy VP Hammes HP Brownlee M 《The Journal of biological chemistry》2007,282(42):31038-31045
Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease. 相似文献
957.
Specific inhibitor of MEK-mediated cross-talk between ERK and p38 MAPK during differentiation of human osteosarcoma cells 总被引:2,自引:0,他引:2
Shimo T Matsumura S Ibaragi S Isowa S Kishimoto K Mese H Nishiyama A Sasaki A 《Journal of cell communication and signaling》2007,1(2):103-111
Osteosarcoma is the most common primary malignant bone tumor, accounting for approximately 20% of all primary sarcomas in
bone. Although treatment modalities have been improved over the past decades, it is still a tumor with a high mortality rate
in children and young adults. Based on histological considerations, osteosarcoma arises from impaired differentiation of these
immature cells into more mature types and that correction of this impairment may reduce malignancy and increase the efficiency
of chemotherapy. The purpose of this study was to determine the effect of specific inhibitors of MAPK extracellular signaling-regulated
kinase (ERK) kinase (MEK) and p38 on the differentiation of human osteosarcoma cell line SaOS-2 cells. We found that PD98059,
a specific inhibitor of MEK, inhibited the serum-stimulated proliferation of SaOS-2 cells; whereas SB203580, a specific inhibitor
of p38 MAPK, had little effect on it. SB203580 suppressed ALPase activity, gene expression of type I collagen, and expression
of ALP and BMP-2 mRNAs; whereas PD98059 upregulated them dose dependently. In addition, immunoblot and immunostaining analysis
revealed that phosphorylation of ERK was increased by treatment with SB203580; whereas PD98059 increased the phosphorylation
of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteosarcoma cell differentiation
is regulated by the balance between the activities of the ERK and p38 pathways and that the MEK/ERK pathway negatively regulates
osteosarcoma cell differentiation, whereas the p38 pathway does so positively. MEK inhibitor may thus be a good candidate
for altering the expression of the osteosarcoma malignant phenotype. 相似文献
958.
Sakamoto A Iwabata K Koshiyama A Sugawara H Yanai T Kanai Y Takeuchi R Daikuhara Y Takakusagi Y Sakaguchi K 《Chromosoma》2007,116(6):545-556
The X family DNA polymerases λ (CcPolλ) and μ (CcPolμ) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPolλ encoded a predicted product of 800 amino acid residues and that of CcPolμ of 621 amino acid residues. Both CcPolλ and CcPolμ required Mn2+ ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts,
CcPolλ and CcPolμ had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPolλ was
present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and
recombine. CcPolμ was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination
intermediate substrates, we found that both CcPolλ and CcPolμ could promote primer extension of an invading strand in a D-loop
structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension
is an activity intrinsic to CcPolλ and CcPolμ. Based on these data, we discuss the possible roles of these polymerases in
meiosis. 相似文献
959.
ABCA3 is proposed to function as a lung surfactant lipid transporter. Here we report ABCA3-dependent lipid uptake into intracellular vesicles in lung adenocarcinoma A549 cells. A549 cells stably expressing GFP-tagged wild-type ABCA3 (A549/ABCA3(WT)) had larger LAMP3-positive vesicles than their parental cells as well as A549 cells expressing a Walker A motif mutant (A549/ABCA3(N568D)). The choline-phospholipids level in A549/ABCA3(WT) was increased 1.25-fold compared to that in A549 and A549/ABCA3(N568D) cells, while the cholesterol levels were similar. Sucrose gradient fractionation analysis in A549/ABCA3(WT) cells revealed that choline-phospholipids were enriched in low-density and nile red-positive vesicles. Electronmicroscopic analysis showed multilamellar vesicles in A549/ABCA3(WT) cells. These results indicate that ABCA3 mediates ATP-dependent choline-phospholipids uptake into intracellular vesicles. 相似文献
960.
Matsumura Y Kidokoro T Miyagi Y Marilingaiah NR Sagami H 《Journal of biochemistry》2007,142(4):533-537
Rat geranylgeranyl diphosphate synthase (GGPS) and its deletion mutants from the carboxyl terminus were analysed using Escherichia coli harbouring pACYC-crtIB, which contains crtI and crtB encoding the carotenoid biosynthetic enzymes. Mutants (delta-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (delta-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (delta-4, -8, -12 and -16) were overexpressed in E. coli BL21 (DE3) and purified in a stable form by nickel affinity chromatography except for one mutant (delta-16). The farnesyl-transferring activities of wild-type GGPS, delta-4, -8 and -12 mutants were relatively in a ratio of 1.0, 0.84, 0.26 and 0.0015. Each Km value of the four recombinants were estimated to be 0.71, 2.0 2.8 and 55 microM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 microM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that delta-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and delta-4 mutant. These results suggest that the KMFTEENE residing on the carboxyl-terminal sequence of GGPS stabilizes the active region involved in the substrate binding and catalysis. 相似文献