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Biomedical subjects

Koji Uchida

Publications and source records attributed to Koji Uchida.

At least 37 records · Page 2Linked to original sources
PubMed2006

Prostaglandins from a zoanthid: paclitaxel-like neurite-degenerating and microtubule-stabilizating activities.

Two prostaglandins, PGA2 and PGB2, were isolated from the Okinawan zoanthid, Palythoa kochii, during a search for paclitaxel-like neurite-degenerating compounds from natural sources using a cell-based assay method. In the presence of PGA2 at 30 microM, the neuronal processes induced in PC12 cells by the nerve growth factor (NGF) degenerated over 24 h, whereas PGB2 had no effect on the neuronal processes of PC12 cells. This activity of PGA2 was similar to that of the microtubule-stabilizing agents, paclitaxel (Taxol) and epothilone A, unlike the microtubule-depolymerizing agent, colchicine, which brought about quick neurite degeneration within 3 h. PGA2 stimulated tubulin polymerization, although less potently than paclitaxel. An examination of structure-activity relationships across several PGs suggests that the cyclopentenone ring structure and the orientation of its dipolar moment played an important role in the paclitaxel-like neurite-degenerating activity. These results suggest that the cyclopentenone-type PGs can interact with microtubules to inhibit their function like paclitaxel.

Animals↗
PubMed2006

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis.

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Acrolein↗
PubMed2005

Maintaining the redox-balance intact: gosha-jinki-gan but not insulin activates retinal soluble guanylate cyclase in diabetic rats.

Strategies to prevent hyperglycemia-induced cytotoxic reactive oxygen species in the retina include the prevention of free radical production, activation of radical-scavenging capacities and inhibition of aldose reductase. This study examined the effect of the standardized Japanese herbal extract product gosha-jinki-gan (GJG) in comparison to insulin treatment in the rat retina. Diabetes was induced in male Wistar rats by single injection of streptozotocin (50 mg/kg i.p.). At 6 and 12 weeks, eye-cups were removed for immunohistochemistry. At 12 weeks, lipid peroxidation (tested with the antiacrolein antibody, Ab5F6) was enhanced significantly in the untreated diabetic group. This effect was absent in both treatment groups, notably in the outer retina. A similar result was obtained for nitrotyrosine overproduction. As an early treatment effect, GJG -- but not insulin -- enhanced soluble guanylate cyclase (sGC) activation (using the function-sensing antibody, MoAb 3221). GJG not only reduces nitroxidative stress and lipid peroxidation in the retina, it also ameliorates glucose metabolism within the cells. We propose that the high glucose turnover in the insulin-treated model disturbs the intracellular redox equilibrium, one result of which might be the impaired sGC activation.

Animals↗
PubMed2005

Susceptibility of actin to modification by 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.

Actins↗
PubMed2005

Identification of a lipid peroxidation product as a potential trigger of the p53 pathway.

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 can trigger apoptosis in many cell types, including neurons. We found that this nuclear protein was significantly phosphorylated when human neuroblastoma SH-SY5Y cells were exposed to in vitro oxidized polyunsaturated fatty acids. To identify an oxidized lipid that induces p53 phosphorylation, we conducted a screening of lipid peroxidation products in human neuroblastoma SH-SY5Y cells and identified 4-oxo-2-nonenal (ONE), a recently identified aldehyde originating from the peroxidation of omega6 polyunsaturated fatty acids, as a potential inducer of the p53 phosphorylation. We also found that ONE induced the phosphorylation of ataxia telangiectasia-mutated, which plays an essential role in transmitting DNA damage signals by the phosphorylation of p53. In addition, exposure of the cells to ONE resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that ONE acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. In addition, the observation that the ONE-induced p53 response was associated with the induction of apoptosis suggested that ONE activated the p53-dependent apoptosis mechanism via activation of the p53 signaling pathway and down-regulation of the p53 turnover. Finally, we observed that the ONE-2'-deoxyguanosine adduct, 7-(2-oxo-heptyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine, was accumulated in the spinal cord motor neurons of patients with sporadic amyotrophic lateral sclerosis. These data may suggest the potential critical role for ONE in the induction of a neuronal apoptosis program during oxidative processes.

Aldehydes↗
PubMed2005

Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome.

Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells.

Amino Acid Sequence↗
PubMed2005

Alpha-tocopherol-mediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli.

Although alpha-tocopherol is known as an essential micronutrient involved in various oxidative stress-related processes, its non-antioxidant activities have only been characterized in recent years. In this study, we reveal that (+)-alpha-tocopherol [RRR-alpha-tocopherol] enhances cellular susceptibility to both oxidative and non-oxidative apoptosis-inducing stimuli through up-regulation of caspase-3/CPP32 expression in several human cell lines. Exposure of (+)-alpha-tocopherol pretreated cells to known apoptosis-inducing stimuli, such as Fas, H(2)O(2), or etoposide, resulted in an increase in cellular apoptotsis. In addition, (+)-alpha-tocopherol also elevated the pro-caspase-3 protein level and mRNA expression in a time- and dose-dependent manner, while other tocopherol analogues showed no effect. Experiments using a GC-specific DNA binding agent, mithramycin A, and an electrophoretic mobility shift assay demonstrated that Sp1 might mediate the enhanced expression of caspase-3. Our results also confirmed that (+)-alpha-tocopherol promotes the expression, but not the activation, of caspase-3 in various human cell lines. These findings provide biological evidence showing that (+)-alpha-tocopherol can amplify the apoptotic response by up-regulating the expression of pro-caspase-3.

Apoptosis↗
PubMed2005

Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I.

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.

ATP Binding Cassette Transporter 1↗
PubMed2005

Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells.

The Nrf2-Keap1 system coordinately regulates cytoprotective gene expression via the antioxidant responsive element (ARE). The expression of several ARE-regulated genes was found to be up-regulated in endothelial cells by laminar shear stress, suggesting that Nrf2 contributes to the anti-atherosclerosis response via the ARE. To gain further insight into the roles that Nrf2 plays in the development of atherosclerosis, we examined how Nrf2 regulates gene expression in response to anti-atherogenic laminar flow (L-flow) or pro-atherogenic oscillatory flow (O-flow). Exposure of human aortic endothelial cells (HAECs) to L-flow, but not to O-flow, induced the expression of cytoprotective genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1) by 5-fold and heme oxygenase-1 by 8-fold. The critical contribution of Nrf2 to the expression induced by L-flow was ascertained in siRNA-mediated knock-down experiments. Two cyclooxygenase-2 (COX-2) specific inhibitors attenuated Nrf2 nuclear accumulation in the acute phase of L-flow exposure. A downstream product of COX-2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), activated the Nrf2 regulatory pathway in HAECs through binding to the cysteines of Keap1. These results demonstrate that 15d-PGJ2 is essential for L-flow to activate Nrf2 and induce anti-atherosclerotic gene expression. Whereas both L-flow and O-flow induced the nuclear accumulation of Nrf2 to comparable levels, chromatin immunoprecipitation analysis revealed that Nrf2 binding to the NQO1 ARE was significantly diminished in the case of O-flow compared with that of L-flow. These results suggest that O-flow inhibits Nrf2 activity at the DNA binding step, thereby suppressing athero-protective gene expression and hence predisposing the blood vessels to the formation of atherosclerosis.

Aorta↗
PubMed2005

Selective induction of the tumor marker glutathione S-transferase P1 by proteasome inhibitors.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs). Transcription factor Nrf2, which is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein-1) under unstimulated conditions, regulates the induction of phase 2 enzymes. In this study, to explore the role of the proteasome in the detoxification response, we tested the effect of proteasome inhibitors such as MG132, clasto-lactacystin beta-lactone, and lactacystin on the induction of GST isozymes and found that these inhibitors selectively induced the class Pi GST isozyme (GST P1). Down-regulation of the proteasome by antisense oligonucleotides or RNA interference indeed resulted in significant up-regulation of GST P1, suggesting that a decline in the proteasome activity could be directly or indirectly linked to the induction of GST P1. From the functional analysis of various deletion constructs of the upstream regulatory region of the GST P1 promoter, GST P1 enhancer I was identified as the response element for proteasome inhibition. Overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 not only by the proteasome inhibitor, but also by phase 2-inducing isothiocyanate, suggesting that there may be a process of GST P1 induction distinct from other phase 2 gene induction mechanisms. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.

Acetylcysteine↗
PubMed2005

Role of 15-deoxy delta(12,14) prostaglandin J2 and Nrf2 pathways in protection against acute lung injury.

RATIONALE: Acute lung injury (ALI) is a disease process that is characterized by diffuse inflammation in the lung parenchyma. Recent studies demonstrated that cyclooxygenase-2 (COX-2) induced at the late phase of inflammation aids in the resolution of inflammation by generating 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2). Transcription factor Nrf2 is activated by electrophiles and exerts antiinflammatory effects by inducing the gene expression of antioxidant and detoxification enzymes. OBJECTIVES: Because 15d-PGJ2 is an endogenous electrophile, we hypothesized that it protects against ALI by activating Nrf2. METHODS: To test this hypothesis, we generated a reversible ALI model by intratracheal injection of carrageenin, an inducer of acute inflammation, whose stimulation has been known to induce COX-2. MAIN RESULTS: We found that ALI induced by carrageenin was markedly exacerbated in Nrf2-knockout mice, compared with wild-type mice. Analysis of bronchoalveolar lavage fluids also revealed that the magnitude and the duration of acute inflammation, indicated by albumin concentration and the number of neutrophils, were significantly enhanced in Nrf2-knockout mice. Treatment of wild-type mice with NS-398, a selective COX-2 inhibitor, significantly exacerbated ALI to the level of Nrf2-knockout mice. In the lungs of NS-398-treated wild-type mice, both the accumulation of 15d-PGJ2 and the induction of Nrf2 target antioxidant genes were significantly attenuated. Exogenous administration of 15d-PGJ2 reversed the exacerbating effects of NS-398 with the induction of antioxidant genes. CONCLUSIONS: These results demonstrated in vivo that 15d-PGJ2 plays a protective role against ALI by exploiting the Nrf2-mediated transcriptional pathway.

Animals↗
PubMed2005

Accumulation of acrolein-protein adducts after traumatic spinal cord injury.

Reactive oxygen species and resultant lipid peroxidation (LPO) have been associated with central nervous system trauma. Acrolein (2-propenal) and 4-hydroxynonenal (HNE) are the most toxic byproducts of LPO, with detrimental effects in various types of cells. In this study, we used immunoblotting techniques to detect the accumulation of protein-bound acrolein and HNE. We report that protein-bound acrolein and HNE were significantly increased in guinea pig spinal cord following a controlled compression injury. The acrolein and HNE protein-adducts increased in the damaged spinal cord as early as 4 h after injury, reached a peak at 24 h after injury, and remained at a significantly high level up to 7 days after injury. Such increase of protein adducts was also observed in the adjacent segments of the injury site beginning at 24 h post injury. These results suggest that products of lipid peroxidation, especially acrolein, may play a critical role in the secondary neuronal degeneration, which follows mechanical insults.

Acrolein↗
PubMed2005

Effects of N-acetylcysteine, quercetin, and phytic acid on spontaneous hepatic and renal lesions in LEC rats.

The effects of anti-oxidants were examined in Long-Evans Cinnamon (LEC) rats, which develop acute hepatic injury, and subsequent hepatic and renal tumors due to accumulation of excess Cu. The rats, at the age of 15 weeks, were supplied a diet containing either 1% of N-acetylcysteine (NAC), quercetin (QC), or phytic acid (PA), or basal diet alone. At weeks 2 and 6 posttreatment, animals were sacrificed for collection of blood and tissue samples. In the NAC-treated group, the development of hepatic and renal lesions was dramatically reduced. In addition, accumulation of Cu and Fe in the liver was suppressed. Acrolein-modified protein, a new marker for lipid peroxidation, was not detected in the liver or kidney of NAC treated rats, even though deposition was evident in control. Neither QC nor PA affected the development of spontaneous hepatic lesions. These results indicate that oxidative stress was reduced by NAC in the liver and kidney, and suggest that Cu and Fe may be involved in the generation of oxidative stress in the liver. In addition, it was suggested that the different effects of the anti-oxidants on lesion development in LEC rats might be related to different mechanisms of action with regard to oxidative stress.

Acetylcysteine↗
PubMed2005

Rapid increase in serum lipid peroxide 4-hydroxynonenal (HNE) through monocyte NADPH oxidase in early endo-toxemia.

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.

Aldehydes↗
PubMed2005

Acrolein modifies apolipoprotein A-I in the human artery wall.

Carbonyl stress is implicated in accelerated vascular disease, but little is known about the factors that control the reactions of carbonyls with proteins. Acrolein is a reactive carbonyl generated by the oxidation of lipids and amino acids. It also forms during cigarette smoking. We therefore investigated the possibility that acrolein might react with apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), which plays a critical role in mobilizing cholesterol from artery wall macrophages. Tandem mass spectrometric analysis demonstrated that lysine residues were the only amino acids in apoA-I that were modified by acrolein. Immunohistochemical studies with a monoclonal antibody revealed that acrolein adducts colocalized with apoA-I in human atherosclerotic lesions. Moreover, the ability of apoA-I to remove cholesterol from cultured cells was impaired after exposure to acrolein, suggesting that the carbonyl might interfere with apoA-I's normal function of promoting cholesterol efflux from artery wall cells. Our observations suggest that acrolein may interfere with normal HDL cholesterol transport by modifying apoA-I. This structural damage might play a critical role in atherogenesis by impairing cholesterol removal from artery wall cells.

Acrolein↗
PubMed2005

Effect of ascorbate on acrolein modification of very low density lipoprotein and uptake of oxidized apolipoprotein e by hepatocytes.

Acrolein modification of apolipoprotein (apo) E in human very low density lipoprotein (VLDL) was suppressed by ascorbate. Acrolein-modified apoE in VLDL was not taken up by human hepatoma cell whereas unmodified apoE in the presence of ascorbate was taken up. These results suggest that ascorbate can play an important role in maintaining proper lipoprotein metabolism by the antioxidant effect.

Acrolein↗
PubMed2004

Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.

Apoptosis↗