Biomedical subjects

Koji Uchida

Publications and source records attributed to Koji Uchida.

At least 55 records · Page 3Linked to original sources

PubMed2004

Endogenous formation of novel halogenated 2'-deoxycytidine. Hypohalous acid-mediated DNA modification at the site of inflammation.

A potential role of DNA damage by leukocyte-derived reactive species in carcinogenesis has been suggested. Leukocyte-derived peroxidases, such as myeloperoxidase and eosinophil peroxidase, use hydrogen peroxide and halides (Cl- and Br-) to generate hypohalous acids (HOCl and HOBr), halogenating intermediates. It has been suggested that these oxidants lead to the formation of halogenated products upon reaction with nucleobases. To verify the consequences of phagocyte-mediated DNA damage at the site of inflammation, we developed a novel monoclonal antibody (mAb2D3) that recognizes the hypohalous acid-modified DNA and found that the antibody most significantly recognized HOCl/HOBr-modified 2'-deoxycytidine residues. The immunoreactivity of HOCl-treated oligonucleotide was attenuated by excess methionine, suggesting that chloramine-like species may be the plausible epitopes of the antibody. On the basis of further characterization combined with mass spectrometric analysis, the epitopes of mAb2D3 were determined to be novel N4,5-dihalogenated 2'-deoxycytidine residues. The formation of the dihalogenated 2'-deoxycytidine in vivo was immunohistochemically demonstrated in the lung and liver nuclei of mice treated with lipopolysaccharides, an experimental inflammatory model. These results strongly suggest that phagocyte-derived oxidants, hypohalous acids, endogenously generate the halogenated DNA bases such as a novel dihalogenated 2'-deoxycytidine in vivo. Halogenation (chlorination and/or bromination) of DNA therefore may constitute one mechanism for oxidative DNA damage at the site of inflammation.

Animals↗

PubMed2004

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages.

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.

Aldehydes↗

PubMed2004

Accumulation of protein-bound 4-hydroxy-2-hexenal in spinal cords from patients with sporadic amyotrophic lateral sclerosis.

4-Hydroxy-2-hexenal (HHE) is a toxic, reactive aldehydic intermediate formed by nonenzymatic peroxidation of n-3 polyunsaturated fatty acids. The aim of this study was to determine the implication for HHE in the pathomechanism of amyotrophic lateral sclerosis (ALS) by immunohistochemical and enzyme-linked immunosorbent assay (ELISA) techniques using a mouse monoclonal IgG(1) antibody mAbHHE53 specific for protein-bound HHE. Immunohistochemical analysis on formalin-fixed, paraffin-embedded sections and frozen sections of spinal cords obtained at autopsy from 10 sporadic ALS patients and 10 age-matched control subjects demonstrated that protein-bound HHE immunoreactivity was seen and was prominent in the entire gray matter in the ALS cases and localized in the neurons, reactive astrocytes, microglial cells, and the surrounding neuropil, while the immunoreactivity was obscure or undetectable in the control cases. No significant protein-bound HHE immunoreactivity was seen in sections processed with omission of mAbHHE53 or in sections incubated with the antibody with an excess of the respective antigen. Competitive ELISA analysis on trypsin-digested protein extracts of fresh-frozen spinal cord samples disclosed a significant increase in protein-bound HHE level in the ALS cases compared with the control cases. Our results indicate that enhanced HHE formation occurs in the entire gray matter of sporadic ALS spinal cords and suggest that the selective vulnerability of motor neurons to HHE mediates the pathomechanism of this disease.

Aged↗

PubMed2004

Zerumbone, a tropical ginger sesquiterpene, activates phase II drug metabolizing enzymes.

Zerumbone (ZER), a sesquiterpene compound occurring in tropical ginger Zingiber zerumbet Smith, has been implicated as one of the promising chemopreventive agents against colon and skin cancer. In the present study, we investigated the phase II detoxification enzymes induction of ZER using a cultured rat normal liver epithelial cell line. Exposure of RL34 cells to ZER resulted in the significant induction of glutathione S-transferase, while the reduced analogues of ZER (alpha-humulene and 8-hydroxy-alpha-humulene) did not show any inducing effect. Therefore, the electrophilic property, characterized by the reactivity with intracellular nucleophiles including protein sulfhydryls as well as low molecular weight thiols, at the 8-position alpha,beta-unsaturated carbonyl group plays an important role in the induction of phase II enzymes. ZER induced nuclear localization of the transcription factor Nrf2 that binds to antioxidant response element (ARE) of the phase II enzyme genes, suggesting that ZER is a potential activator of the Nrf2/ARE-dependent detoxification pathway. This is consistent with the observation that ZER potentiated the gene expression of several Nrf2/ARE-dependent phase II enzyme genes, including gamma-glutamylcysteine synthetase, glutathione peroxidase, and hemeoxygenase-1. The present study also implied the antioxidant role of this detoxification system activation by ZER in the neutralization of lipid peroxidation in hepatocytes, providing a new insight for cancer prevention.

Animals↗

PubMed2004

Rapid formation of 4-hydroxy-2-nonenal, malondialdehyde, and phosphatidylcholine aldehyde from phospholipid hydroperoxide by hemoproteins.

4-Hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) are well-known toxic products of lipid peroxidation. Phosphatidylcholine aldehydes are also known as oxidation products of phosphatidylcholine. The mechanism of the formation of these compounds in vivo has been a long-standing question. We observed that the rapid reaction of hemoproteins (methemoglobin, metmyoglobin, and cytochrome c) with 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), having a hydroperoxylinoleoyl residue, generated HNE, MDA, and the phosphatidylcholine aldehyde 1-palmitoyl-2-(9-oxononanoyl) phosphatidylcholine. The efficiencies (mol% yield) of the formation of HNE and MDA from decomposed PLPC-OOH by methemoglobin, metmyoglobin, and cytochrome c after incubation for 10 min were 1.6, 1.0, and 1.0% for HNE and 1.2, 0.6, and 0.9% for MDA, respectively. When 1-palmitoyl-2-linoleoyl phosphatidylcholine was incubated with lipoxidase and methemoglobin, the formation of HNE and the phosphatidylcholine aldehyde 1-palmitoyl-2-(9-oxononanoyl) phosphatidylcholine was observed. When 1-palmitoyl-2-arachidonyl phosphatidylcholine was used instead of 1-palmitoyl-2-linoleoyl phosphatidylcholine, the phosphatidylcholine aldehyde 1-palmitoyl-2-oxovaleroyl phosphatidylcholine was obtained. These data suggest that HNE and phosphatidylcholine aldehydes might be rapidly formed from phosphatidylcholine by lipoxygenase and hemoproteins. Furthermore, hemichrome, converted from methemoglobin by deoxycholic acid and ursodeoxycholic acid, showed marked decomposition of HNE. These results suggest that hemoproteins are related to both the formation and the decomposition of HNE.

Aldehydes↗

PubMed2004

A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation.

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G(2)-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G(2)-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G(2)-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G(2)-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G(2)-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.

Apoptosis↗

PubMed2004

Expression and function of PPARgamma in rat placental development.

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor known to regulate adipogenesis. Deletion of the PPARgamma gene in the mouse results in death by embryonic day 10.0 (E10.0) due to the failure of establishment of a labyrinth layer in the placenta, which suggests that PPARgamma is involved in trophoblast differentiation. To define PPARgamma function further in placental development, the expression and localization of the PPARgamma gene in the rat placenta was investigated. RT-PCR analysis shows the presence of PPARgamma mRNA in the placenta of day 11 of pregnancy (d11). The expression level is higher at d13 and then later decreased. Immunohistochemistry detects both PPARgamma and its putative intrinsic ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), in the trophoblast of layer I which lined the maternal sinus. Oral administration of troglitazone, an agonist of PPARgamma, to pregnant rats between d9 and d11 increases the expression level of PPARgamma in the placenta and reduces the mortality of the fetuses by half. These results suggest that PPARgamma is required not only for trophoblast differentiation but also trophoblast maturation to establish maternal-fetal transport.

Administration, Oral↗

PubMed2004

2'-deoxycytidine in free nucleosides and double-stranded DNA as the major target of lipid peroxidation products.

Lipid peroxidation generates a variety of reactive products that covalently modify DNA, yielding several types of adducts with nucleobases. In the present study, we characterized the modification of nucleobases during peroxidation of linoleate and found that 2'-deoxycytidine (dC) could be a major target of the modification by lipid peroxidation reactions. Upon incubation with oxidized linoleate, dC and 2'-deoxyguanosine (dG) were significantly modified among four 2'-deoxynucleosides. The major product in dG/linoleate was identical to the 2-oxo-heptyl-substituted 1,N(2)-etheno-dG that had been previously identified as a 4-oxo-2-nonenal (ONE)-dG adduct. On the basis of spectroscopic and chemical characterization, we identified the major product in dC/linoleate as the 2-oxo-heptyl-substituted 3,N(4)-etheno-dC. The same adduct was also produced upon reaction of dC with ONE, suggesting that ONE might represent the major reactive species that modifies DNA during lipid peroxidation. Indeed, this proposition was supported by the observation that ONE was far more reactive with dC and dG than other genotoxic aldehydes, such as 4-hydroxy-2-nonenal. More strikingly, we found that, in contrast to the similar reactivity of ONE toward free nucleobases (dC and dG), ONE preferentially reacted with dC residues in double-stranded DNA. These results suggest that ONE and other 4-oxo-2-alkenals may possess by far the strongest electrophilic potential vs. dC and that the formation of 4-oxo-2-alkenal-adducted dC may thus serve as one mechanism for oxidative damage to DNA in vivo.

Aldehydes↗

PubMed2004

Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids.

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases.

Aldehydes↗

PubMed2004

Esterified lipid hydroperoxide-derived modification of protein: formation of a carboxyalkylamide-type lysine adduct in human atherosclerotic lesions.

We have recently identified Nepsilon-azelayllysine (AZL) as a carboxyalkylamide-type novel lysine adduct in the reaction of linoleic acid hydroperoxides with the lysine derivative. To examine the formation of AZL in vivo, a novel monoclonal antibody (mAb19D5) specific to AZL moiety was prepared. The mAb19D5 scarcely recognized oxidized low-density lipoprotein (oxLDL), whereas the treatment of oxLDL with alkali or phospholipase A2 significantly increased the immunoreactivity. Similarly, the immunopositive materials were detected in alkali- or phospholipase A2-treated sections from human atherosclerotic aorta but not in untreated sections. These results suggest that esterified lipid hydroperoxide-derived modification of protein may serve as one mechanism for the oxidative modification of LDL and subsequent formation of atherosclerotic lesions in vivo.

Alkalies↗

PubMed2004

Benzyl isothiocyanate modifies expression of the G2/M arrest-related genes.

Naturally occurring isothiocyanates are effective chemoprotective agents against chemical carcinogenesis in experimental animals. In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis. The exposure of HL-60 cells to BITC resulted in the inhibition of the G2/M progression that coincided with the apoptosis induction. We demonstrated that BITC significantly up-regulated expression of the G2/M cell cycle arrest-regulating genes including p21, GADD45, and 14-3-3sigma. Thus, these gathered data further supported that BITC has a potential to induce apoptosis selectively in the proliferating pre-cancerous cells through a cell cycle arrest-dependent mechanism.

Anticarcinogenic Agents↗

PubMed2004

Kupffer cells alter organic anion transport through multidrug resistance protein 2 in the post-cold ischemic rat liver.

Although Kupffer cells (KCs) may play a crucial role in post-cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown. This study examined reveal the role of KC in post-cold ischemic liver grafts. Rat livers treated with or without liposome-encapsulated dichloromethylene diphosphonate, a KC-depleting reagent, were stored in University of Wisconsin (UW) solution at 4 degrees C for 8 to 24 hours and reperfused while monitoring biliary output and constituents. The ability of hepatocytes to excrete bile was assessed through laser-confocal microfluorography in situ. Cold ischemia-reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury. This event coincided with impaired excretion of glutathione and bilirubin-IXalpha (BR-IXalpha), suggesting delayed transport of these organic anions. We examined whether intracellular relocalization of multidrug resistance protein-2 (Mrp2) occurred. Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi. The KC-depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8-hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane. Furthermore, thromboxane A(2) (TXA(2)) synthase in KC appeared involved as blocking this enzyme improved 5-carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC-depleting grafts. In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post-cold ischemic grafts.

ATP-Binding Cassette Transporters↗

PubMed2004

Induction of reversible cysteine-targeted protein oxidation by an endogenous electrophile 15-deoxy-delta12,14-prostaglandin J2.

We have previously shown that a prostaglandin D(2) metabolite, 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), 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 investigate the correlation between the redox regulation and the 15d-PGJ(2)-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins using biotinylated cysteine as a molecular probe. In addition, the reversible regulation of protein function by S-oxidation/thiolation was characterized in vitro. When human neuroblastoma SH-SY5Y cells were exposed to 15d-PGJ(2), followed by treatment with biotinylated cysteine, 26 proteins, including glycolytic enzymes, cytoskeletal proteins, redox enzymes, and stress proteins, were identified as substrates for reversible cysteine-targeted oxidation. To investigate the regulatory mechanism of protein function by S-oxidation/thiolation, the binding of a low molecular weight thiol (glutathione) to a glycolytic enzyme alpha-enolase was characterized. Treatment of alpha-enolase with the thiol oxidant diamide in the presence of glutathione in vitro resulted in the binding of glutathione to the protein and concomitant loss of the enzymatic activity, whereas the glutathiolation and inactivation of alpha-enolase were fully reversed by dithiothreitol. Mass spectrometric analysis of the tryptic fragments from native and oxidized alpha-enolase identified two cysteine residues, Cys-118 and Cys-388, as the S-oxidation sites, which may play a role in the regulation of the biological activities of the protein and may be regulated by a reversible S-oxidation/thiolation reaction. These results suggest that cysteine-targeted oxidation/thiolation plays a critical role in the regulation of protein function under conditions of electrophile-induced oxidative stress.

Binding Sites↗

PubMed2004

Transcription factor Nrf2 regulates inflammation by mediating the effect of 15-deoxy-Delta(12,14)-prostaglandin j(2).

Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), it was observed that macrophages from pleural lavage accumulate 15d-PGJ(2). We show that in mouse peritoneal macrophages 15d-PGJ(2) can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ(2) accumulation and PrxI expression. Administration of 15d-PGJ(2) into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ(2) by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Animals↗

PubMed2003

Carbonyl stress and detoxification ability in the male genital tract and testis of rats.

Carbonyl compounds, which are naturally produced and augmented under oxidative stress, have deleterious effects on the reproductive system. The aldo-keto reductase (AKR) family of enzymes catalyze the reductive detoxification of various carbonyl compounds in an NADPH-dependent manner. To elucidate involvement of AKR in detoxification of endogenously produced carbonyls in the male reproductive system, we investigated the differential expression and tissue localization of aldehyde reductase (ALR) and protein adducts produced by reaction with lipid peroxidation products. A strong immunoreactivity to an anti-ALR antibody was observed in the epithelia of the epididymis, vas deferens, seminal vesicle, and prostate gland. Virtually the same cells were stained with a monoclonal antibody (mAb) 5F6, raised against an acrolein-modified protein. In the testis, however, mAb5F6 specifically stained the nuclei of somatic cells and less differentiated spermatogenic cells. While acrolein inactivated glutathione reductase, an enzyme involved in recycling oxidized glutathione, AKR activity was affected at the high concentration only. The colocalization of lipid peroxidation products and AKR in the epithelia of the male genital tract indicates that these tissues are exposed to oxidative stress and possess a protective system coordinately.

Acrolein↗

PubMed2003

An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome.

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We have previously shown that 15d-PGJ(2) potently induces apoptosis of SH-SY5Y human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ(2)-induced accumulation of p53. It was observed that 15d-PGJ(2) potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ(2) resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ(2) acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ(2) on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ(2) virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ(2), and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ(2) indeed resulted in the formation of a 15d-PGJ(2)-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ(2).

Animals↗

PubMed2003

Hypothermia attenuates delayed cortical cell death and ROS generation following CO inhalation.

Carbon monoxide (CO) is the most popular cause of poisoning. The bilateral basal ganglia lesion characterizes the delayed neuronal cell death (DCD). We demonstrated there were both apoptosis and necrosis in the cortex, basal ganglia and hippocampus in a case of human CO accident. To elucidate the mechanism of DCD after CO inhalation, histological studies on the rat brain were conducted. Rats were ventilated with nitrous oxide (sham group), 10% O(2) (hypoxia group) or 1005 ppm CO (CO group) for 90 min, while the pericranial temperature was controlled at either 32, 37, or 39 degrees C during CO inhalation. After reoxygenation for 30 min, the rats were allowed to recover for 48 h. The ratio of eosinophilic and HNE-positive neurons in the cortex were higher in the CO group than in the hypoxia group at 37 degrees C, while the PaO(2) was much lower in the hypoxia than in the CO group. The damage was alleviated in the hypothermia (32 degrees C) as compared with normothermia, while the hyperthermia (39 degrees C) did not significantly increased it. CO inhalation injures neuron by reactive oxygen species (ROS), independent of hypoxia, as can be concluded from the histological comparison of DCD with HNE immunoreactivity.

Aldehydes↗

PubMed2003

Formation of acrolein-derived 2'-deoxyadenosine adduct in an iron-induced carcinogenesis model.

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2' -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2' -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2' -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2' -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.

Acrolein↗