Biomedical subjects

Takeshi Kumagai

Publications and source records attributed to Takeshi Kumagai.

At least 19 recordsLinked to original sources

PubMed2006

Influences of drugs on the oxygen uptake rate and biosorption of activated sludge.

To prevent pollution of the water environment by drugs, we evaluated factors affecting the elimination of drugs by inducing reactions between the pharmaceutical chemicals originating from the drugs and activated sludge (AS) in test tubes. Of 30 pharmaceutical chemicals examined, ibuprofen (IBP) as an anti-inflammatory drug most markedly inhibited the oxygen uptake rate (OUR) of AS, and its IC50 was 172 mg/l. The IBP elimination from the wastewater was due to the significant biosorption by AS and was based on the time-response and dose-response relationships. In the pharmaceutical chemical group (1) (diclofenac Na, ketoprofen, indomethacin, salicylic acid, mefenamic acid, phenylbutazone, chlorpromazine.HCl, furosemide, tolbutamide and warfarin K) showing a relatively significant OUR inhibition (IC50, 200-500 mg/l), protein binding (75-99.7%), and the biosorption by AS also tended to be significant. In group (2) (acetaminophen, kanamycin.H2SO4, antipyrine, ethenzamide, gentamicin.H2SO4, cyclophosphamide.HCl, aminophylline, procainamide.HCl and cimetidine) showing a negligible OUR inhibition (IC50 > or =2000 mg/l), the protein binding was slight (0-74%), and biosorption by AS was also negligible. For the IBP and group (1), AS was pretreated with a certain excessive amount of each pharmaceutical chemical, and the qualitative OUR curves of the AS-synthetic sewage-II after washing showed a significant OUR inhibition immediately after the start of the reaction. Group (2), as well as the control group showed no OUR inhibition. These results suggest that the degree of OUR inhibition of AS by pharmaceutical chemicals is affected by the protein binding and the degree of biosorption. This suggests that pharmaceutical chemicals with a significant protein binding possibility can be eliminated from the wastewater by binding to AS.

Absorption↗

PubMed2006

Oxidized low-density lipoprotein-binding specificity of the Asp-hemolysin-related synthetic peptides from Aspergillus fumigatus.

Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. In the present study, in order to clarify the structure-binding activity relationship of Asp-hemolysin-related peptides to OxLDL, we investigated the interaction between Asp-hemolysin-related peptides consisting of 4 to 29 amino acid residues and OxLDL. The incubation of OxLDL with each Asp-hemolysin-related peptide resulted in the formation of an Asp-hemolysin/OxLDL complex. In particular, the tetrapeptide, YKDG (P-4), bound to OxLDL and inhibited the OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, we demonstrated that lysophosphatidylcholine (LysoPC) extracted from OxLDL inhibited the binding of P-21 to OxLDL in a dose-dependent manner and synthetic [14C]LysoPC bound to P-21. We propose here that the YKDG region is one of the important sites for the binding of these peptides to OxLDL, and LysoPC as a typical lipid moiety of OxLDL is attributed to the binding of OxLDL to these peptides.

Amino Acid Sequence↗

PubMed2006

Synthetic peptide (P-21) derived from Asp-hemolysin inhibits the induction of apoptosis on HUVECs by lysophosphatidylcholine.

Lysophosphatidylcholine (LPC), formed during low-density lipoprotein (LDL) oxidation and located within atherosclerotic plaques, regulates a variety of cellular functions, some of which could be construed to promote atherosclerotic lesion development, including vascular muscle cell proliferation, monocyte attraction, and endothelial cell apoptosis. We have previously reported that the synthetic peptide derived from Asp-hemolysin, named P-21, inhibits oxidized LDL (OxLDL)-induced macrophage proliferation through binding of P-21 to OxLDL. In this study, to clarify the interaction between P-21 and LPC as a typical lipid moiety of OxLDL, we examined the influence of P-21 on LPC-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Based on flow cytometric analysis, using annexin V-fluorescein isothiocyanate and propidium iodide as probes to assess apoptosis, LPC induced the apoptosis of HUVECs, and P-21 significantly inhibited this activity by 82.4%. Furthermore, dissociation-enhanced lanthanide fluorometric immunoassay indicated that LPC inhibited the binding of P-21 to OxLDL in a dose-dependent manner. A 50% inhibition dose was estimated to be 4.65 microM of LPC. These results suggest that P-21 inhibits LPC-induced HUVEC apoptosis through binding of P-21 to LPC.

Annexin A5↗

PubMed2006

[Biological activity of Asp-hemolysin as a regulation factor to atherogenic effect by oxidized low-density lipoprotein].

Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. Asp-hemolysin, a hemolytic toxin from Aspergillus fumigatus, is a binding protein for OxLDL. This study was undertaken to clarify the biological activity of OxLDL and the potentially of Asp-hemolysin as a regulation factor to atherogenic effect by OxLDL. We first analyzed the interaction between OxLDL and blood coagulation factors, which are involved in the blood coagulation pathway. OxLDL caused prolongation of activated partial thromboplastin time (APTT) as a parameter of the intrinsic pathway of blood coagulation in a dose- and oxidation time-dependent manner. In addition, OxLDL significantly inhibited blood coagulation factor VIII, IX, and XI activity. Furthermore, we demonstrated that factor VIII binds to OxLDL. These results indicate that the binding of factor VIII to OxLDL affects the intrinsic pathway of the blood coagulation cascade. Next, to clarify the structure-function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography. The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody. In addition, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin. Furthermore, to investigate the effect of the Asp-hemolysin-related peptide (P-21), a synthetic peptide derived from a region of Asp-hemolysin that is rich in positive charges, on macrophage proliferation induced by OxLDL. P-21 inhibited OxLDL-induced macrophage proliferation in a dose-dependent manner. In addition, the binding analysis of P-21 to OxLDL indicated that P-21 binds to OxLDL. These results indicate that P-21 inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL. In conclusion, we have shown that OxLDL affects the intrinsic pathway of blood coagulation, and its mechanism is dependent on the binding of factor VIII to OxLDL. Furthermore, we indicate the possibility that Asp-hemolysin is a useful tool to investigate the pathophysiological significance of OxLDL. In particular, since the P-21, an Asp-hemolysin-related peptide, inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL, further study on the binding mechanism between Asp-hemolysin-related peptide and OxLDL may provide important information on the prevention and treatment of atherosclerosis.

Aspergillus fumigatus↗

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

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

Central hypothyroidism in infants who were born to mothers with thyrotoxicosis before 32 weeks' gestation: 3 cases.

We describe 3 infants who were born to mothers with Graves' disease and developed central hypothyroidism that persisted for >6 months after birth. Two were preterm infants, and the other was a term infant who was born to a euthyroid mother who had been treated with an antithyroid drug since week 31 of gestation. These cases suggest that passage of thyroid hormones can occur from a thyrotoxic mother to the fetus and that the gestational period earlier than 32 weeks may be the critical time for development of central hypothyroidism.

Female↗

PubMed2005

A synthetic peptide (P-21) derived from asp-hemolysin inhibits the induction of macrophage proliferation by oxidized low-density lipoprotein.

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (OxLDL) induces macrophage proliferation via the specific uptake of lysophosphatidylcholine (LysoPC) of OxLDL by class A, type I and type II macrophage scavenger receptors. We have previously shown that Asp-hemolysin from Aspergillus fumigatus binds to LysoPC as a typical lipid moiety of OxLDL. This study investigated the effect of the Asp-hemolysin-related peptide (P-21), a synthetic peptide derived from a region of Asp-hemolysin that is rich in positive charges, on macrophage proliferation induced by OxLDL. Mouse peritoneal macrophages were used for proliferation study. OxLDL induced macrophage proliferation in an oxidation time-dependent manner, and P-21 inhibited OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, the binding analysis of P-21 to OxLDL by dissociation-enhanced lanthanide fluorometric immunoassay indicated that P-21 binds to OxLDL. These results indicate that P-21 inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL.

Amino Acid Sequence↗

PubMed2005

Inhibition of plasma coagulation through interaction between oxidized low-density lipoprotein and blood coagulation factor VIII.

Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. Thrombosis is the major mechanism underlying acute complications of atherosclerosis. In the present study, we analyzed the interaction between OxLDL and blood coagulation factors, which are involved in the blood coagulation pathway. We investigated the effect of OxLDL on plasma coagulation by measuring prothrombin time (PT) as a parameter of the extrinsic pathway of blood coagulation and activated partial thromboplastin time (APTT) as a parameter of the intrinsic pathway of blood coagulation following the addition of OxLDL to plasma. OxLDL, but not native LDL, caused prolongation of APTT in a dose- and oxidation time-dependent manner. In addition, the oxidatively modified product of acetylated LDL (AcLDL), but not AcLDL, also caused prolongation of APTT. The inhibition of lysophosphatidylcholine production in OxLDL by phenylmethylsulfonyl fluoride or Pefabloc pretreatment of LDL resulted in a prolongation of APTT, which was equivalent to the effect of OxLDL. Moreover, OxLDL significantly inhibited blood coagulation factor VIII, IX, and XI activity. Furthermore, we demonstrated that recombinant factor VIII binds to OxLDL and that factor VIII associated with OxLDL is detected in the incubation mixture of OxLDL and plasma. These results indicate that the binding of factor VIII to OxLDL affects the intrinsic pathway of the blood coagulation cascade. The present study suggests that the interaction between OxLDL and factor VIII may provide important information on the initiation and progression of atherosclerosis.

Blood Coagulation↗

PubMed2005

[Development of the new desiccator system for measuring the removal effect of the formaldehyde as an indoor air pollutant by the adsorbent].

The new desiccator system with measures for the prevention of dew drops and the processing of the formaldehyde (FA) gas discharged from the final desiccator was produced, and the FA removal rate for various adsorbents was examined. For the prevention of dew drops in the desiccator, a hygroscopic bottle containing silica gel was used next to the FA gas generator, and humidity was adjusted by adjusting the interval between the FA gas outlet (a) and the desiccant (b). The removal of the harmful FA gas discharged from the final desiccator (n=5) is an important in the environmental preservation. To solve this problem, the FA gas was passed through an oxidation bottle containing KMnO(4)-H(2)SO(4) solution, and it was possible to confirm the complete decomposition of the FA by increase of the CO(2) and elimination of the FA. For the determination of the FA concentration in the desiccator, 100 ml air was beforehand collected using a gas collector into a 100 ml vial bottle containing 2 ml distilled water, and 50 ml of air from each desiccator was injected using a glass syringe. This was left under a slightly reduced pressure for 20 min, and the FA concentration was determined by the AHMT method. The FA removal rate after 1 h for each adsorbent (0.5 g) was 50% or more for chitin, KIMCO and silica gel. The removal efficacy for activated carbon was higher for fine particles than for coarse particles, and a dose-response relationship was established.

Adsorption↗

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↗

PubMed2003

Correlation of antimutagenic activity and suppression of CYP1A with the lipophilicity of alkyl gallates and other phenolic compounds.

Alkyl gallates are widely used as food antioxidants. Methyl, ethyl, propyl, lauryl, and cetyl gallates showed antimutagenicity to activated 2-aminoanthracene (2AA)-induced SOS responses in Salmonella typhimurium TA1535/pSK1002. They also exhibited a suppressive effect on 3-methylcholanthrene (3-MC)-induced cytochrome P450 1A (CYP1A) in human hepatoma HepG2 cells, as indexed by the 7-ethoxyresorufin-O-deethylase (EROD) activity, and on CYP1A protein level. Both antimutagenicity and suppression of CYP1A appeared to be dependent on alkyl chain lengths, which suggested lipophilicity dependence. Based on those results, we investigated 26 other phenolic compounds for their lipophilicity, antimutagenicity and inhibition of EROD activity. The lipophilicity correlated well with the inhibition of EROD activity (r=0.78), and the inhibition of EROD activity correlated with the antimutagenicity of those compounds (r=0.71). The results suggest that the lipophilicity of the phenolic compounds may be an important factor in their ability to inhibit EROD activity.

Anthracenes↗

PubMed2003

Pivotal role of electrophilicity in glutathione S-transferase induction by tert-butylhydroquinone.

Although the induction of glutathione S-transferase (GST) activity by tert-butylhydroquinone (tBHQ) has been well-documented in several cell culture systems and rodent experiments, the exact mechanism responsible for its inducibility is still not thoroughly understood. To more precisely define the molecular mechanism of GST induction by tBHQ, we examined the one-electron oxidation and glutathione (GSH) reaction potentials of tBHQ as compared to its analogue, 2,5-di-tert-butylhydroquinone (DtBHQ). tBHQ and DtBHQ showed similar one-electron oxidation potentials, including free radical quenching (antioxidant), oxidative conversion of both compounds to a benzoquinone form, and Cu(2+)-dependent superoxide generation. On the other hand, the reduced GSH level was observed by the addition of tBHQ, but not DtBHQ, suggesting that tBHQ acts as an electrophile while DtBHQ does not. The data were consistent with the observation that tBHQ more potently induced the GSTP1 gene expression in RL34 cells than DtBHQ did. Moreover, we indeed detected the GSH-tBHQ conjugates in the cells exposed to tBHQ using an electrochemical detector-high-performance liquid chromatography technique. Thus, we conclude that an electrophilic quinone oxidation product that reacts with intracellular nucleophiles including protein thiol or GSH plays a major role in the GSTP1 gene expression.

Cell Line↗

PubMed2003

Color appearance in the entire visual field: color zone map based on the unique hue component.

To provide the fundamental data for a color zone map, the color appearances of nearly unique hue stimuli presented over the entire visual field were qualitatively and quantitatively evaluated by hue and saturation judgments, blackness evaluation, and categorical color naming. The hue of red and green stimuli shifts toward a unique yellow, while that of yellow and blue does not change with an increase in eccentricity. The saturation of all the stimuli falls with an increase in the eccentricity in all directions. On the basis of the unique hue component, color zone maps for red, dark yellow, yellow, green, and blue stimuli are drawn. All the color zone maps extend over a wider region in the temporal and lower directions than in the nasal and upper directions of the visual field. The results are compared with the color zones of previous studies. The relationship between the color zones and the color categorization, as well as the underlying mechanisms of reduced saturation and hue shift, is discussed.

Adult↗

PubMed2002

15-Deoxy-Delta(12,14)-prostaglandin J(2): the endogenous electrophile that induces neuronal apoptosis.

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J(2)-series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). The observation that the level of 15d-PGJ(2) increased in the tissue cells from patients with sporadic amyotrophic lateral sclerosis suggested that the formation of 15d-PGJ(2) may be closely associated with neuronal cell death during chronic inflammatory processes. In vitro experiments using SH-SY5Y human neuroblastoma cells revealed that 15d-PGJ(2) induced apoptotic cell death. An oligonucleotide microarray analysis demonstrated that, in addition to the heat shock-responsive and redox-responsive genes, the p53-responsive genes, such as gadd45, cyclin G1, and cathepsin D, were significantly up-regulated in the cells treated with 15d-PGJ(2). Indeed, the 15d-PGJ(2) induced accumulation and phosphorylation of p53, which was accompanied by a preferential redistribution of the p53 protein in the nuclei of the cells and by a time-dependent increase in p53 DNA binding activity, suggesting that p53 accumulated in response to the treatment with 15d-PGJ(2) was functional. The 15d-PGJ(2)-induced accumulation of p53 resulted in the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand.

Adult↗

PubMed2002

Role of p38 mitogen-activated protein kinase in the 4-hydroxy-2-nonenal-induced cyclooxygenase-2 expression.

COX-2 is rapidly expressed by various stimuli and plays a key role in conversion of free arachidonic acid to prostaglandins (PGs). 4-Hydroxy-2-nonenal (HNE), one of the lipid peroxidation end-products, has been recently identified as a potent COX-2 inducer in rat epithelial cell RL34 cells (Kumagai et al. (2000) Biochem. Biophys. Res. Commun. 273, 437-441). Here we investigated the molecular mechanism underlying the COX-2 induction by HNE mainly focusing on the activation of p38 mitogen-activated protein kinase (MAPK) pathways. The observations that (i) HNE induced phosphorylation of p38 MAPK and MKK3/MKK6 within 5 min and that (ii) SB203580, a p38 MAPK-specific inhibitor, suppressed the HNE-induced COX-2 expression suggested that the p38 MAPK pathway was involved in the HNE-induced COX-2 expression. Overexpression of p38 MAPK enhanced the HNE-induced COX-2 expression, whereas the overexpression of dominant negative p38 MAPK suppressed it. Furthermore, we also found that HNE upregulated the COX-2 expression by the stabilization of COX-2 mRNA via the p38 MAPK pathway.

Aldehydes↗

PubMed2002

Expression of a synthetic gene encoding the Asp-hemolysin from Aspergillus fumigatus in Escherichia coli.

Asp-hemolysin is a hemolytic toxin from Aspergillus fumigatus and is a specific binding protein with high affinity for oxidized low density lipoprotein (Ox-LDL). As a first step in clarifying the structure-function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli (E. coli) as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography on an amylose resin. The apparent molecular size of the protein produced by E. coli was approximately 57 kDa, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This is consistent with the predicted molecular size of 56.9 kDa for a fusion protein which includes 14.2 kDa of Asp-hemolysin and 42.7 kDa from MBP. The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody as revealed by western blot analysis. Furthermore, in dot blot analysis, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin.

ATP-Binding Cassette Transporters↗

PubMed2002

A novel oxidized low-density lipoprotein-binding protein, Asp-hemolysin, recognizes lysophosphatidylcholine.

Oxidized low-density lipoprotein (Ox-LDL) plays an important role in the initiation and progression of atherosclerosis. Asp-hemolysin, a hemolytic toxin from Aspergillus fumigatus, is a specific, high affinity binding protein for Ox-LDL. We have previously shown that Ox-LDL strongly inhibits the hemolytic activity of Asp-hemolysin, and that the removal of lysophosphatidylcholine (lysoPC) from Ox-LDL abolished the inhibition. In the present study, to clarify the binding mechanism of Asp-hemolysin to Ox-LDL, we investigated the interaction between Asp-hemolysin and lysoPC as a typical lipid moiety of Ox-LDL. Based on western blot analysis, the binding of Asp-hemolysin to LDL, oxidized for different times, depended on the lysoPC content in each Ox-LDL. In addition, the inhibition of lysoPC production in Ox-LDL by phenylmethylsulfonyl fluoride (PMSF) pretreatment of LDL resulted in a marked decrease of Asp-hemolysin binding to PMSF-pretreated Ox-LDL. Furthermore, the binding analysis of Asp-hemolysin to lysoPC using ion-exchange chromatography revealed that Asp-hemolysin directly binds to lysoPC.

Blotting, Western↗