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.

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.

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.

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.

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.

[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.

[Relation between oxygen uptake rate and biosorption of activated sludge against chemical substance].

In this study, the elucidation of the toxicity mechanism was undertaken regarding the IC(50) of the oxygen uptake rate (OUR) with relevance to the biosorption as a toxicity evaluation of chemical substances for activated sludge (AS). At the IC(50) of<100 mg/l, malachite green (MG) and crystal violet (CV) were confirmed in the group showing relatively strong OUR inhibition. These dyes were markedly biosorbed by AS in a short time. The biosorption for AS showed a weak tendency in linear alkyl benzene sulfonate (LAS), alkyl ethoxy sulfonate (AES), alpha-olefine sulfonate (AOS), sodium dodecyl sulfate (SDS), formaldehyde (FA), benzalkonium chloride (BZaC), benzethonium chloride (BZeC), rhodamine 6G (R-6G) and fuchsine (Fuc) in which the IC(50) belonged to the 100-1000 mg/l group, when it was compared with CV and MG. In ethanol (EtOH), isopropanol (PrOH), nile blue (NB), evans blue (EB), methylene blue (MB), methyl orange (MO), paraquat (PQ), chlorophyllin (Chl) and auramine (Aur), the IC(50) was large, and the biosorption of AS was weak at 0-15%. The biosorption of MG for AS followed the adsorption isotherm equation Y=0.002X(0.511) of Freundrich. The correlation coefficient was gamma=0.998 (n=8), and a very high correlation was obtained. In the qualitative OUR curve by AS pretreated with MG or CV which belonged to the IC(50) small group, the inhibition of remarkable OUR was observed. Therefore, the findings of the present investigation suggest that the inhibition of the OUR for AS by the tested chemical substances was markedly affected by the biosorption.

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.

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.