Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H 2 O 2 . The apoxin I-induced apoptosis was inhibited by catalase, a H 2 O 2 scavenger. These results indicate that the H 2 O 2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom.Apoptosis is a physiological process by which cells undergo controlled cell death, accompanied by nuclear condensation and fragmentation prior to loss of membrane integrity (1-3). Many kinds of stimuli have been reported to induce apoptosis in a variety of cell systems. These include ligation of Fas and tumor necrosis factor receptors by their ligands (4 -8), deprivation of growth factors (9), and cytotoxic stimuli such as anticancer drugs (10 -12) and ionized radiation (13). In addition to these well characterized stimuli, hemorrhagic snake venoms from the western diamondback rattlesnake (Crotalus atrox), mamushi (Agkistrodon halys blomhottii), puff adder (Bitis arietans), and habu (Trimeresurus flavoviridis) induce apoptosis in vascular endothelial cells (14), although the active component in the venom is not identified. We report here the identification of the apoptosis-inducing factor apoxin I from venom of the western diamondback rattlesnake that has Lamino acid oxidase (LAO, 1 EC 1.4.3.2) activity. EXPERIMENTAL PROCEDURESMaterials-Lyophilized crude venom from the western diamondback rattlesnake was purchased from Sigma. Benzyloxycarbonyl-Val-AlaAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-VAD) and benzyloxycarbonylAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-Asp) were synthesized at Kirin Brewery Co. Ltd (Gunma, Japan) as described (15) and dissolved in Me 2 SO at 10 mg/ml. Trolox (Aldrich) was dissolved in 1 M NaHCO 3 at a concentration of 300 mM, and the pH was adjusted to 7.0. For the experiments, the solution was diluted to the desired concentration with medium. Catalase and all other chemicals were purchased from Wako Pure Chemicals (Tokyo, Japan) and Sigma.Purification of Apoxin I-Lyophilized crude venom (100 mg) from the western diamondback rattlesnake (Sigma) was dissolved in 5 ml of distilled water, and the insoluble material was removed by centrifugation (1,000 ϫ g) for 10 min at 4°C. The supernatant was applied to Sephadex G-100 column (Pharmacia Biotech Inc.; 3 ϫ 60 cm) equilibrated with 50 mM sodium phosphate buffer containing 0.15 M sodium chloride (pH 7.0), and the proteins were eluted ...
We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B, and mouse interleukin 4-induced F1G1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I. In addition, deleting the amino-terminal region flanking the signal sequence, the FAD-binding domain and the carboxy-terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active, these regions and posttranslational modification, such as N-glycosylation, are required.
Aflatoxin produced by Aspergillus flavus is known to be strongly related to liver injury (hepatocellular carcinoma) and immune system damage involving leukocytes. This toxin suppresses both the cell-mediated immune system and macrophage function, and decreases the production of complement and interferon molecules. Purpose: To evaluate the presence of aflatoxin in infectious lesions as well as how the toxin is taken up by leukocytes. Method: Pathological specimens from a patient who died from aspergillosis caused by aflatoxin-producing A. flavus were used. Anti-aflatoxin B1 antibody was reacted with paraffin-embedded lesion specimens from the heart, kidney, and thyroid gland of the patient and observed microscopically. Result: Positive reactions were detected in fungal elements and leukocytes (neutrophils and macrophages) in inflammatory lesions. Conclusion: Within the patient' s body, A. flavus likely produced aflatoxin, which then was taken up by neutrophils and macrophages. These results suggest that leukocyte function and the immune mechanism are locally suppressed by aflatoxin.
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