Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos.

The teratogenicity of two fungal ribosome-inactivating proteins, hypsin from Hypsizigus mamoreus and velutin from Flammulina velutipes, was examined in this investigation using microinjection and postimplantation whole-embryo culture. The results demonstrated that hypsin induced abnormal embryonic development at 2.5 microM during the organogenesis period from E8.5 to E9.5. As its dosage increased, there was an increase in the total number of abnormal embryos, a drop in the final somite number, and a rise of abnormal structures. Structural abnormalities were detected: open cranial neural tube, abnormal branchial arches, absence of forelimb buds and twisted body axis. The otic and optic placodes were, however, less affected. Histological study of the abnormal embryos revealed a correlation of increased cell death with abnormal structures, suggesting that induction of cell death by hypsin may account for its teratogenicity. In contrast, velutin did not exert any adverse influence on mouse development.

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro.

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 microg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 microg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.

A napin-like polypeptide with translation-inhibitory, trypsin-inhibitory, antiproliferative and antibacterial activities from kale seeds.

A heterodimeric napin-like polypeptide with translation-inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion-exchange chromatography on dielthylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. The napin-like polypeptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and Mono S. Its 7-kDa large subunit differs in N-terminal amino acid sequence from the 4-kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC50 of 37.5 nM. This activity was preserved between pH 5 and pH 11, and between 10 and 40 degrees C. It fell to a low level at pH 3 and pH 13 and at 70 degrees C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.

A napin-like polypeptide from dwarf Chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities.

Napins are 1:1 disulfide-linked complexes of a smaller (ca. 4kDa) subunit and a larger (ca. 10kDa) subunit. The intent of the present study was to ascertain the production of napin by the seeds of a Brassica species that has not been examined previously, and also to explore new biological activities of the napin. A heterodimeric 11-kDa napin-like polypeptide has been isolated from Chinese white cabbage (Brassica chinensis cv dwarf) seeds with a protocol comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The N-terminal sequence of the 7-kDa subunit manifests striking similarity to napin large chain, albumin and trypsin inhibitor. The N-terminal sequence of the 4-kDa subunit is homologous to napin large chain and an antimicrobial peptide. The napin-like polypeptide inhibited translation in the rabbit reticulocyte system with an IC50 of 18.5nM. This translation-inhibitory activity was stable between pH 4 and 11, and between 10 and 40 degrees C. The polypeptide inhibited trypsin with a higher potency ( IC50 = 8.5 microM) than it inhibited chymotrypsin (IC50 = 220 microM), but was devoid of ribonuclease and antifungal activities. It manifested antibacterial activity against Pseudomonas aeruginosia, Bacillus subtilis, Bacillus cereus, and Bacillus megaterium. The results revealed that the napin-like polypeptide from Chinese white cabbage seeds exhibited some potentially exploitable activities.