In this study, to investigate the secondary function of Rpl10a in zebrafish development, morpholino antisense oligonucleotides (MOs) were used to knock down the zebrafish ribosomal protein L10a (rpl10a). At 25 hpf (hours post-fertilization), embryos injected with the rpl10a MO showed an abnormal morphology, including short bodies, curved tails, and small yolk sac extensions. We observed pigment reductions, edema, larger yolk sacs, smaller eyes and smaller yolk sac extensions at 50 hpf. In addition, reductions in the expression of primordial germ cell (PGC) marker genes (nanos1 and vasa) were observed in rpl10a knockdown embryos. A rescue experiment using a rpl10a mRNA co-injection showed the recovery of the morphology and red blood cell production similar to wild-type. Moreover, the CRISPR-Cas9 system was used to edit the sequence of rpl10a exon 5, resulting in a homozygous 5-bp deletion in the zebrafish genome. The mutant embryos displayed a morphology similar to that of the knockdown animals. Furthermore, the loss of rpl10a function led to reduced expression of gata1, hbae3, and hbbe1 (erythroid synthesis) and increased tp53 expression. Overall, the results suggested that Rpl10a deficiency caused delays in embryonic development, as well as apoptosis and anemia, in zebrafish.

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During studies on the oxidative cyanation reaction catalyzed by a variant of D-amino acid oxidase from porcine kidney (pkDAO) (Y228L/R283G), an unexpected formation of 1-phenyl-N-(1-phenylethylidene)ethanamine (PPEA) was detected. The optimal reaction conditions for the synthesis of PPEA and the reaction mechanism were investigated using the pkDAO variant. The highest PPEA synthesis was obtained in the reaction with 150 mM (R)-MBA at pH 9.0 and at 20 °C. Since PPEA synthesis proceeded by trapping the intermediate 1-phenylethanimine (1-PEI) by 15N-labeled n-hexylamine, which is not a substrate for the pKDAO variant, it was deduced that PPEA would be synthesized by a nucleophilic substitution of 1-PEI by another molecule of (R)-MBA. PPEA was further identified by its conversion to bis(1-phenylethyl)amine (BPEA) through reduction with NaBH4. Thus, a new enzymatic method of imine synthesis by oxidation of primary amine by the variant pkDAO was achieved for the first time.

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