Melamine ELISA Kit

1-phenyl 2-thiourea (PTU) is a Tyr (tyrosinase) inhibitor that is extensively used to block pigmentation and improve optical transparency in zebrafish (Danio rerio) embryo. Here, we reported a previously undescribed effect of PTU on macroautophagy/autophagy in zebrafish embryos. Upon 0.003% PTU treatment, aberrant autophagosome and autolysosome formation, accumulation of lysosomes, and elevated autophagic flux were observed in various tissues and organs of zebrafish embryos, such as skin, brain, and muscle. Similar to PTU treatment, autophagic activation and lysosomal accumulation were also observed in the somatic tyr mutant zebrafish embryos, which suggest that Tyr inhibition may contribute to PTU-induced autophagic activation. Furthermore, we demonstrated that autophagy contributes to pigmentation inhibition, but is not essential to the PTU-induced pigmentation inhibition. With the involvement of autophagy in a wide range of physiological and pathological processes and the routine use of PTU in zebrafish research of autophagy-related processes, these observations raise a novel concern in autophagy-related studies using PTU-treated zebrafish embryos.

In order to understand the regulation of a gene, it is important to understand the mechanism of DNA-protein interaction at the molecular level. Recognition of zinc finger domains is one approach to discern the target sites of DNA protein interactions. Utilizing this information, zinc finger proteins (ZFPs), most common DNA binding proteins in mammals, can be synthetically designed and engineered for the modulation of endogenous genes expression and for targeted genome engineering. Here, we discovered zinc finger target sites in an important regulatory DNA sequence tyrosinase 5' upstream regulatory sequence (TYR 5'URS) using online bioinformatics tool 'Zinc Finger Tools' (ZF Tools). TYR 5'URS acts as a transcriptional cis regulatory/locus control region (LCR) of a multifactorial tyrosinase (TYR) gene. A total of 79 top scoring zinc finger target sites could be detected in TYR 5'URS. Top score of the identified target sites ranged from 40.87 to 61.01. Further, we generated protein coding sequences for the top scoring target sites which contained canonical amino acid linker between the zinc finger modules. Amino acid sequences of designed ZFPs, when subjected to identity analysis, did not correspond significantly to any known human protein indicating the specificity of the zinc fingers for the target sites. Information and data generated in this work will be further helpful to design synthetic ZFPs and to assemble the zinc finger-DNA binding domain coding sequence to target TYR 5'URS. Designing ZFPs specific to TYR gene regulatory elements will allow us to explore and understand the mutation spectrum of this gene in a population. This will further pave the way to therapeutic interventions.