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    • We observed PACAP immunoreactivity in the molecular layer of

    2024-03-13

    We observed PACAP2 immunoreactivity in the molecular layer of the cerebellar cortex in zebrafish. This result is consistent with the reporting of PACAP immunoreactivity in the soma and fibers of Purkinje cells and the presence of PACAP mRNA in the Purkinje-cell and granular-cell layers in rats [43]. PACAP immunoreactivity has also been found in the teleost (stargazer) cerebellum [21]. PAC1R mRNA has been detected in the cerebellum of rodents [44]. In humans, the cerebellum contributes to motor, emotional, and cognitive associative learning [45]. Cerebellar lesions impair motor function in rodents but not in teleosts [46]. Moreover, cerebellar lesions and cooling impair classical fear-conditioned learning in goldfish [47,48]. These reports support the hypothesis that PACAP2 is associated with learning in teleosts. The distribution of PACAP immunoreactivity in the zebrafish Imiquimod was reported previously [22]. In that study, PACAP immunoreactivity was observed mainly in the hypothalamus, pituitary gland, and rhombencephalon, with less in the telencephalon and cerebellum [22]. The contradiction between this observation and our results may result from the specificity of the PACAP antibody used for immunostaining. PACAP is known as a hypophysiotropic factor in rodents, frogs, and teleosts [25,49,50]. PACAP2 immunoreactivity was not observed in the zebrafish pituitary in our study, suggesting that the zebrafish pituitary may instead express PACAP1. Unfortunately, we did not obtain a PACAP1-specific antibody in this study. In future studies, we will try again to obtain such an antibody so that we can finally determine the distribution of PACAP1 and PACAP2 and the relationship between them in teleosts.
    Conflict of interest
    Acknowledgments This work was supported in part by a Toyama University Grant-in Aid for Scientific Research (T.N., K.M), a grant from Toyama First Bank (T.N.), and grants from the Japan Society for the Promotion of Science (KAKENHI Grant Numbers JP15H04394, JP15H04395, and JP16H02684).
    Introduction Enzymatic catalysis leading the third wave of biocatalysis is realized in parallel with the development of chemical syntheses, allowing biocatalysis to develop as an important tool in industrial synthesis [1]. Compared to many heterogeneous chemical catalysts, enzymes produce highly-selective reaction outcomes, in both structural and stereochemical terms, under relatively mild conditions [[2], [3], [4], [5]]. Enzymes in insoluble form are essentially a specialized form of heterogeneous catalyst in that they can be recovered and reused, often retain activity for long time periods and are amenable to a wide variety of process formats [[6], [7]]. With the development of protein-based biotechnology, a new trend in enzyme immobilization is to form affinity bonds between an affinity tag present on the protein and porous supports [[8], [9], [10]]. Generally, it is achieved according to the immobilized metal ion affinity chromatography (IMAC). IMAC is a technology that separate and purify proteins by means of the coordination interaction between the specific amino acids (histidine or cysteine) on the surface of the protein and metal ions immobilized on the matrix [[11], [12], [13]]. Due to the fact that metal chelate carriers have the feature of simple ligands and display a large amount of adsorption, it is applied in a variety of protein purifications and isolations, such as lectins [14], green fluorescent protein [15] and soybean protein [16]. In recent years, the immobilization method of affinity between histidine tags and metal ions has attracted more and more attention, which is based on the IMAC [[17], [18], [19], [20]]. This presents several advantages including: (1) the protection of the enzyme activity and conformation in the process of immobilization (2) the one-step purification and immobilization of the enzyme and (3) the possibility of re-using the supports and reloading enzyme. The immobilization by histidine tags has been successfully used for many enzymes e.g., for lipase [[21], [22]], O-acetylserine sulfhydrylase [23], aminopeptidase [[24], [25]], L-arabinitol 4-dehydrogenase [26], β-glucosidase [[27], [28]], cellulolytic enzyme [29] and lysozyme [30]. Thus, the successful application of affinity adsorption provides a new direction for developing a novel immobilization strategy of other enzymes.