Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Effects of ascorbic acid and uracil on exo-polysaccharide production with Hericium erinaceus in liquid culture

Hericium erinaceus is a well known edible and medicinal mushroom used in East-Asia. Recently, H. erinaceus has attracted a lot of attention owing to its antitumor, immuno-modulatory, and cytotoxic effect. It has been postulated that the fruiting body of H. erinaceus contains a polysaccharide that is similar to β-D-glucan, which is known to have antitumor activity against Sarcoma 180. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, the aim of this study was to determine the optimal liquid culture conditions for maximum polysaccharide production. In shake flask cultures, the optimal concentration of ascorbic acid was found to be 2.0 g/L, which prevented the broth from changing color from yellow to black. The optimal culture conditions were determined to be 23°C, 200 rpm, and a 10% inoculum size, at an uncontrolled initial pH. In addition, the modified medium contained 20 g/L glucose, 10 g/L yeast extract, and 2.0 g/L ascorbic acid. The maximum mycelial biomass and exo-polysaccharide (EPS) production in the modified medium containing uracil was 13.43 and 1.26 g/L, respectively.     

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.     

Sequence analysis of genomic DNA (680 Mb) by GS-FLX-Titanium sequencer in the monogonont rotifer, Brachionus ibericus

The monogonont rotifer, Brachionus ibericus (S type), is considered to be a promising model species for developmental biology, evolution, and environmental genomics. In an attempt to accelerate the molecular understanding of B. ibericus, we sequenced 680.5 Mb of genomic DNA using the genome sequencer GS-FLX-Titanium. We obtained 2,062,621 reads (average read length 329.9 bp) and 145,418 contigs (total contigs length 125.7 Mb) after excluding small reads (less than 200 bp) from the assembly, and finally obtained 10,133 unigenes (E value ?? 9.00E?04) after non-redundant (NR) BLAST search. In this article, we summarize the genomic DNA sequences of B. ibericus and discuss their potential use in the study of reproductive biology, endocrinology, environmental genomics, and ecotoxicological studies, and for providing insight into the genetic basis of mechanisms such as egg formation, antioxidant stress defense, and xenobiotic metabolism.     

Occurrence and molecular identification of the zoysiagrass mite Aceria zoysiae (Acari: Eriophyidae) in turfgrasses in Korea

Mites are one of the serious pests of turfgrass. Our survey of turfgrass fields from 2013 to 2015 in Korea showed that the occurrence of leaf chlorotic symptom has gradually extended to at least 60% of the examined golf courses. We identified the zoysiae mite Aceria zoysiae in most damaged zoysiagrasses. Artificial infestation of A. zoysiae into zoysiagrasses in pots resulted in symptoms of chlorosis and marginal rolling of the leaves within 3 weeks. We firstly determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA region of A. zoysiae. The variations in COI and ITS2 between A. zoysiae and other species of the genus were 20.9%–43.0% and 7.5%–67.3%, respectively, suggesting significant genetic divergence within the genus. Our study provides valuable information for the rapid diagnosis and infestation monitoring of A. zoysiae in turfgrass fields.