Degradation of antifungal anthraquinone compounds is a probable physiological role of DyP secreted by Bjerkandera adusta.

Alizarin is an anti-fungal compound produced by the plant, Rubia tinctorum. The parasitic fungus Bjerkandera adusta Dec 1 was cultured in potato dextrose (PD) medium with or without alizarin. Alizarin was a good substrate for the dye-decolorizing peroxidase (DyP) from B. adusta Dec 1 and hampered B. adusta growth at the early stage of plate culture. During liquid shaking culture, DyP activity in cultures supplemented with 100 µM alizarin was greater than that in controls cultured without alizarin. In particular, DyP activity per dry cell mass increased approximately 3.5-, 3.1-, and 2.9-fold at 24, 30, and 36 h after inoculation, respectively, compared with control cultures. These data suggest that alizarin stimulates the expression of DyP. Interestingly, alizarin rapidly decomposed at an early stage in culture (24-42 h) in PD medium supplemented with 100 µM alizarin. Thus, alizarin appears to induce DyP expression in B. adusta Dec 1, and this DyP, in turn, rapidly degrades alizarin. Collectively, our findings suggest that the physiological role of DyP is to degrade antifungal compounds produced by plants.

Genetic Code Expansion of the Silkworm Bombyx mori to Functionalize Silk Fiber.

The genetic code in bacteria and animal cells has been expanded to incorporate novel amino acids into proteins. Recent efforts have enabled genetic code expansion in nematodes, flies, and mice, whereas such engineering is rare with industrially useful animals. In the present study, we engineered the silkworm Bombyx mori to synthesize silk fiber functionalized with azidophenylalanine. For this purpose, we developed a bacterial system to screen for B. mori phenylalanyl-tRNA synthetases with altered amino-acid specificity. We created four transgenic B. mori lines expressing the selected synthetase variants in silk glands, and found that two of them supported the efficient in vivo incorporation of azidophenylalanine into silk fiber. The obtained silk was bio-orthogonally reactive with fluorescent molecules. The results showed that genetic code expansion in an industrial animal can be facilitated by prior bacterial selection, to accelerate the development of silk fiber with novel properties.

Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes.

The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.

Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

[Clinical effects of Lactobacillus acidophilus strain L-55-contained yogurt on symptoms of Japanese cedar pollen allergy].

BACKGROUND: It has been reported that oral administration of Lactobacillus acidophilus strain L-55 (L-55) suppressed nasal symptom and antigen-specific IgE induced by antigen challenge in mice. We investigated clinical effects of L-55-contained yogurt on symptoms and IgE production in the patients with Japanese cedar pollinosis. METHOD: The study was performed as a randomized double blind placebo-controlled trial. L-55-contained yogurt (L-55 yogurt) or placebo yogurt was started to administer to each subject from 4 weeks prior to the onset of Japanese cedar pollen release for total 13 weeks. RESULT: The mean value of symptom score and symptom medication score were lower in L-55 yogurt group compared with placebo yogurt group during 5th week to 9th week from the first week of Japanese cedar pollination. Especially, in medicated subjects, total symptom score and throat symptom score were significantly lower in L-55 yogurt group compared with placebo yogurt group at 5th and 4th week from the first week of Japanese cedar pollination, respectively. Moreover, in medicated subjects, change ratio of serum total IgE was significantly lower in L-55 yogurt group compared with placebo yogurt group at 1st week from the first week of Japanese cedar pollination. CONCLUSION: Intake of L-55 during Japanese cedar pollinating season may be effective in alleviating the allergic symptoms related to Japanese cedar pollinosis. From these finding, it was suggested that L-55 is a possible candidate as a complementary medicine for Japanese cedar pollinosis.