Yasushi Sugimoto

Galectin-9 Is a High Affinity IgE-binding Lectin with Anti-allergic Effect by Blocking IgE-Antigen Complex Formation

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.

Egg-yolk trypsin inhibitor identical to albumen ovomucoid

Abstract Two preparations of trypsin inhibitor were purified from yolk, one from unincubated hens eggs and the other from those at day 16 of incubation. Both were indistinguishable from albumen ovomucoid with respect to molecular weight, isoelectric point, peptide map after limited proteolysis, immunological reactivity and so forth. Although a gap in specific activity was observed, it was concluded that the yolk inhibitors were species of ovomucoid. This suggested a flow of ovomucoid from egg white to yolk during embryonic development.

Flow of egg white ovalbumin into the yolk sac during embryogenesis

Western blot analyses of yolk proteins of the White Leghorn hens showed that an ovalbumin-like molecule was included in day 16 eggs but not in the ovarian follicles, and very little in newly deposited eggs. Northern hybridization, as well as in vitro translation, of poly(A)+ RNAs prepared from the yolk sac membranes of developing embryos gave no signal for ovalbumin messages. These results imply that the present ovalbumin-like protein of yolk has its origin in egg white, not being a de novo synthesis product in the yolk sac membranes.

A proteinase inhibitor from egg yolk of hen is an ovoinhibitor analog

A proteinase inhibitor, tentatively termed vitelloinhibitor, was purified from yolk of hens ovarian follicles. It resembled egg-white ovoinhibitor not only in inhibitory spectrum (active for bovine trypsin and bovine chymotrypsin) but also in thermal stability, pH stability, antiserum reactivity and amino-acid composition. However, vitelloinhibitor had different molecular weight from that of ovoinhibitor. An alpha 2-proteinase inhibitor preparation, isolated from laying hens serum in the present study, was found to exhibit two bands, and the larger one of the latter corresponded to vitelloinhibitor in molecular weight. The partial N-terminal amino-acid sequence of vitelloinhibitor was the same as those of the two components of serum inhibitor and all three agreed with that of ovoinhibitor. Vitelloinhibitor is likely to be an ovoinhibitor analog derived from a serum precursor, which might be the larger component of alpha 2-proteinase inhibitor.

Isolation and characterization of cDNA and genomic promoter region for a heat shock protein 30 from Aspergillus nidulans

A cDNA encoding for a heat shock protein 30 (HSP30) of Aspergillus nidulans and the promoter region of its gene were analyzed for their primary structures. The promoter region had no heat shock element but possessed three inverted repeat sequences. Northern blot hybridization indicated that the expression of the HSP30 gene was high at a normal temperature and was slightly accelerated at elevated temperatures in A. nidulans cells. Although the deduced amino acid sequence of the A. nidulans HSP30 had a domain highly conserved among other small HSPs from different species, it showed a sequence homology of only 42% even in comparison with the most closely related molecular species, Neurospora crassa HSP30. These findings suggest that the present HSP30 belongs to a novel subfamily of low-molecular-weight HSPs.

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8

Background: TTHA0420 is a flavin reductase, which makes free reduced flavin involved in a variety of fields. Results: We determined the dual binding mode of the substrate and co-factor flavins of TTHA0420. Conclusion: A specific motif YGG in the C terminus functions to regulate the alternative binding of NADH and substrate flavin. Significance: Our results have mechanistic implications for the reductase with two flavins. Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20–40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.

Occurrence and distribution of aldolase isozymes during embryonic and post-embryonic development of the silkworm, Bombyx mori

Abstract Fructose 1,6-bisphosphate aldolase (EC4.1.2.13) occurred as two distinct molecular varieties in Bombyx mori in zymogram analysis: a slowly migrating (S) band observed in the ovary and in yolk of the eggs during embryonic development until the stage of head pigmentation and a fast migrating (F) band appearing after this stage in the embryo. Organs during post-embryonic development exhibited three additional bands between the S and F bands on zymograms. The composition of the five bands was organ-specific, with S predominating in the fat bodies and F in the muscles. The partially purified S and F preparation differed in the activity ratio towards the two substrates fructose 1,6-bisphosphate/fructose 1-phosphate, the ratios being 3 for S and 10 for F. In vitro mixing of the S and F preparations produced intermediary migrating materials, indicating that the occurrence of five components in the organs may be due to the formation of heterotetramers of the S and F subunits.

Presence of translatable mRNA in diapause eggs of Bombyx mori

Abstract Total RNA extracted from diapause eggs of Bombyx mori was found to be translatable in a cell-free heterologous protein synthesis system derived from wheat germ or rabbit reticulocyte lysate. This supports the idea that active mRNAs are contained in the polysomes of diapause eggs.

Comparison of egg and embryo proteins and a trial to detect proteolytic activities in eggs of Bombyx mori

Abstract 1. 1. One- or two-dimensional gel electrophoresis of proteins in the Bombyx eggs at various stages revealed spots corresponding to vitellin heavy chain (VH), vitellin light chain (VL), egg specific proteins (ESPs) and 30 kDa proteins. 2. 2. Isolated embryos showed many other minor components and abundant proteins at 30 kDa. 3. 3. Incubation of crude egg extracts at pH 3 and 4 at 37°C caused a rapid decrease of electrophoresis spots corresponding to VH, VL and ESP. 4. 4. At pH 5, a preferential decrease of ESP spots was seen. 5. 5. Under all acid, neutral and alkaline conditions tested proteins remained intact.