Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori

We previously reported the purification of an ommin-binding protein (OMBP) from an acid-methanol extract of diapause eggs of the silkworm and that OMBP reacted with the anti-30K proteins antiserum. In order to clarify the relationship between OMBP and the 30K proteins, we attempted to determine the sequence of the N-terminal amino acid of OMBP, which was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We observed ten protein spots of various isoelectric points; the spots corresponded with 30 kDa. Based on the sequence of the N-terminal amino acid (20 residues), the spots belonged to two kinds of 30K proteins (6G1 and 19G1), which are known as the major plasma proteins in the larval hemolymph of the silkworm. The proteins are expected to attach to polysaccharide because they reacted with concanavalin A and elderberry bark lectin. Immunohistochemical observations clarified that the proteins were localized in yolk granules and serosa in the diapause egg. These results suggest that OMBP is composed of 30K proteins which were modified with polysaccharides. In addition, the expression of 30K proteins mRNA was observed at early embryonic stage in diapause eggs by RT-PCR analysis. The 30K proteins as OMBP may play an important role in the transport and accumulation of tryptophan metabolites and ommochrome during the formation of serosa.     

A simple two-transition model for loss of infectivity of phages on exposure to organic solvent

The effect of water-miscible organic solvents on the stability of filamentous phages displaying a random peptide library was investigated. A kinetic analysis of the time course of viral infectivity indicated that the transition between the noninfectious and infectious form was directly related to the concentration of organic solvent.     

DNA methylation errors in cloned mice disappear with advancement of aging

Cloned animals have various health problems. Aberrant DNA methylation is a possible cause of the problems. Restriction landmark genomic scanning (RLGS) that enabled us to analyze more than 1,000 CpG islands simultaneously demonstrated that all cloned newborns had aberrant DNA methylation. To study whether this aberration persists throughout the life of cloned individuals, we examined genome-wide DNA methylation status of newborn (19.5 dpc, n=2), adult (8-11 months old, n=3), and aged (23-27 months old, n=4) cloned mice using kidney cells as representatives. In the adult and aged groups, cloning was repeated using cumulus cells of the adult founder clone of each group as nucleus donor. Two newborn clones had three with aberrantly methylated loci, which is consistent with previous reports that all cloned newborns had DNA methylation aberrations. Interestingly, we could detect only one aberrantly methylated locus in two of the three adult clones in mid-age and none of four senescent clones, indicating that errors in DNA methylation disappear with advancement of animals' aging.     

Three antinematodal diterpenes from Euphorbia kansui

Three compounds, 20-O-acetyl-[3-O-(2'E,4'Z)-decadienoyl]-ingenol (1), 20-O-acetyl-[5-O-(2'E,4'Z)-decadienoyl]-ingenol (2) and 3-O-(2'E,4'Z)-decadienoylingenol (3), were isolated from Euphorbia kansui under the bioassay-guided method. Each compound showed the same antinematodal activity against the nematode, Bursaphelenchus xylophilus, at a minimum effective dose (MED) of 5 microg/cotton ball.     

Lessons from studies of Symbiobacterium thermophilum, a unique syntrophic bacterium

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a cognate Bacillus sp. We have been studying the unique features of S. thermophilum in terms of taxonomy, ecology, genome biology, and physiology. Here we overview current knowledge of this bacterium. Although S. thermophilum shows several physiological properties of Gram-negative bacteria, 16S rRNA gene-based phylogeny indicated that it represents a distinct lineage of Gram-positive bacteria with deep branching between the clades for the high-G+C (Actinobacteria) and the low-G+C (Firmicutes) groups. Ecological study has revealed that S. thermophilum and its relatives are widely distributed in the natural environment, including soil, animal intestines and seawater. A whole genome sequencing study uncovered its unusual features, which overall indicate that this bacterium is a member of Firmicutes despite of its high G+C content (68.7%). The genome appeared to retain fully the genes for primary metabolism, except for carbonic anhydrase. We discovered that carbon dioxide is a marked inducer of the mono-growth of S. thermophilum, and speculated that this is due to a lack of carbonic anhydrase. The lines of evidence suggest that S. thermophilum requires additional conditions for full growth, including not only the supply of an unknown positive factor but also the elimination of oxygen and self-growth inhibitory substances. We conclude that the role of the cognate Bacillus is to establish a complex environment suitable for the growth of S. thermophilum, which is achieved by supplying and removing multiple factors. Understandings of this type of mutualism should provide new insight into microbial physiology as well as the issue of uncultivability.     

Effects of salicylic acid on mushroom tyrosinase and B16 melanoma cells

Salicylic acid slightly inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase noncompetitively without being oxidized. In contrast, 4-hydroxybenzoic acid did not inhibit this enzymatic oxidation if a longer reaction time was observed, although it suppressed the initial rate of the oxidation to a certain extent. Neither acid showed noticeable effects on cultured murine B16-F10 melanoma cells except weak cytotoxicity.     

Enzymatic conversion-based method for screening cyclic dipeptide-producing microbes

We developed a method for screening cyclic dipeptide-producing microbes by enzymatic conversion. In this method, cyclic dipeptides are detected by the combination of: (i) conversion of cyclic dipeptides to dehydro cyclic dipeptides by cyclo(Leu-Phe) oxidase and (ii) detection of the dehydro derivative by UV spectrophotometry using TLC or HPLC analysis based on the absorbance change caused by the conversion. Using this method, the actinomycete strain A8 was isolated as a cyclic dipeptide-producing strain. The cyclic dipeptides were purified from the microbial extract by enzymatic detection-guided fractionation, and their structures were determined to be cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) by spectroscopic methods.