Influence of light on the morphological changes that take place during the development of the <Emphasis Type="Italic">Flammulina velutipes</Emphasis> fruit body

We show that fruit bodies of Flammulina velutipes can be induced in complete darkness after a sharp temperature reduction (23° to 16°C). However, the fruit bodies that form in complete darkness have a long stipe with an undeveloped pileus on the top (pinhead fruit bodies) and are thinner and whiter than the normal fruit bodies which are formed in the light. This finding suggests that F. velutipes fruit bodies cannot mature in complete darkness. However, when we irradiated the fruit bodies that had formed in complete darkness, a pileus developed immediately, and 4 days later the separation between the stipe and the pileus could be observed. Immediately after light exposure, the stipe also thickened and became increasingly pigmented. The stipe elongation was inhibited until 8 days after light exposure, although stipe elongation progressed very quickly thereafter. Basidospores were also visible in the gills 8 days after light exposure. We consider that the basidiospore development is involved in this rapid stipe elongation, which aids the effective dispersal of basidiospores.     

Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).     

Novel relationship between the antifungal activity and cytotoxicity of marine-derived metabolite xestoquinone and its family

Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.     

Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of ?-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

Background

Bioluminescence in fireflies and click beetles is produced by a luciferase-luciferin reaction. The luminescence property and protein structure of firefly luciferase have been investigated, and its cDNA has been used for various assay systems. The chemical structure of firefly luciferin was identified as the ᴅ-form in 1963 and studies on the biosynthesis of firefly luciferin began early in the 1970’s. Incorporation experiments using ¹⁴C-labeled compounds were performed, and cysteine and benzoquinone/hydroquinone were proposed to be biosynthetic component for firefly luciferin. However, there have been no clear conclusions regarding the biosynthetic components of firefly luciferin over 30 years.

Methodology/Principal Findings

Incorporation studies were performed by injecting stable isotope-labeled compounds, including ʟ-[U-¹³C₃]-cysteine, ʟ-[1-¹³C]-cysteine, ʟ-[3-¹³C]-cysteine, 1,4-[D₆]-hydroquinone, and p-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly Luciola lateralis. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was identified by LC/ESI-TOF-MS. The positions of the stable isotope atoms in firefly luciferin were determined by the mass fragmentation of firefly luciferin.

Conclusions

We demonstrated for the first time that ᴅ- and ʟ-firefly luciferins are biosynthesized in the lantern of the adult firefly from two ʟ-cysteine molecules with p-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of ʟ-cysteine.     

Phosphodiesterase inhibitors. Part 6: Design,synthesis, and structure–activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity

We previously identified KCA-1490 [(?)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.     

Rodent alpha-chymases are elastase-like proteases.

Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.     

Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization

The human thyroxine-binding globulin (TBG) gene has been localized to X chromosome (Xq22.2) by in situ hybridization using a biotinylated gDNA probe. This is consistent with previous mapping of the TBG gene to chromosome Xq21-q22.