Purification, crystallization and X-ray analysis of Pf-SCP (sarcoplasmic Ca-binding protein), related to storage and transport of calcium in mantle of Pinctada fucata.

Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and ß = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.

cDNA Cloning and Functional Analyses of Ashitaba (Angelica keiskei) Sesquiterpene Synthase Genes.

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus.

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors.

Inhibitory Activities of Alkyl Syringates and Related Compounds on Aflatoxin Production.

Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin production of Aspergillus flavus became stronger as the length of the alkyl chains on the esters became longer. Pentyl, hexyl, heptyl, and octyl syringates showed strong activity at 0.05 mM. Heptyl and octyl parabens, and octyl gallate also inhibited aflatoxin production as strongly as octyl syringate. Alkyl parabens and alkyl gallates inhibit the complex II activity of the mitochondrial respiration chain; thus, whether alkyl syringates inhibit complex II activity was examined. Inhibitory activities of alkyl syringates toward complex II also became stronger as the length of the alkyl chains increased. The complex II inhibitory activity of octyl syringate was comparable to that of octyl paraben and octyl gallate. These results suggest that alkyl syringates, alkyl parabens, and alkyl gallates, including commonly used food additives, are useful for aflatoxin control.

Search for aflatoxin and trichothecene production inhibitors and analysis of their modes of action.

Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action. In this article, we review aflatoxin and trichothecene production inhibitors, including our works on blasticidin S, methyl syringate, cyclo(L-Ala-L-Pro), respiration inhibitors, and precocene II.

Identification and characterization of a matrix protein (PPP-10) in the periostracum of the pearl oyster, Pinctada fucata.

The periostracum is a layered structure that is formed as a mollusk shell grows. The shell is covered by the periostracum, which consists of organic matrices that prevent decalcification of the shell. In the present study, we discovered the presence of chitin in the periostracum and identified a novel matrix protein, Pinctada fucata periostracum protein named PPP-10. It was purified from the sodium dodecyl sulfate/dithiothreitol-soluble fraction of the periostracum of the Japanese pearl oyster, P. fucata. The deduced amino acid sequence was determined by a combination of amino acid sequence analysis and cDNA cloning. The open reading frame encoded a precursor protein of 112 amino acid residues including a 21-residue signal peptide. The 91 residues following the signal peptide contained abundant Cys and Tyr residues. PPP-10 was expressed on the outer side of the outer fold in the mantle, indicating that PPP-10 was present in the second or third layer of the periostracum. We also determined that the recombinant PPP-10 had chitin-binding activity and could incorporate chitin into the scaffolds of the periostracum. These results shed light on the early steps in mollusk shell formation.

Spatiotemporal expression of the dermatopontin gene in zebrafish Danio rerio.

There has been significant interest in the expression and function of dermatopontin (DPT) in mammals owing to recent evidence pointing to its critical role in collagen fibrillogenesis. Despite this interest, limited information is available about the site/s of DPT mRNA expression or changes in expression in vivo. We used reverse-transcription polymerase chain reaction and in situ hybridization to evaluate the spatial and temporal pattern of DPT mRNA expression in zebrafish, Danio rerio, a widely used vertebrate model. We observed that DPT transcripts were expressed in zebrafish embryos at all developmental stages in a range of tissues, including the brain and optic neuron cells. Based on our results, we hypothesize that DPT may also play a role in neural functions in vivo.

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus.

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.