Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.     

Preliminary Study on Linkage Mapping Based on Microsatellite DNA and AFLP Markers Using Homozygous Clonal Fish in Ayu (<Emphasis Type="Italic">Plecoglossus altivelis</Emphasis>)

A mapping referential family (F₁) of ayu was produced by crossing a normal diploid male with a homozygous clonal female. A genetic linkage map was constructed using 191 amplified fragment length polymorphism (AFLP) and 4 microsatellite DNA markers. A total of 178 loci were mapped in 36 linkage groups comprising 1659.6 cM, which includes approximately 77.3% to 81.8% of the total genome. As the markers were randomly distributed over the genome, they showed high efficiency for the construction of a wide linkage map.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution

Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) BIOSCI: Biotechnol. Biochem. 65, 1627-1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.     

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.     

Targeted disruption of the Tab1 gene causes embryonic lethality and defects in cardiovascular and lung morphogenesis

The transforming growth factor-beta (TGF-beta) superfamily consists of a group of secreted signaling molecules that perform important roles in the regulation of cell growth and differentiation. TGF-beta activated kinase-1 binding protein-1 (TAB1) was identified as a molecule that activates TGF-beta activated kinase-1 (TAK1). Recent studies have revealed that the TAB1-TAK1 interaction plays an important role in signal transduction in vitro, but little is known about the role of these molecules in vivo. To investigate the role of TAB1 during development, we cloned the murine Tab1 gene and disrupted it by homologous recombination. Homozygous Tab1 mutant mice died, exhibiting a bloated appearance with extensive edema and hemorrhage at the late stages of gestation. By histological examinations, it was revealed that mutant embryos exhibited cardiovascular and lung dysmorphogenesis. Tab1 mutant embryonic fibroblast cells displayed drastically reduced TAK1 kinase activities and decreased sensitivity to TGF-beta stimulation. These results indicate a possibility that TAB1 plays an important role in mammalian embryogenesis and is required for TAK1 activation in TGF-beta signaling.