Development of a novel heterologous gene expression system using earthworms.

In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and animal proteins. The aim of the present study was to develop the earthworms Eisenia fetida Waki and Eisenia andrei Sagami as next-generation animal protein production hosts. These earthworms have been approved as model animals for acute toxicity tests by the Organization for Economic Co-operation and Development, and they have post-translational modification systems. However, so far, none of the studies have used earthworm transfection techniques. Thus, we developed a transfection method for E. fetida and E. andrei using microinjection and electroporation systems. The maximum survival rates and transfection efficiencies were 79.2% and 29.2% for E. fetida, and 95.8% and 50.0% for E. andrei, respectively. Furthermore, human erythropoietin was detected in the transformed earthworm tail fragments using an enzyme-linked immunosorbent assay. These results contribute to the development of a potential earthworm-based novel animal protein production system.

Characterization of earthworm α-amylases for dietary supplement development and biomass utilization.

Earthworms are useful soil-decomposing animals that possess various saccharification enzymes such as cellulases and amylases. Earthworms have also been traditionally used as antipyretic agents and medicines for preventing thrombotic diseases such as brain infarction. We previously developed a novel earthworm dietary supplement with fibrinolytic, cellulase, and amylase activities using high-pressure technology. However, the optimal temperature and pH required for amylase activity in bioindustry have not yet been investigated. In the present study, we purified and characterized two α-amylases of Eisenia fetida Waki, EfAMY1 and EfAMY2, which were monomeric enzymes of 63.8 kDa and 64.0 kDa, with specific activities of 69.2 and 40.4 units/mg, respectively. The optimal pH was 5.5 for both enzymes, and the optimal temperatures were 45 °C and 35 °C for EfAMY1 and EfAMY2, respectively; however, the enzymes were stable over a wide pH range (5-10) and at high temperature (up to 40 °C). These amylases showed higher specific activity and cold tolerance than those previously reported. These data should help to promote the development of E. fetida AMYs as functional dietary supplements and in biomass utilization.

High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), ß-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment.

Characterization of two endoglucanases for the classification of the earthworm, Eisenia fetida Waki.

Eisenia fetida and Eisenia andrei are vermicomposting species that are used as model animals for testing chemical material toxicology. Eisenia spp. are grown commercially in various fields in Japan. However, these two species have not been classified because it is difficult to distinguish them morphologically; thus, all bred earthworms are called E. fetida. However, it has been proposed that these two species have different expression regulation mechanisms. Here, we classified a sample of earthworms purchased from several farms, confirming that both E. fetida and E. andrei are present in Japanese earthworm breeding programs. We also characterized two highly active endoglucanases (EfEG1 and EfEG2) from the E. fetida Waki strain, which contained strong fibrinolytic enzymes for improving human health. We confirmed that EfEG1 is 1371 bp long and belongs to GHF9. Thus, E. fetida Waki may have commercial application for biomass utilization and as a dietary health supplement.

An extract from brown rice inhibits signal transduction of angiotensin II in vascular smooth muscle cells.

BACKGROUND: Health benefits of brown rice over white rice have been described previously. However, whether the outer bran of rice contains an ingredient useful to prevent cardiovascular diseases remains unknown. The subaleurone layer of rice, which is usually lost by milling brown rice for whitening, is rich in varied nutrients, suggesting that some ingredient contained within this layer may be beneficial for the cardiovascular system. METHODS: To assess potential benefits of the subaleurone layer toward pathological vascular remodeling, we examined the effects of the layer extracts from Japanese rice (Oryza sativa var. japonica) on angiotensin II (Ang II)-induced signal transduction and hypertrophy in cultured rat vascular smooth muscle cells (VSMCs). RESULTS: Pretreatment of the ethyl acetate extract (100 µg/ml), but not other extracts, inhibited Ang II (100 nmol/l)-induced immediate signal transduction events. Also, the extract diminished c-Fos expression and hypertrophic protein accumulation induced by Ang II in the cells. CONCLUSION: These data suggest that an ingredient in the ethyl acetate extract from the subaleurone layer of rice has a protective effect toward cardiovascular diseases by interfering with signal transduction induced by Ang II.

[Survey of difference in appearance between multiple-specifications press-through-package drug products and an attempt to evaluate their effectiveness based on dispensing incidents].

To survey the difference in appearance between multiple-specification press-through-package (PTP) drug products and to attempt to evaluate their effectiveness as discriminating factors based on dispensing incidents. Front and back sides of, respectively, 153 and 134 PTP drug products of multiple specifications stockpiled in the author's pharmacy were surveyed for differences in wording and appearance between specifications of the same type of drug. Fifty six dispensing incidents with 40 sets occurred over a year and they were analyzed for the appearance similarity of the front side. The difference factors detected in the 40 sets of "mix-ups" were also reviewed after similarity-omitted counting. We identified six factors with difference in appearance: color-related (letter front or patterns, sheet, medicine) and shape- or pattern-related details (sheet and medicine sizes, patterns). Multiple differences on the front packaging of the same type of drug were identified in 93% of the sets, while only one difference was found in about half of the sets on the back, indicating that pharmaceutical companies placed more emphasis on the front side to discriminate their features. When reviewed by similarity-omitted counting, the ratio of sets with only one difference in the 40 mix-ups was higher than those to 128 sets of non-mix-ups, the total sets except the mix-ups, while the ratio of sets with two differences was lower. In addition, the ratio of sets in which only color-related factors differed in the 40 mix-ups was higher than that in the corresponding category to the 128 sets of non-mix-ups. Various discriminating factors were used in combination on the front side of multiple-specification PTP drug products. A combined use of shape- or pattern-related and color-related factors probably reduces dispensing incidents among products with multiple specifications. However, further accumulation of incident data and multifactor analysis of those data seem necessary to clarify the function of difference in appearance in dispensing incidents.

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.

Fructosyl-amino acid oxidases of Aspergillus oryzae are induced by the reaction product, glucosone.

Aspergillus oryzae has two fructosyl-amino acid oxidase (FAOD) isozymes (AoFao1 and AoFao2), which are different in the substrate specificities. Northern blot analysis showed both FAO genes were induced by autoclave-browned medium containing l-lysine or l-valine. Studies with a mutant, that had a disrupted AoFAO2 gene, revealed that the expression of AoFAO1 by fructosyl l-valine depended on the expression of AoFAO2. Both genes were also induced by one of the FAOD-reaction products, glucosone. In contrast, other alpha-dicarbonyl compounds, which display a similar structure to that of glucosone were not able to induce the genes expression. These results imply that glucosone may contribute to the expression of FAO genes.

Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae.

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N(epsilon)-fructosyl N(alpha)-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain Delta faoAo2 did not grow. Addition of glucose or (NH(4))(2)SO(4) to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO(2) as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.

Characterization of two fructosyl-amino acid oxidase homologs of Schizosaccharomyces pombe.

Two putative fructosyl-amino acid oxidase genes, FAP1 and FAP2, found in the Schizosaccharomyces pombe genome were cloned and expressed. Both of the gene products (Fap1 and Fap2) were flavoproteins and have no activity for fructosyl-amino acids. It was suggested that Fap1 and Fap2 are an L-pipecolic acid oxidase and L-saccharopine oxidase, respectively.