Authentication of flying-fish-meal content of processed food using PCR-RFLP

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flying-fish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of ~200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of ~530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.     

东亚日本农书的形成及特征

本研究主要考察了东亚的日本农书的形成及其特征。作者认为在东亚，中国农书对日本农书的形成产生过影响。同时作者指出，日本农书有两大特征：其一是强调植物也有灵性：其二是强调了神的作用。     

Quantification of relative flying fish paste content in the processed seafood ago-noyaki using real-time PCR

Real-time polymerase chain reaction (PCR) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence was used to determine flying fish paste in ago-noyaki. We quantified the amount of flying fish paste in ago-noyaki samples using flying fish-specific primers (Tobi16SF3/Tobi16SR) and universal primers (Univ16SF2/Univ16SR2). Using real-time PCR of standard ago-noyaki, a standard equation was obtained (y = 1.08x − 3.20; R ² = 0.977). This equation was then used to estimate the relative flying fish paste contents of eight commercially available ago-noyaki and two similar products. These results verified that the ago-noyaki products that had already been labeled with the E-mark deserved this status.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.     

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells

The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B₄ isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.     

Ethylenediurea (EDU) spray effects on willows (Salix sachalinensis F. Schmid) grown in ambient or ozone?enriched air: implications for renewable biomass production

Ground-level ozone -(O3) is a widespread air pollutant causing extensive injuries in plants. However, its effects on perennial energy crops remain poorly under-...     

Possible implication of metalloproteinases in post-mortem tenderization of fish muscle

ABSTRACT: It is suspected that the proteolytic breakdown of extracellular matrix proteins is responsible for the postmortem tenderization of fish muscle during chilled storage. In order to identify the type(s) of proteinases involved in this phenomenon, the effect of proteinase inhibitors, EDTA (ethylenediamine tetraacetic acid), 1,10-phenanthroline, ρ-APMSF [(ρ-amidinophenyl) methanesulfonyl fluoride hydrochloride] and E-64 [ L - trans -epoxysuccinyl-leucylamido-(4-guanidinobutane)] on tenderization was investigated by using Japanese flounder. Proteinase inhibitor solution was injected into a blood vessel in a caudal portion of live flounder and the firmness of muscle was then evaluated as a shear force value at 0 h and 6 h after death. Metalloproteinase inhibitors, EDTA and 1,10-phenanthroline, significantly suppressed postmortem tenderization. These findings suggest that metalloproteinases are candidates for proteinases involved in the postmortem tenderization of fish muscles. Although not significantly, p -APMSF, a serine proteinase inhibitor, partially suppressed muscle tenderization, which suggests that serine proteinases are also implicated in postmortem tenderization. A cysteine proteinase inhibitor, E-64, showed no effect, suggesting that cysteine proteinases are not involved.     

Tolerance of Japanese larch to drought is modified by nitrogen and water regimes during cultivation of container seedlings

Improving drought tolerance of container seedlings of Japanese larch is of high importance to afforestation. We hypothesized that adequate nitrogen (N) and limited water supply would increase the tolerance of container seedlings to water-deficit stress, circumventing photoinhibition, by means of (i) enhanced photosynthetic capacity with higher leaf N and (ii) decreased water loss from leaves with lower biomass allocation into aboveground parts. Container seedlings of Japanese larch were grown under the treatment combinations of adequate (+?N: 300 mg N container^?1) or limited (??N: 150 mg N container^?1) N and adequate (+?W: daily irrigation) or limited (??W: twice-a-week irrigation) water. Then, seedlings were subjected to a progressive drought treatment. Higher leaf N was observed in container seedlings grown under?+?N and???W. During progressive drought, lower stomatal conductance and net photosynthetic rate were observed in leaves with higher leaf N at a given predawn leaf water potential. Furthermore, the maximum efficiency of PSII photochemistry (F_v/F_m) was lower in leaves with higher leaf N, suggesting that higher leaf N might impair intrinsic tolerance to drought at the leaf level contrary to expectations. Conversely,???N and???W seedlings with lower shoot biomass delayed soil drying as a whole-plant response via a reduction in leaf transpiration, leading to delayed photoinhibition as indicated by a decline in F_v/F_m. To circumvent stress at the initial stage of water deficit, lower leaf N via limited N regime and smaller shoot biomass driven by limited N and water regimes would be important.