Quantification of genetically modified soybean by quenching probe polymerase chain reaction

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.     

Application of time-of-flight near infrared spectroscopy for detecting sugar and acid contents in apples

A newly constructed optical measurement system was introduced to nondestructively measure the composition of the inside of an apple by time-of-flight near-infrared spectroscopy (TOF-NIRS). As sugar content increased, optical parameters concerned with time-resolved profile of transmitted pulsed light (the attenuance of peak maxima, At, the time delay of peak maxima, Deltat, and the variation of full width at half-maximum, Deltaw) decreased gradually. When the acid content increased, At and Deltaw increased; however, a significant tendency could not be found for Deltat. At, Deltat, and Deltaw were employed as the explanatory variables for multiple linear regression, principle component regression, and partial least-squares analysis. It was possible to predict both sugar and acid contents in an apple with high precision by TOF-NIRS. Especially, the superiority of TOF-NIRS lied in more precise determination of acid content.     

Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.     

Porcine skeletal muscle troponin is a good source of peptides with Angiotensin-I converting enzyme inhibitory activity and antihypertensive effects in spontaneously hypertensive rats

In the search for novel peptides that inhibit the angiotensin I-converting enzyme (ACE), porcine skeletal troponin was hydrolyzed with pepsin, and the products were subjected to various types of chromatography to isolate active peptides. Glu-Lys-Glu-Arg-Glu-Arg-Gln (EKERERQ) and Lys-Arg-Gln-Lys-Tyr-Asp-Ile (KRQKYDI) were identified as active peptides, and their 50% inhibitory concentrations were found to be 552.5 and 26.2 microM, respectively. These are novel ACE inhibitory peptides, and the activity of KRQKYDI was the strongest among previously reported troponin-originated peptides. KRQKYDI was slowly hydrolyzed by treatment with ACE, and kinetic studies indicated that this peptide was a competitive inhibitor of the enzyme. When KRQKYDI was administered orally to spontaneously hypertensive rats (SHR) at a dose of 10 mg/kg, a temporary antihypertensive activity was observed at 3 and 6 h after administration.     

Development of singlet oxygen absorption capacity (SOAC) assay method. 2. Measurements of the SOAC values for carotenoids and food extracts

Recently a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of antioxidants was proposed. In the present work, kinetic study of the reaction of singlet oxygen ((1)O(2)) with carotenoids and vegetable extracts has been performed in ethanol/chloroform/D(2)O (50:50:1, v/v/v) solution at 35 °C. Measurements of the second-order rate constants (k(Q)(S)) and the SOAC values were performed for eight kinds of carotenoids and three kinds of vegetable extracts (red paprika, carrot, and tomato). Furthermore, measurements of the concentrations of the carotenoids included in vegetable extracts were performed, using a HPLC technique. From the results, it has been clarified that the total (1)O(2)-quenching activity (that is, the SOAC value) for vegetable extracts may be explained as the sum of the product {Σ k(Q)(Car-i)(S) [Car-i](i)} of the rate constant (k(Q)(Car-i)(S)) and the concentration ([Car (i)]) of carotenoids included in vegetable extracts.     

Applicability of the quantification of genetically modified organisms to foods processed from maize and soy

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.     

Influences of feedstock and pyrolysis temperature on the nitrate adsorption of biochar

Biochar (BC), charcoal produced through the pyrolysis of biomass, is reported to adsorb dissolved nitrate-nitrogen (NO₃-N). The NO₃-N adsorption properties of BC differ depending on the feedstock and the pyrolysis conditions, and the influences have not been systematically clarified. Therefore, we evaluated the dependence of feedstock and pyrolysis temperature on the NO₃-N adsorption properties of BC. Wood chips [Japanese cedar [Cryptomeria japonica] (CE) and Japanese cypress [Chamaecyparis obtusa] (CY)], moso bamboo [Phyllostachys edulis] chips (MB), rice [Oryza sativa] husks (RH), sugarcane [Saccharum officinarum] bagasse (SB), poultry manure (PM) and domestic wastewater sludge (WS) were air-dried and heated in a batch-type carbonization furnace at pyrolysis temperatures of 400, 600 and 800°C, with a hold time of 2 h. Among the BC produced from each feedstock, the one produced at 800°C had the greatest NO₃-N adsorption. The NO₃-N adsorption by BC produced from wood-based biomass at 800°C was significantly higher than that of the BC produced from non-wood-based biomass at 800°C. Therefore, BC made from wood-based biomass at higher temperature can be adequate as soil amendment material for adsorption of NO₃-N.     

家蚕35K蛋白酶基因的组织表达

用RT－PCR方法分析了家蚕（Bombyz mori)35K蛋白酶基因在家蚕幼虫期的组织及发育经时性表达。结果表明：35K蛋白酶基因只在幼虫的中肠中部有特异性的表达，在中肠后部有微弱的表达，而在中肠前部及脂肪体，丝腺，精巢，卵巢和血球细胞等中不表达；在幼虫期，该基因在中肠组织中的表达量与幼虫摄食活动相关联，认为受该基因控制的35K蛋白酶与营养物质的消化吸收有关，在家蚕的生长发育过程中起重要作用。     

Estimation of mycorrhizal colonization of the roots of oak seedlings inoculated with an ectomycorrhizal fungus,Laccaria amethystea

Six-month-old seedlings of Quercus serrata and Quercus glauca in a nursery were inoculated with the ectomycorrhizal fungus Laccaria amethystea encapsulated in alginate gel and grown in the nursery. The seedlings were collected at 1, 3, and 5 months after the inoculation and examined for colonization of the root system with ectomycorrhizal fungi. The roots within 5 months after the inoculation showed rudimentary ectomycorrhizal colonization. The level of colonization of the root system was estimated based on the intensity of hyphal covering on the root tips by staining with a fluorescent dye and expressed as an index of mycorrhizal colonization (IMC). IMC increased with the time after inoculation and reached values of 4 and 12% in Q. serrata and Q. glauca, respectively at 5 months after the inoculation. The determination of IMC may enable to assess the development of mycorrhizal colonization of the root system that shows rudimentary ectomycorrhizas after the inoculation.