Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Acylated and non-acylated flavonol monoglycosides from the Indian minor spice Nagkesar (Mammea longifolia)

A methanol extract of nagkesar (buds of Mammea longifolia), which showed strong radical scavenging activity, yielded 13 compounds by separations using column chromatography and HPLC. Structure elucidation of these compounds was achieved by (1)H and (13)C NMR, including DQF-COSY, TOCSY, DEPT, HMQC, HSQC, and HMBC. They include two new compounds, quercetin 3-O-(2' ',4' 'di-E-p-coumaroyl)-alpha-L-rhamno-pyranoside and quercetin 3-O-(3' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, along with known compounds kaempferol, quercetin, the isopropylidenedioxy derivative of shikimic acid, kaempferol 3-O-(2' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, kaempferol 3-O-(3' ',4' '-di-E-p-coumaroyl)-alpha-L-rhamnopyranoside, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, shikimic acid, kaempferol 3-O-beta-D-glucopyranoside, quercetin 3-O-beta-D-glucopyranoside, and beta-sitosterol 3-O-beta-D-glucopyranoside.     

Relationship between antibacterial activity of (+)-catechin derivatives and their interaction with a model membrane

(+)-Catechin derivatives with different alkyl chain lengths were synthesized from (+)-catechin and various straight chain alkylaldehydes in the presence of methyl mercaptan, and their antibacterial activities against Gram-positive bacteria were evaluated. The antibacterial activity increased markedly with elongation of the alkyl chain lengths of the derivatives and reached a maximum at a chain of four to seven carbons. Subsequently, interaction of the (+)-catechin derivatives with a model membrane using liposome was investigated. The derivatives with a chain of three carbons or more were found to have very strong affinity for the membrane. The injury action of the derivatives against the membrane was examined with liposome in which calcein was enclosed as a fluorescent indicator. The leakage was observed in the derivatives with chain lengths of four carbons or more. Particularly the derivatives with chains longer than five carbons are considered to destroy the liposome membrane judging from the degree of the fluorescent leakage. These results implied that the lipophilicity and disrupting ability of the (+)-catechin derivatives to the liposome membrane participate in their antibacterial activity.     

Long-Term Observation of Fogwater Composition at two Mountainous Sites in Gunma Prefecture, Japan

We made serial observations on acid fog at Mt. Akagi and Mt. Haruna, Japan for 5 years. The altitudes of the sampling sites were 1500 m (Mt. Akagi) and 1200 m (Mt. Haruna) above sea level, and the sites were approximately 30 km apart. The average liquid water content (LWC) at Mt. Akagi and Mt. Haruna was 74 mg m^?3 and 63 mg m^?3, respectively. The pH of fogwater was 2.72–7.14 (mean 3.71) at Mt. Akagi and 2.94–6.58 (mean 3.73) at Mt. Haruna. Our long-term observations indicate that there was no significant difference in the chemical components in fogwater at both sites except for ammonium ion. However, there were some cases where the chemical components of fogwater at each site were differed remarkably even in the concurrent fog event. Nitrate and sulfate ions contributed to acidification of fogwater at Mts. Akagi and Haruna and 95% of sulfate ion in the fogwater originated from air pollution. Ammonia gas in the air was the main neutralizer of acidity in fogwater. When absorption of excessive nitric acid gas over ammonia gas in the air occurred, the pH of fogwater was lowered. Our back trajectory analysis indicated that the fogwater at Mt. Akagi was mainly affected by an air mass from the Kanto Plain, including Tokyo, while the fogwater at Mt. Haruna was influenced by an air mass from large, western cities, such as Nagoya and Osaka, as well as Tokyo.     

Analysis of hop-derived terpenoids in beer and evaluation of their behavior using the stir bar-sorptive extraction method with GC-MS

Hop aroma components, which mainly comprise terpenoids, contribute to the character of beers. However, pretreatments are necessary before analyzing these components because of their trace levels and complicated matrixes. Here, the stir bar-sorptive extraction (SBSE) method was used to detect and quantify many terpenoids simultaneously from small samples. This simple technique showed low coefficients of variation, high accuracy, and low detection limits. An investigation of the behavior of terpenoids identified two distinct patterns of decreasing concentration during wort boiling. The first, which was seen in myrcene and linalool, involved a rapid decrease that was best fitted by a quadratic curve. The second, which was observed in beta-eudesmol, humulene, humulene epoxide I, beta-farnesene, caryophyllene, and geraniol, involved a gentle linear decrease. Conversely, the concentration of beta-damascenone increased after boiling. As the aroma composition depended on the hop variety, we also examined the relationship between terpenoid content and sensory analysis in beer.     

Forecast of Sulfur Deposition in Japan for Various Energy Supply and Emission Control Scenarios

In order to contribute to the analysis and solution of regional scale environmental problems in East Asia, we developed a tool for the comprehensive assessment of alternative policy options to improve air quality. This tool projects the future regional energy supply, calculates the emission levels of sulfur dioxide and estimates the geographical pattern of sulfur deposition resulting from emissions. Sulfur deposition in Japan through 2030 was forecasted for various energy supply and emission control scenarios using the analysis tool. Future sulfur depositions were calculated from the source-receptor matrix for 1995 and the growth rate of emission for the source subregion. In the case of the current legislation scenario, anthropogenic SO₂ emissions in East Asia would grow by 34 percent and sulfur deposition in Japan would increase by approximately 20 percent between 1995 and 2030. This increase in sulfur deposition over these 35 years is sligthly less than the contribution from volcanic emission to sulfur deposition in Japan. In the case of the hypothetical dirty scenario for China, sulfur deposition in several grids which face the Sea of Japan would double by 2030.     

Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats

To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 microg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 microg/kg) and 164 (8.0 microg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.