Screening method for determination of high levels of cadmium, lead, and copper in foods by polarized Zeeman atomic absorption spectrometry using discrete nebulization technique

A screening method for determination of cadmium, lead, and copper in foods was developed. The sample (1-3 g) is digested with HNO3-H2SO4-HClO4 in a centrifuge tube attached to a straight glass tube that prevents loss of HNO3 by volatilization. After digestion, potassium iodide, H2SO4, and MIBK (4-methyl 2-pentanone) are added, and the metals are extracted with MIBK as metal iodides. The MIBK solution is injected and the metals are determined by flame polarized Zeeman atomic absorption spectrometry using a discrete nebulization technique. Recoveries of metals from fortified milk powder, unpolished rice, fish, beef, peanut butter, apple, and cabbage were satisfactory. The analytical results for NBS Oyster Tissue and NIES Pepperbush, Chlorella, and Mussel agreed with certified or reference values except lead in Pepperbush. The limits of quantitation for cadmium, lead, and copper were 0.01, 0.09, and 0.02 ppm, respectively. This method is simple and safe for routine analysis of high levels of cadmium, lead, and copper in foods.     

Risk factors for Campylobacter colonization in broiler flocks in Japan

The present study was conducted to estimate the prevalence of and to identify the risk factors for Campylobacter colonization in broiler flocks in Japan. Campylobacter colonization status in flock was evaluated by culturing pooled caecal excrement from 124 broiler flocks. Potential exposure to risk factors was evaluated with a questionnaire for the broiler producers. Odds ratios (ORs) were calculated using a multivariable logistic regression analysis. The prevalence of Campylobacter-positive flocks was 43.5% (upper and lower limits of 95% confidence interval (CI(95%) ): 34.8, 52.3). Multivariable logistic regression model identified two variables as risk factors for Campylobacter colonization. The ORs of Campylobacter colonization were higher in flocks in western Japan (OR=2.68; CI(95%) : 1.04, 6.91) than in eastern Japan, and in flocks supplied with undisinfected drinking water (OR=7.41; CI(95%) : 3.11, 17.66) than in those supplied with disinfected drinking water. These findings indicate that water may play an important role in Campylobacter colonization in broiler flocks in Japan and the use of disinfected water may reduce the risk of Campylobacter colonization.     

Administration of two oils rich in n-3 long-chain polyunsaturated fatty acids to rat pups of dams fed a diet high in fat and low in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated fatty acids (LCPUFA) with 20 or 22 carbons are considered important to the development of infants and sometimes added to infant formulae. In this study, two characteristic sources of n-3 LCPUFA (fish oil and microalgal oil) were orally administrated to rat pups of mildly n-3 PUFA — deficient dams to compare the consequences of the administration. The milk from the dams fed a n-3 PUFA — restricted diet contained less n-3 LCPUFA than that of the dams fed a control diet. Pups were administered 1 mg/g weight of the test oil at the age of 5–7 days. At the age of 7 days, they were sacrificed before or after the administration and fatty acid compositions of the stomach and serum lipid were studied. The administration changed docosahexaenoic acid (DHA; 22:6n−3) levels in the stomach contents and serum lipids with time. Eicosapentaenoic acid (EPA; 20:5n−3) levels increased immediately after the administration of fish oil. The administration of microalgal oil also affected the serum lipid EPA level, in spite of a lack of EPA. In this study, both oils effectively supplemented DHA. Fish oil returned the serum EPA level close to the control value while microalgae oil had little effect.     

Preparation of calibration standards of N1-H paralytic shellfish toxin analogues by large-scale culture of cyanobacterium Anabaena circinalis (TA04)

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ± 0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50-90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.     

Cloning and characterization of the antigenic membrane protein (Amp) gene and in situ detection of Amp from malformed flowers infected with Japanese hydrangea phyllody phytoplasma

A Japanese hydrangea phyllody (JHP) disease found throughout Japan causes economic damage to the horticultural industry. JHP phytoplasma-infected Japanese hydrangea plants show several disease symptoms involved in floral malformations, such as virescence, phyllody and proliferation. Here, we cloned and characterized the antigenic membrane protein (Amp) gene homolog from the JHP phytoplasma (JHP-amp), expressed the JHP-Amp protein in Escherichia coli cells, and then obtained an antibody against JHP-Amp. The antibody against JHP-Amp had no cross-reactions with the antibody against the Amp protein from a closely related onion yellows phytoplasma. This serologic specificity is probably due to the high diversity of the hydrophilic domains in the Amp proteins. The in situ detection of the JHP-Amp protein revealed that the JHP phytoplasma was localized to the phloem tissues in the malformed flower. This study shows that the JHP-Amp protein is indeed a membrane protein, which is expressed at detectable level in the JHP phytoplasma-infected hydrangea.     

Spatiotemporal catch distribution of age-0 Pacific bluefin tuna Thunnus orientalis caught by the Japanese troll fishery in relation to surface sea temperature and seasonal migration

This study used a delta-lognormal model to analyze monthly catches of age-0 Pacific bluefin tuna by the troll fishery. The model included fixed effects of month, area, and month–area interaction, and random effects of port, year and port–year interaction. The catch patterns by month and area predicted by the statistical model (standardized catch) revealed that main fishing grounds along the Tsushima Warm Current generally shifted from north to south as the season turned from autumn to winter. In contrast, the standardized catch along the Kuroshio Current did not show such clear spatiotemporal patterns. The standardized catch along the Tsushima Warm Current is significantly associated with average monthly sea surface temperatures in the fishing grounds and consistent with migration routes revealed by tagging experiments in previous studies. These associations indicate the spatiotemporal catch pattern in the Tsushima Warm Current region partly reflects seasonal migration. Knowledge of the possible associations among fish migration, environmental factors and spatiotemporal distribution of the catch will contribute to future management of this species.     

Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C₂C₁₂ myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.