Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.     

Practicable group testing method to evaluate weight/weight GMO content in maize grains

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.     

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.     

Artificial insemination with canine semen stored at a low temperature

Cooled canine semen solutions for storage were investigated with three stock solutions: egg yolk-citrate-glycine-glucose solution, egg yolk Tris-fructose citrate solution (EYT-FC), and egg yolk sodium citrate dihydrate solution (EYCD). For the control group, the second fraction of semen was examined. Nine male beagles and 37 female (47 experimental cases) beagles for artificial insemination (AI) were used. The qualities of semen stored at 4 degrees C deteriorated earlier in the control and EYCD groups. In the other two groups, sperm motility was 60% or higher after storage for 6 days and 20% or higher after storage for 12 days. On a comparison of these two groups, the sperm motility and viability were slightly higher in the EYT-FC group. A high conception rate was obtained by AI using semen stored at a low temperature for a maximum of two days in the control group and four days in the EYT-FC group.     

Cold-induced ascites in broiler chickens and its improvement by temperature-controlled rearing

Pulmonary hypertension syndrome (PHS), also known as ascites, in broiler chickens prevailed in the local area of Ibaraki prefecture, Japan, and was investigated epidemiologically, serologically, and pathologically. PHS developed in chickens older than 35 days of age when rapid increase of body weight started. Approximately 90% of affected birds were males, in which weight increase was greater than in females. Serologic test revealed that PHS broilers had an increase of hematocrit value. Pathologic studies indicated that the heart of affected birds had an obese-induced pressure and cold exposure triggered congestion in the right ventricle/cava and an increase in peritoneal fluid. These changes were consistent with the previous reports of PHS, so we designed the experiment of effects on cold-induced PHS birds in a temperature-controlled house. After the 10 PHS birds at 55 days were reared for 14 days in a temperature-controlled house at 20 +/- 5 C, ascites disappeared in eight birds and hematocrit values decreased to normal range in nine birds. Our finding indicated that temperature-controlled environment may be one solution to reduce mortality in PHS birds.     

Development of a method for forest cafeteria tests for evaluating chemical deterrents against bark stripping by sika deer (Cervus nippon yesoensis)

Damage caused by sika deer (Cervus nippon yesoensis) is a serious problem in commercial and environmental (non-harvested) forests in Hokkaido, northern Japan. Cafeteria tests in forests may be useful for evaluating the efficacy of chemical deterrents against bark stripping by deer. To develop a method for forest cafeteria tests in the continuous snow cover period, two experiments were carried out. In the experiments, logs were produced from tree trunks, and used as carriers of chemical deterrents. Carriers were installed in forests and fed to deer. The first experiment was to find suitable sites and installation methods for carriers. Criteria for the local suitability and the installation methods were as follows: a) Sites where deer are active should be selected; b) Carriers should be installed along actively used deer trails; c) Installation sites of carriers should be changed in response to deer movement; d) Carriers should be produced from tree species that deer naturally prefer; and e) Each carrier should be partially buried in the snow. The second experiment evaluated the feasibility of a cafeteria test method based on the results of the first experiment. The method was used for 13 sets of the cafeteria test, in which the deterrent effectiveness of 5 chemicals (wood tar, rosin, wood vinegar, and 2 pyroligneous liquors) was examined. We obtained results from all the sets. The chemicals tested did not deter bark stripping by deer. Nevertheless, the method used in the present study was practical for the cafeteria tests.     

Isolation and expression of five-amino-acid-deleted variant of feline hepatocyte growth factor (HGF) cDNA

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and hematopoietic cells. We have cloned a different form of cDNA, with a deletion of 15 base pairs predicted to result in the loss of 5 amino acids from the first kringle domain. To investigate the biological activity, original and deleted variant of feline HGF cDNAs were transiently expressed in COS-7 cells. Both recombinant feline HGFs showed almost the same dose-response curves in the stimulation of the growth of BNL CL.2 cells (a mouse hepatocyte cell line) and scatter activity of Madin-Darby canine kidney (MDCK) cells. The findings reported here suggest that the deleted variant of feline HGF has almost the same biological activity as the original in terms of the proliferation and scatter activity.     

Effect of Ca on Al-induced activation of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa)

The effect of CA(NO₃)₂ on the active oxygen scavenging system in hinoki cypress (Chamaecyparis obtusa) seedlings cultured in a nutrient solution containing aluminum was examined. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The growth in height and dry matter allocation to each organ was little influenced over a period of 12 weeks by either Al or the concentration of Ca(NO₃)₂. The activity of superoxide dismutase (SOD) in the needles was stimulated by Al, and the effect of Al was lowered significantly by simultaneous application of 25 mM Ca(NO₃)₂. At week 1, the activity of catalase (CAT) in the needles was increased by Al, but the effect was no longer observed at week 12. The Al concentration in the roots was increased by treatment with Al, whereas the Al concentration in needles was not. These results indicate that rhizospheric Al stress stimulates antioxidative enzyme activities in hinoki cypress needles and the activation of the enzymes is suppressed by addition of Ca. The transmission of Al stress to the needles, which induced a change in the enzyme activity, is not caused by the transfer of the Al ion itself from roots to needles. This work was supported in part by funding from the Japan Science and Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.     

Effects of phosphorus addition with and without ammonium, nitrate, or glucose on N2O and NO emissions from soil sampled under Acacia mangium plantation and incubated at 100 % of the water-filled pore space

An incubation experiment was conducted to examine the effects of phosphorus (P) addition with and without ammonium, nitrate, or glucose on N₂O and NO emissions from soil taken under Acacia mangium plantation and incubated at 100 % water-filled pore space (WFPS). Additions of NO ₃ ^? stimulated the N₂O and NO emissions while NH ₄ ⁺ did not, showing that denitrification was the main process of N₂O and NO production in the study condition. When NO ₃ ^? was added with P significantly (P?<?0.05) increased N₂O emissions regardless of the ratio of the added nitrogen and carbon, suggesting that P addition stimulated denitrification activity. The activation of denitrification by P addition is possibly attributed to two mechanisms: (1) the added-P stimulated denitrification by relieving P shortage for denitrifying bacteria and (2) the added-P stimulated activity of heterotrophic soil microflora with increased O₂ consumption promoting the development of anaerobic conditions with stimulation of denitrification.