Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

Internal Distribution of Acid Materials within Snow Crystals

We found that river water is acidified not only in the first stage but also in the later stage of the snowmelt season in Japan, which differs from the so called acid shock occurring in the first stage of the snowmelt in the northern Europe. The acid shocks depend on the regional characteristics of the melt-refreeze processes forming the internal distribution of acid materials within snow crystals under the warm metamorphism. In a warm climatic region like the central Japan, there are possibilities to have the complicated distribution structure of acid materials within granular-snow crystals due to the repeated melt-refreeze processes even in midwinter. Consequently, the pH value of meltwater does not always increase as the snowmelt proceeds. Then, we showed the possibility by using the X-ray computed tomography that the domains with the acid materials exist in the inner parts of snow crystals. So, the acidification of the river water may occur even in the later stage of the snowmelt.     

Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells

The presence of endothelin, a vasoconstrictor peptide, in the hypothalamus and posterior pituitary suggests that it also regulates neural and other nonvascular target cells. In pituitary gonadotrophs, low doses of endothelin evoked oscillations in the intracellular calcium concentration, and high doses induced a biphasic calcium response. Mobilization of intracellular calcium predominated during the spike phase of the calcium response to endothelin, whereas calcium entry through dihydropyridine-sensitive channels contributed to both the spike and plateau phases of the calcium response. Endothelin was a potent as hypothalamic gonadotropin-releasing hormone (GnRH) in stimulation of gonadotropin release in perifused pituitary cells. Endothelin bound specifically to pituitary cells with a dissociation constant of 70 picomolar, and induced rapid formation of inositol trisphosphate and diacyglycerol. Although intracellular calcium concentration and gonadotropin secretory responses to endothelin were independent to the GnRH receptor, endothelin and GnRH appeared to have a common signal transduction mechanism. These observations suggest that endothelin can act as a neuropeptide to regulate anterior pituitary function.     

Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus

Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.     

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.     

Bioengineering of Leuconostoc mesenteroides glucansucrases that gives selected bond formation for glucan synthesis and/or acceptor-product synthesis

The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from Leuconostoc mesenteroides B-1299CB4 were constructed. A triple amino acid mutation (S642N/E643N/V644S) immediately next to the catalytic D641 (putative transition state stabilizing residue) converted DSRBCB4 enzyme from the synthesis of mainly α-(1→6) dextran to the synthesis of α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. The subsequent introduction of mutation V532P/V535I, located next to the catalytic D530 (nucleophile), resulted in the synthesis of an α-glucan containing increased branched α-(1→4) glucosidic linkages (approximately 11%). The results indicate that mutagenesis can guide glucansucrase toward the synthesis of various oligosaccharides or novel polysaccharides with completely altered linkages without compromising high transglycosylation activity and efficiency.     

Reverse-phase liquid chromatographic determination of paraquat and diquat in agricultural products

A simple, rapid, highly sensitive liquid chromatographic method is described for the quantitative determination of paraquat and diquat residues in agricultural products. Paraquat and diquat are extracted with hot dilute hydrochloric acid and are cleaned up on an Amberlite CG-50 column, followed by reverse-phase liquid chromatography on an NH2 column, with ultraviolet detection at 257 nm (paraquat) and 310 nm (diquat). The minimum detectable concentration of both paraquat and diquat was 0.5 ng per injection, which corresponds to a lower detection limit of approximately 0.02 microgram/g in the original samples. Recoveries of paraquat and diquat added to various samples were greater than 79%, and averaged 91 and 90%, respectively, at the 0.1 and 1.0 microgram/g spiking levels.