Biomedical subjects
Publications and source records attributed to Hiroshi Akiyama.
A sensitive qualitative detection method for soybeans in foods by using the polymerase chain reaction (PCR) was developed. For specific detection of soybeans with high specificity, the primer pair of Gym 81/Gym 82 was designed on the gene encoding the Glycine max repetitive sequence. The trace amount of soybeans in commercial food products could be qualitatively detected by this method.
For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established. To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective. The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.
Chondroitin sulfate (CS) was administered orally to BALB/c mice immunized intraperitoneally with ovalbumin (OVA) and/or dinitrophenylated OVA. The titers of antigen-specific IgE and IgG1 in mouse sera were determined. The antigen-specific IgE production by mice fed ad libitum with CS was significantly inhibited. We also examined the effect of feeding CS on immediate-type hypersensitivity. One hour after antigen stimulation, the ears of mice fed with CS swelled less than those of the control mice. Furthermore, the rise in serum histamine in the mice fed with CS under active systemic anaphylaxis was significantly lower than that in the controls. We next examined the pattern of cytokine production by splenocytes from mice followed by re-stimulation with OVA in vitro. The splenocytes from the mice fed with CS produced less interleukin (IL)-5, IL-10, and IL-13 than those from the control group. In contrast, the production of interferon-gamma and IL-2 by the splenocytes of mice fed with CS was not significantly different from those in the control mice. In addition, the production of transforming growth factor-beta from the splenocytes of mice fed with CS was significantly higher than that of the control mice. Furthermore, we showed that the percentages of CD4(+) cells, CD8(+) cells, and CD4(+)CD25(+) cells in the splenocytes of mice fed with CS are significantly higher than those of the control. These findings suggest that oral intake of CS inhibits the specific IgE production and antigen-induced anaphylactic response by up-regulating regulatory T-cell differentiation, followed by down-regulating the Th2 response.
BACKGROUND: Wheat protein hydrolysates have been traditionally used as food additives and are now being used in cooking worldwide. There have been a few studies on the relationship between the molecular mass distribution and the immunoglobulin E (IgE) reactivity of the wheat protein hydrolysates. METHOD: We analyzed the peptide profile of commercial wheat protein hydrolysate samples from enzymatic or acid hydrolysis of wheat protein using size exclusion chromatography. We further investigated the IgE reactivity of the wheat protein hydrolysates using the inhibition ELISA method and sera of 5 patients sensitive to wheat. RESULTS: The wheat protein enzymatic hydrolysate samples showed high concentrations of peptides with molecular masses greater than 1,050 Da, whereas in contrast, the wheat protein acid hydrolysates showed extremely low concentrations of peptides with molecular masses greater than 1,050 Da. Tested wheat protein acid hydrolysates hardly inhibited the patient IgE binding ability to wheat proteins in the five patient sera. On the contrary, some tested wheat protein enzymatic hydrolysate samples inhibited the IgE binding ability to wheat proteins. CONCLUSION: These results suggested that the uptake of wheat protein enzymatic hydrolysates might still have the possibility of causing food allergic reactions in patients allergic to wheat and the processed foods containing them.
A sensitive and specific method for quantifying a genotoxic hydrazine, agaritine, has been developed using liquid chromatography-electrospray ionization tandem mass spectrometry (MS). Synthetic agaritine was structurally assigned by (1)H, (13)C and two-dimensional nuclear magnetic resonance (NMR) analysis (heteronuclear multiple-bond correlation [HMBC] and heteronuclear multiple-quantum coherence [HMQC]), high-resolution fast-atom-bombardment (HR-FAB) MS. Agaritine was separated on an ODS column using 0.01% AcOH-MeOH (99:1) as an eluent with a simple solid-phase-extraction cleanup for mushroom samples and with acetonitrile and methanol deprotenization for plasma samples. There were no interference peaks in any of the mushrooms or mouse plasma samples. The recoveries of agaritine from the spiked mushroom samples and spiked mouse plasma were 60.3-114 and 74.4%, respectively. The intra-day precision values for the spiked mushrooms were 5.5 and 4.2%, and the inter-day precision values were 15.0 and 23.0%, respectively. The limit of quantification was 0.01 microg/g (in mushrooms) and 0.01 microg/ml (in plasma). A precursor ion scan confirmed that agaritine derivatives, which can exert a similar toxicity, were absent. These results indicate that this analytical method for quantifying agaritine could help to evaluate the risk of mushroom hydrazines to humans.
In this study, we investigated the absorption of apple procyanidins, namely, apple condensed tannins (ACTs), in rats using the Porter method and high-performance liquid chromatography/tandem mass spectrometry. The apple procyanidin concentrations in the rat plasma reached a maximum 2 h after administration and decreased thereafter. To investigate the limits of the absorption of apple procyanidins in the polymerization degree, we administered the procyanidin oligomer fraction, which was separated from ACT using normal-phase chromatography according to the degree of polymerization. Procyanidins from each dimer to pentamer group were detected in the plasma by the Porter method. Moreover, by the study using reconstituted procyanidins, polymeric procyanidins influenced the absorption of procyanidin oligomers. These results suggest that ACTs are absorbed and directly involved in physiological functions in the rats.
A new method with good sensitivity and specificity for detecting and quantifying genotoxic hydrazines, agaritine and 4-(hydroxymethyl)phenylhydrazine (HMPH), was developed using liquid chromatography-electrospray tandem mass spectrometry (MS). Synthetic agaritine and HMPH were structurally assigned by 1H-, 13C- and two-dimensional nuclear magnetic resonance (NMR) analysis (HMBC and HMQC), high-resolution fast-atom bombardment (HR-FAB) MS and time of flight (TOF) MS. The polar molecule agaritine was separated on an ODS column using 0.01% AcOH-MeOH (99:1, v/v) as an eluent with a simple solid-phase extraction cleanup. There were no interference peaks for any of the mushrooms. Agaricus spp. contained 1247 and 2017 microg g-1 agaritine. Other species of mushroom had no agaritine. Recoveries of agaritine from spiked mushroom samples were 60.3-114%. Intra-day precision values were 5.5 and 4.2%, and the inter-day precision values were acceptable (15.0 and 23.0%), as agaritine is unstable. The limit of quantification was 0.003 microg g-1. Even a trace amount of agaritine in mushrooms can, therefore, be determined using this method. We also directly analysed HMPH, an active free hydrazine form of genotoxic agaritine, and obtained direct evidence of its absence from mushrooms. A precursor ion scan confirmed that agaritine derivatives, which could exert similar toxicity, were absent. The results indicate that this specific and sensitive analytical method for detecting and quantifying agaritine and its derivatives could help evaluate the risk of mushroom hydrazines to humans.
In autumn 2004, many Japanese patients with renal failure developed cryptogenic encephalopathy by consuming sugihiratake mushroom, a Japanese delicacy. To elucidate the relationship between the cryptogenic cases and this mushroom, we conducted a multivariate analysis of metabolites in 'Probably Toxic' sugihiratake collected from the area of encephalopathy outbreaks, and 'Probably Safe' sugihiratake collected from unaffected areas using UPLC/ToF MS. The results indicate that the presence of milligram quantities of vitamin D-like compounds per 10 g of dried sugihiratake from the areas of encephalopathy outbreaks. Two hypotheses to induce the encephalopathy are proposed: the found metabolites are (1) vitamin D agonists, which induce acute and severe hypercalcemia and/or hyperammonemia and/or vitamin D toxicity, or (2) vitamin D antagonists, which induce acute and severe hypocalcemia.
Three novel conjugated long-chain fatty acids (1-3) were obtained from aqueous methanol extracts of Pleurocybella porrigens together with nine known constituents including (8E,10E)-7,12-dioxo-8,10-octadecadienoic acid (ostopanic acid) (4). The structures of the new fatty acids were characterized as (14RS)-(10E,12E)-14-hydroxy-9-oxo-10,12-octadecadienoic acid (1), (12RS)-(8E,10E)-12-hydroxy-7-oxo-8,10-octadecadienoic acid (2), and (10E,12E)-9,14-dioxo-10,12-octadecadienoic acid (3) by spectroscopic methods.
Agaricus blazei MURRILL mushroom products are sold as so-called health foods in Japan. However, a part of Agaricus is known to contain hydrazines. A sensitive and specific method for analyzing a genotoxic hydrazine, agaritine, and its derivatives was developed to assess the safety of Agaricus products. beta-N-(gamma-L(+)-Glutamyl)-4-(hydroxymethyl)phenylhydrazine (agaritine, AGT), 4-hydrazinylbenzylalcohol (HMPH), 4-hydrazinylbenzoic acid (CPH), 4-methylphenylhydrazine (MPH) and phenylhydrazine (PH) were converted to their correspond-ing fluorescent derivatives with 3,4-dihydro-6,7 dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride (DMEQ-COCl) as the fluorescence derivatization reagent. The detection limits (S/N=3) for CPH, AGT, PH and MPH were 422, 45.3, 16.5 and 138 fmol, respectively, in a 20 microl injection volume. Recoveries, achieved by adding known AGT amounts to the Agaricus sample and Agaricus products, ranged from 92.8 to 102%. By using this method which does not require partial purification of the Agaricus sample, the amounts of AGT in several types of foods were found to be 112-1836 microg/g dry weight.
Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.
BACKGROUND: We have already reported that antigen-specific IgG1 antibody production in WBB6F1-W/Wv (W/Wv) mice after oral administration of ovalbumin (OVA) was extremely high. Active systemic anaphylaxis (ASA) was induced in these mice after intraperitoneal (i.p.) administration of OVA, and Th2-dominant helper T-cell activation occurred. In this study, we examined the effect of CpG oligodeoxynucleotide (ODN) conjugation of OVA on oral immunization of W/Wv mice. METHODS: W/Wv mice were sensitized by administration of 0.1 mg OVA or CpG ODN-OVA by gavage every day for 4 weeks, and the serum titers of OVA-specific IgG1, IgE, and IgG2a antibody were determined. ASA was induced by i.p. injection of OVA, and the changes in body temperature were monitored. In vitro production of Th1- and Th2- type cytokines by splenocytes re-stimulated with antigen was also measured. RESULTS: The antigen-specific IgG1 antibody titer in the CpG ODN-OVA-sensitized W/Wv mice was lower than in the OVA-sensitized group, but the IgG2a titer was higher. ASA was not induced by i.p. OVA challenge. There were significant increases in the production of Th1-type cytokine (IFN-gamma) by splenocytes in the CpG ODN-OVA-sensitized mice, but the Th2-type cytokine (IL-4) level in the splenocyte culture medium was lower. CONCLUSIONS: These results indicated that oral administration of CpG ODN-OVA conjugate significantly induced antigen-specific Th1 responses and reduced Th2 responses (allergic reactions) on re-stimulation. These findings suggest that CpG ODN-antigen conjugate may be useful as an oral vaccine.
We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.
A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.
A laboratory-performance study was carried out to investigate factors affecting the reliability of the quantitative PCR method to analyze an approved genetically modified (GM) maize (Mon810 line). Test maize powdered samples were prepared as blind samples containing a high (assigned value; 5.45%) or low (assigned value; 0.35%) concentration of the Mon810 line. After confirmation of their homogeneity, they were provided to 27 laboratories participating in the collaborative study. The data were collected from all laboratories and statistically analyzed. Two laboratories, which used a Roche LightCycler (LC), reported significantly high test values. A further examination showed that the LC method is greatly affected by the equipment itself or PCR reagents, resulting in poor repeatability. On the other hand, some laboratories, which used ABI quantitative PCR equipment, reported erroneous test values. In these laboratories, the errors appeared to have been due to inadequate quality and/or yield of DNA. To identify factors affecting the test values, analysis of the measured values for the taxon-specific gene will be useful. Furthermore, the modified silica-gel membrane DNA extraction method made it possible to extract the required amounts of DNA more easily and in a shorter time than before.
As part of an investigation on the chemical constituents and contaminants of the basidiomycete Pleurocybella porrigens (Japanese name: Sugihiratake), we analyzed the UV-detected constituents of this mushroom using HPLC. One of the major UV peaks detected was isolated and identified as a-eleostearic acid, a long-chain fatty acid with a conjugated triene moiety, based on the results of spectroscopic methods. alpha-Eleostearic acid was concluded to be a characteristic fatty acid of P. porrigens, because it was not detected in eight other edible mushrooms examined. Free long-chain fatty acids in P. porrigens and other edible mushrooms were analyzed by HPLC after derivatization with acidic 2-nitrophenylhydrazine hydrochloride. Oleic acid was the main fatty acid in P. porrigens, and saturated long-chain fatty acids such as linoleic acid, palmitic acid, and stearic acid, together with a-eleostearic acid, were also detected.
In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.
Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.