Masahiko Kitayama

Simultaneous Determination of Residual Veterinary Drugs in Bovine, Porcine, and Chicken Muscle Using Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.

Differential gene expression profiles of red and green forms of Perilla frutescens leading to comprehensive identification of anthocyanin biosynthetic genes

Differential screening by PCR‐select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin‐accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid‐derived metabolism. The cDNAs encoding glutathione S‐transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST‐like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2S)‐naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla.

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.

Metabolome analysis of Ephedra plants with different contents of ephedrine alkaloids by using UPLC-Q-TOF-MS.

Metabolome analysis of four varieties of Ephedra plants, which contain different amounts of ephedrine alkaloids, was demonstrated in this study. The metabolites were comprehensively analyzed by using ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and the ephedrine alkaloids were also profiled. Subsequently, multivariate analyses of principal component analysis (PCA) and batch-learning self-organizing mapping (BL-SOM) analysis were applied to the raw data of the total ion chromatogram (TIC). PCA was performed to visualize the fingerprints characteristic for each Ephedra variant and the independent metabolome clusters were formed. The metabolite fingerprints were also visualized by BL-SOM analysis and were displayed as a lattice of colored individual cells which was characteristic for each Ephedra variant. BL-SOM analysis was also used for identification of chemical marker peaks because the information assigned to a cell represented either increases or decreases in peak intensities. Using this analysis, ephedrine alkaloids were successfully selected from the TICs as chemical markers for each Ephedra variant and this result suggested that BL-SOM analysis was an effective method for the selection of marker metabolites. We report our study here as a practical case of metabolomic study on medicinal resources.

Effect of high temperature on anthocyanin composition and transcription of flavonoid hydroxylase genes in ‘Pinot noir’ grapes (Vitis vinifera)

Summary Anthocyanin accumulation in grape berry skins is influenced by temperature during the maturation period. Although it has been reported that high temperatures result in lower anthocyanin concentrations in berry skins, their effects on anthocyanin composition remain unclear. To study the effect of high temperature on anthocyanin composition, the accumulation of individual anthocyanins in the skin of ‘Pinot noir’ berries was investigated in two experiments: Experiment 1, high morning and evening temperature treatment; Experiment 2, high day-time temperature treatment. High temperatures (30°C) in the morning and evening did not the affect the total anthocyanin content in the skin, but decreased the levels of delphinidin 3-glucosides, petunidin 3-glucosides and malvidin 3-glucosides, which are 3’,5’-hydroxylated, or methylated following 3’,5’-hydroxylation. A high day-time temperature (35°C) also decreased the levels of delphinidin, petunidin and malvidin 3-glucosides. In both Experiments, grape berries grown under high temperature had a low abundance of mRNA for flavonoid 3’, 5’-hydroxylase (F3’5’H) in the skin.These results suggest that changes in the accumulation of individual anthocyanins in the skins of ‘Pinot noir’ grape berries, due to high temperature, are regulated at the level of transcription of the F3’5’H gene.

Purification and cDNA Isolation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

Chloroplastic phosphoglycerate kinase (PGK) was purified to homogeneity from a soluble fraction of chloroplasts of a cell-wall-deficient mutant strain of Chlamydomonas reinhardtii (cw-15) using ammonium sulfate fractionation, Reactive Blue-72 column chromatography, and native polyacrylamide gel electrophoresis. PGK activity was attributed to a single polypeptide with a molecular mass of 42 kD. Relative purity and identity of the isolated enzyme was confirmed by N-terminal amino acid sequence determination. Antiserum against this enzyme was raised and a western blot analysis of whole-cell lysate from cw-15 cells using this anti-chloroplastic PGK serum detected a single polypeptide with a molecular mass of 42 kD. The cDNA clone corresponding to the Chlamydomonas chloroplastic PGK was isolated from a Chlamydomonas cDNA expression library using the anti-PGK serum. The cDNA sequence was determined and apparently codes for the entire precursor peptide, which consists of 461 codons. The results from Southern and northern blot analyses suggest that the chloroplastic PGK gene exists as a single copy in the nuclear genome of C. reinhardtii and is expressed as a 1.8-kb transcript. The C. reinhardtii chloroplastic PGK cDNA has 71 and 66% homology with wheat chloroplastic PGK and spinach chloroplastic PGK, respectively. Based on the deduced amino acid sequence, the chloroplastic PGK of C. reinhardtii has more similarity to plant PGKs than to other PGKs, having both prokaryotic and eukaryotic features.

A cDNA clone encoding a ferredoxin-NADP+ reductase from Chlamydomonas reinhardtii.

FNR (EC 1.18.1.2) is a flavoenzyme that plays an important role in the metabolism of photosynthetic organisms. FNR catalyzes the final step of the linear photosynthetic electron transfer chain by mediating the passage of electrons from reduced Fd to NADP+. FNR is situated at a branch point in electron flow, playing a key role in regulating the relative amounts of cyclic and noncyclic electron flow to meet the demands of the plant for ATP and reducing power. Besides these photosynthetic functions, recently FNR has been implicated in the defense response mechanisms against oxidative stress in Escherichia coli (Liochev et al., 1994). We report here the isolation of a cDNA clone encoding an FNR of the unicellular green alga Chlamydomonas reinhardtii (Table I). This clone was obtained as a false-positive signal from a cDNA library (Merchant and Bogorad, 1987) by antiphosphoglycerate kinase (EC 2.7.2.3) antibodies (Kitayama and Togasaki, 1992). The size of the cDNA insert of Chlamydomonas FNR is 1565 bp. The DNA sequence of Chlamydomonas FNR has 63, 57, and 58% homology with rice (Aoki and Ida, 1994), pea (Newman and Gray, 1988), and spinach FNR (Jansen et al., 1988), respectively. The deduced amino acid sequence of Chlamydomonas FNR has 59, 46, and 45% identity with rice, pea, and spinach FNR, respectively. Studies on the three-dimensional x-ray structure of FNR have focused on spinach FNR and the functions of amino acid residues have been assigned by Karplus et al. (1991). A11 of the amino acid residues that have been assigned specific functions, such as flavin adenine dinucleotide-binding residues and NADP-binding residues, have been well conserved in the FNR of Chlamydomonas. The homology within the putative N-terminal amino acid regions are very low when compared with N-terminal amino acid regions of a11 other species of FNRs. In addition, there is no significant identity of the putative transit peptide regions to the organellar FNR from spinach, pea, rice, and Cyanophora paradoxa (Jakowitsch et al., 1993).

Characterization of Transformant of Chlamydomonas Reinhardtii with Antisense Directed cDNA of Phosphoglycerate Kinase

In green algae, including C. reinhardtii more than 70% of the rubisco present in the chloroplast is confined to a crystal-like body called the pyrenoid (1), unlike the situation in the chloroplasts of flowering plants wherein rubisco is freely distributed throughout. Thus, green algae apparently exhibit an unique system for maintenance and/or regulation of calvin cycle enzyme(s). Recently, Henk et al. studied rubisco deficient mutants by electron microscopy and found that the appearance of the pyrenoid body and the activity of rubisco were correlated (2).