Biomedical subjects

Muneharu Esaka

Publications and source records attributed to Muneharu Esaka.

13 recordsLinked to original sources

PubMed2006

Rapid quantification methods for genetically modified maize contents using genomic DNAs pretreated by sonication and restriction endonuclease digestion for a capillary-type real-time PCR system with a plasmid reference standard.

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.

DNA Restriction Enzymes↗

PubMed2006

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

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.

Food, Genetically Modified↗

PubMed2005

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants.

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.

Arabidopsis↗

PubMed2005

Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco.

Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and -independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA2 was at pH 8-10 when PC was used as a substrate.

Amino Acid Sequence↗

PubMed2005

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus.

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.

Amino Acid Sequence↗

PubMed2004

Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase.

L-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative L-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.

Ascorbic Acid↗

PubMed2004

The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain.

The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.

Amino Acid Sequence↗

PubMed2003

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor.

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.

Base Sequence↗

PubMed2003

A simple and eficient method for high fidelity PCR cloning using antibody-neutralizing technology.

We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.

Antibodies, Bacterial↗

PubMed2003

Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1.

Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.

Amino Acid Sequence↗

PubMed2003

Improvement of reverse transcription PCR by RNase H.

An improvement in the method of the Reverse Transcription PCR (RT-PCR) using RNase H is proposed here. We succeeded in RT-PCR amplification against the full sequence of the coding region (8.9 kb) of the Insulin-like growth factor II receptor gene which has the area called the GC-block of about 90% GC contents at the 5' terminal. Furthermore, the RNase H treatment improved the sensitivity of RT-PCR amplification against a general target.

Base Sequence↗

PubMed2002

Gene expression of ascorbic acid-related enzymes in tobacco.

GDP-D-mannose pyrophosphorylase (GMPase) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) are key enzymes in L-ascorbic acid (AsA) biosynthesis of plants, and a full-length cDNA for GMPase was isolated from tobacco using PCR. Additionally, expression of GMPase, GalLDH and other AsA-related enzymes was examined in tobacco tissues and cultured BY-2 cells, and the relationship between their expression patterns and AsA content is discussed. It was found that the expression of GalLDH and GMPase mRNAs was markedly suppressed by loading AsA, suggesting that AsA concentration in the cells may regulate AsA biosynthesis. Moreover, the expression of GMPase and GalLDH mRNAs in tobacco leaf also suggested that AsA biosynthesis may be induced by light.

Ascorbic Acid↗

PubMed2002

Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1.

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.

Cloning, Molecular↗