• Parasites, Hosts and Diseases
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• v.39 no.2
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• pp.151-160
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• 2001

Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes enconding surface Proteins , enzymes for DNA, energy Production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba.

• Journal of Plant Biotechnology
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• v.42 no.2
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• pp.93-103
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• 2015

The isolation, cloning, and characterization of an R2R3-MYB transcription factor gene (MtMYB70) from the model legume Medicago truncatula is reported. MtMYB70 consists of a 768-bp coding sequence corresponding to 255 amino acids. Sequence alignment revealed that MtMYB70 cDNA contains conserved R2R3-type MYB domains with highly divergent C terminal regions. MtMYB70 was found to have relatively low sequence homology with known R2R3-MYB genes. Phylogenetic analysis placed the R2R3-MYB domain of MtMYB70 closest to PtMYB1, a known activator of lignin biosynthesis. Overexpression of MtMYB70 under the control of the 35S promoter in transgenic poplar did not cause a significant difference in total lignin content relative to the control, but glucan content was significantly increased in transgenic poplar. Therefore, MtMYB70 might have regulatory role in the biosynthesis of cell wall structural carbohydrates.

• Journal of Microbiology
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• v.37 no.3
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• Natural Product Sciences
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• v.17 no.1
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• pp.26-32
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• 2011

Novel tyrosinase inhibitors are important for pigmentation in the skin. Following extraction of tyrosinase inhibitors from edible vegetables or fruits, we found that the Prunus persica flesh extract (PPFE) exhibited potential inhibitory activity for melanogenesis. PPFE showed tyrosinase inhibitory activity in an enzymatic assay and PPFE also significantly inhibited the melanin formation in cultured mouse melan-a cells. Moreover, real-time RT-PCR analysis revealed that the inhibition of melanin production by PPFE was closely related to marked suppression of mRNA expression of tyrosinase and tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) in melan-a cells. Further investigation found that the modulation of tyrosinase expression by PPFE was associated with the transcriptional regulation of the microphthalmia-associated transcription factor (MITF). PPFE inhibited the promoter activity of MITF and suppressed MITF mRNA expression in melan-a cells. These results indicate that PPFE down-regulates melanogenesis-associated gene expression through MITF-mediated transcriptional regulation and these events might be related to the hypopigmentary effects of PPFE.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.1
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• pp.111-118
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• 2017

Objective: Adipose tissue plays a key role in the development of obesity and diabetes. We previously reported that lignosulfonic acid suppresses the rise in blood glucose levels through the inhibition of ${\alpha}$-glucosidase activity and intestinal glucose absorption. The purpose of this study is to examine further biological activities of lignosulfonic acid. Methods: In this study, we examined the effect of lignosulfonic acid on differentiation of 3T3-L1 cells. Results: While lignosulfonic acid inhibited proliferation (mitotic clonal expansion) after induction of differentiation, lignosulfonic acid significantly increased the size of accumulated lipid droplets in the cells. Semi-quantitative reverse transcription polymerase chain reaction analysis showed that lignosulfonic acid increased the expression of the adipogenic transcription factor, peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$), leading to increased glucose transporter 4 (Glut-4) expression and 2-deoxyglucose uptake in differentiated 3T3-L1 cells. Additionally, feeding lignosulfonic acid to diabetic KK-Ay mice suppressed increase of blood glucose level. Conclusion: Lignosulfonic acid may be useful as a functional anti-diabetic component of food.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.880-886
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• 2015

Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. coli-synthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results.

• Journal of Microbiology
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• v.39 no.3
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• pp.186-194
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• 2001

Scrub typhus, caused by Orientia tsutsugamushi infection, is clinically and histopathologically characterized by local as well as systemic inflammatory reactions, indicating that orientiae induce mechanisms that amplify the inflammatory response. To reveal underlying mechanisms of chemoattraction and activation of responding leukocytes, expression of chemokine and tumor necrosis factor alpha (TNF-$\alpha$) genes in murine peritoneal macrophages after infection with the obligate intracellular bacterium Ο.tsutsugamushi was investigated. The genes that were unregulated included macrophage inflammatory proteins l$\alpha$/$\beta$(MIP-l$\alpha$/$\beta$), MIP-2, monocyte chemoattractant protein 1(MCP-1), RANTES (regulated upon activation, normal T-cell expressed and secreted), gamma-interferon-inducible protein 10(IP-10) and TNF-$\alpha$. Peak expression of these chemokines and TNF-$\alpha$ was observed between 1 and 3 h after infection. These responses returned to or approached baseline preinfection levels 6 h after challenge. Semiquantitative reverse transcription (RT)-PCR analysis revealed dramatic Increases during infection in the steady-state levels of mRNA ceding for the inhibitory subunit of NF-kB (IkB$\alpha$), whose transcription is enhanced by binding of NF-kB within the IkB$\alpha$promoter region. Thus, Ο. tsutsugamushi appears to be a stung inducer of chemokines and TNF-$\alpha$ which may significantly contribute to inflammation and tissue damage observed in scrub typhus by attracting and activating phagocytic leukocytes.

• Journal of Aquaculture
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• v.17 no.1
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• pp.62-68
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• 2004

A cDNA encoding the masu salmon, Oncorhynchus masou, estrogen receptor $\alpha$ (msER$\alpha$) was cloned from the pituitary gland by polymerase chain reaction (PCR). This cDNA contains an open reading frame encoding 513 amino acid residues, and the calculated molecular weight of this protein is about 56,430 Dalton. The amino acid sequences of the DNA binding and ligand binding domains of msER$\alpha$ showed high homology to those of other fish species (84-100%). Reverse transcription PCR analysis showed that the mRNA level of msER$\alpha$ in the pituitary was slightly higher in estradiol-17$\beta$(E2) injected masu salmon than that of control fish. To test the biological activity of msER$\alpha$, the cDNA was ligated to a mammalian expression vector and transfected into a gonadotrope-derived cell line, L$\beta$T2, with a reporter plasmid including estrogen responsive element. Expression of the reporter protein, luciferase, was E2 and msER$\alpha$-dependent. The masu salmon ER$\alpha$ is structurally conserved among teleost species and functions as a transcriptional activator in the pituitary cells.

• BMB Reports
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• v.30 no.4
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• pp.292-295
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• 1997

Exposure of seedling of rice (Oriza sativa cv.Dongin) to cold stress ($6^{circ}C$, 7day) induced differential gene expression. Differentially expressed polyadenylated RNA induced by low temperature were isolated and identified from the leaves of rice (Oriza sativa cv.Dongin) seedling by using the technique, differential display of reverse transcription through polymerase chain reaction (DDRT-PCR). Four bands of cDNAs were differentially displayed on the PAGE gel through DDRT-PCR, and among them three bands were those of overexpressed genes while one band was of an underexpressed gene One of the overexpressed cDNA was characterized. The size of the DDRT-PCR product was found to be about 200 bp. The sequence of the cloned DNA was compared with those of GenBank through a BLAST E-Mail server, and it was found to have no homologies in the nucleotide sequence with that of any known DNA: therefore, it was designated as RC101 The expression of the cold-stress induced-gene, RC101, was sustained with Northern Blot analysis by using the cloned DDRT-PCR product as a probe.

• The Plant Pathology Journal
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• v.15 no.3
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• pp.131-136
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• 1999

In fungi known as ascomycetes, ability to mate is controlled by a single mating type (MAT) locus with two dissimilar sequences called idiomorphs carrying genes encoding transcription factors that are unrelated to each other. Fungi requiring strains with different MAT genes to complete the sexual process are heterothallic (self-sterile); species in which as single strain is able to undergo sexual reproduction are homothallic (self-fertile). Previous analysis of sequences from several heterothallic and homothallic species of the ascomycete genus Cochliobolus showed that homothallics evolve from heterothallics and that each known Cochliobolus homothallic species arose independently, from a different heterothallic ancestral species. Here we report detailed comparative analyses of MAT sequences ad their flanking regions, and show that: (1) The level of MAT gene similarity is not correlated with reproductive life style; (2) MAT proteins from all Cochliobolus species are conserved within the transcription factor signature sequences; they are not conserved in the carboxy terminal half of MAT-1, or third of MAT-2, except in those from very closely related species; (3) A gene (ORF1) of unknown function, consistently found on the MAT flank, is more conserved than are the MAT genes themselves; (4) The intergenic sequences diverge sharply among species.