• Archives of Pharmacal Research
• /
• v.28 no.3
• /
• pp.269-273
• /
• 2005

A furocoumarin derivative, psoralen (7H-furo[3,2-g][1]benzopyran-7-one), was isolated from the n-hexane fraction of Heracleum moellendorffii Hance. We examined the effects of psor-alen on a human Kv1.5 potassium channel (hKv1.5) cloned from human heart and stably expressed in Uk- cells. We found that psoralen inhibited the hKv1.5 current in a concentration-, use- and voltage-dependent manner with an IC$_{50}$ value of 180 $\pm$ 21 nM at +60 mV. Psoralen accelerated the inactivation kinetics of the hKv1.5 channel, and it slowed the deactivation kinetics of the hKv1.5 current resulting in a tail crossover phenomenon. These results indicate that psoralen acts on the hKv1.5 channel as an open channel blocker. Furthermore, psoralen prolonged the action potential duration of rat atrial muscles in a dose-dependent manner. Taken together, the present results strongly suggest that psoralen may be an ideal antiarrhythmic drug for atrial fibrillation.

• BMB Reports
• /
• v.35 no.2
• /
• pp.178-185
• /
• 2002

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine $Cys^{50}$, $Cys^{83}$, and $Cys^{230}$, and $lys^{81}$ residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the $Cys^{50}$ and $Cys^{230}$ mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the $Cys^{50}$ and $Cys^{230}$ mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the $Cys^{50}$ and $Cys^{230}$ residues are structurally important.

• BMB Reports
• /
• v.32 no.5
• /
• pp.436-439
• /
• 1999

Shigella sonnei is an important cause of human enteric infections. S. sonnei KNIH104S was previously reported to be isolated from Korean shigellosis patients. We cloned a 2.8-kb KpnI fragment containing the recA gene encoding a recombinase from the chromosomal DNA of S. sonnei KNIH104S. This recombinant plasmid was named pRAK28. E. coli HB101, a recA mutant, cannot grow on Luria-Bertani medium in the presence of the alkylating agent methylmethane sulfonate, however, E. coli HB101 harboring pRAK28 was found to grow on this medium. As far as we know, we are the first to sequence the recA gene from S. sonnei. This gene is composed of 1062 base pairs with an ATG initiation codon and a TAA termination codon. Nucleotide sequence comparison of the S. sonnei recA gene exhibited 99.7% and 99.5% identity with those of S. flexneri and E. coli, respectively.

• The Journal of Korean Society of Virology
• /
• v.29 no.1
• /
• pp.39-44
• /
• 1999

Warts, or verrucae, are benign epithelial proliferations of the skin and mucosa caused by infection with human papillomaviruses (HPV). It is now recognized that there are many different HPV types. Especially type3 is most frequently observed in flat wart. Other types, such as type2, 10, 14, 27, 28, 29, 38, and 41 are rarely encounted in flat wart. We describe here a simple and economic method for detection and identification of epidermodysplasia verruciformis-associated HPV. The method is based on polymerase chain reaction (PCR) amplification and restriction analysis. The method has been developed with cloned HPV DNA and DNA from clinical samples. Clinical samples are from either frozen tissue or paraffin-embedded tissue. Genomic fragments were obtained from two different HPV types (3 and 10). The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. We have tested 74 clinical samples. Only type3 among these clinical samples is detected, and one sample is involved in neither type3 nor type10.

• Archives of Pharmacal Research
• /
• v.29 no.10
• /
• pp.834-839
• /
• 2006

Torilin was purified from Torilis japonica (Houtt.) DC., and its effects on a rapidly activating delayed rectifier $K^+$ channel (hKv1.5), cloned from human heart and stably expressed in Ltk cells, as well as the corresponding $K^+$ current (the ultrarapid delayed rectifier, $I_{KUR}$) were assessed in human atrial myocytes. Using the whole cell configuration of the patch-clamp technique, torilin was found to inhibit the hKv1.5 current in time and voltage-dependent manners, with an $IC_50$ value of $2.51{\pm}0.34\;{\mu}M$ at +60 mV. Torilin accelerated the inactivation kinetics of the hKv1.5 channel, and slowed the deactivation kinetics of the hKv1.5 current, resulting in a tail crossover phenomenon. Additionally, torilin inhibited the hKv1.5 current in a use dependent manner. These results strongly suggest that torilin is a type of open-channel blocker of the hKv1.5 channel.

• Asian-Australasian Journal of Animal Sciences
• /
• v.25 no.6
• /
• pp.758-763
• /
• 2012

As a member of a subclass of immunophilins, it is controversial that FKBP38 acts an upstream regulator of mTOR signaling pathway, which control the process of cell-growth, proliferation and differentiation. In order to explore the relationship between FKBP38 and mTOR in the Cashmere goat (Capra hircus) cells, a full-length cDNA was cloned (GenBank accession number JF714970) and expression pattern was analyzed. The cloned FKBP38 gene is 1,248 bp in length, containing an open reading frame (ORF) from nucleotide 13 to 1,248 which encodes 411 amino acids, and 12 nucleotides in front of the initiation codon. The full cDNA sequence shares 98% identity with cattle, 94% with horse and 90% with human. The putative amino acid sequence shows the higher homology which is 98%, 97% and 94%, correspondingly. The bioinformatics analysis showed that FKBP38 contained a FKBP_C domain, two TPR domains and a TM domain. Psite analysis suggested that the ORF encoding protein contained a leucine-zipper pattern and a Prenyl group binding site (CAAX box). Tissue-specific expression analysis was performed by semi-quantitative RT-PCR and showed that the FKBP38 expression was detected in all the tested tissues and the highest level of mRNA accumulation was detected in testis, suggesting that FKBP38 plays an important role in goat cells.

• Journal of Life Science
• /
• v.18 no.7
• /
• pp.912-917
• /
• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.1
• /
• pp.56-63
• /
• 2013

We have characterized the putative ${\alpha}$-glucosidase gene (st2525) selected by total genome analysis from the acidothermophilic crenarchaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli, and recombinant ST2525 was purified by Ni-NTA affinity chromatography. Maximum activity was observed at $95^{\circ}C$ and pH 4.0, and the enzyme exhibited stability with half-lives of 40.1 min and 7.75 min at extremely high temperatures of $100^{\circ}C$ and $105^{\circ}C$, respectively. The enzyme retained at least 85% of its maximal activity in the pH range of 4.0-11.0. ST2525 exclusively hydrolyzed ${\alpha}$-1,4-glycosidic linkages of oligosaccharides in an exo-type manner, with highest catalytic efficiency toward maltotriose. The enzyme also displayed transglycosylation activity, converting maltose to isomaltose, panose, maltotriose, isomaltotriose, etc. From these results, ST2525 could be potentially useful for starch hydrolysis as well as novel synthesis of oligosaccharides in industry.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.8
• /
• pp.796-801
• /
• 2011

Corynebacterium tuberculostearicum B146, a strain derived from healthy human skin, contains a medium copy plasmid, p1B146. This plasmid was cloned and its complete nucleotide sequence determined. As a result, p1B146 was found to be 4,2 kb in size with a 53% G+C content, plus six open reading frames (ORFs) were distinguished. According to a computer-assisted alignment, two of the ORFs exhibited significant similarities to already-known common plasmid proteins, the first being the RepA gene, responsible for plasmid replication via a rolling-circle mechanism, and the second being an FtsK-like protein, the function of which remains unclear. The presence and quantity of RNA fragments in the putative ORFs were also evaluated.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.6
• /
• pp.553-559
• /
• 1998

cDNA of human thrombopoietin (hTPO) amplified by polymerase chain reaction from a cDNA library of human fetal liver was cloned. EPO-like domains ($hTPO_{153} \;or\; hTPO_{l63})\; of\; hTPO(hTPO_{332}$) were expressed in Escherichin coli using several kinds of expression systems, such as ompA secretion, thioredoxin fusion, and the $P_L$ and T7 expression systems. To obtain $hTPO_{153}$ in soluble form, $hTPO_{153}$ cDNA was fused in-frame behind the gene encoding ompA signal sequence and thioredoxin protein. When fused with either of the genes, $hTPO_{153}$ was not expressed to the detectable level. However, a high level expression of the EPO-like domain of hTPO was obtained using the PL and T7 expression system. $hTPO_{153} \;or\; hTPO_{l63} cDNA were subcloned into the pLex and pET-28a(＋) vectors under the control of the inducible$ P_L\;T_7$ promoter, respectively. Proteins expressed using pl.ex vector and pET-28a(＋) detected in insoluble forms with an expression level of about 14% and 9% of total cellular proteins, respectively, and the level of expression was rapidly diminished in 2 h after the maximum level of expression was reached.