In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.
This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.
Park, Ik Soo;Sohn, Jang Won;Yoon, Ho Joo;Shin, Dong Ho;Park, Sung Soo
Tuberculosis and Respiratory Diseases
/
v.54
no.4
/
pp.439-448
/
2003
Background : Surfactant protein B(SP-B) and surfactant protein C(SP-C) are important in accelerating surface spreading of surfactant phospholipid. The glucocorticoids accelerate the morphologic differentiation of epithelial cells into type II cells and increase the rate of phosphatidylcholine synthesis. The hydrophobic surfactant protein has been shown to be upregulated by glucocorticoids in vitro, however, its regulation in vivo is not well established. Methods : The authors investigated the effects of glucocorticoid on the accumulation of mRNA encoding SP-B and SP-C protein content of the lung. Adult rats were given different doses of subcutaneous dexamethasone and sacrificed at 24 hours and 1 week. SP-B and SP-C mRNA were measured by a filter hybridization method. Results : 1) The accumulation of SP-B mRNA at 24 hours after 0.2 mg/kg dexamethasone treatment was increased by 23.7%. 2) The accumulation of SP-B mRNA at 1 week after 2 mg/kg dexamethasone treatment was significantly increased by 96.6%(P<0.001). 3) The accumulation of SP-C mRNA at 24 hours after 0.2 mg/kg dexamethasone treatment was significantly increased by 42.7%(P<0.01). 4) The accumulation of SP-C mRNA at 1 week after 2 mg/kg dexamethasone treatment was significantly increased by 60.0% (P<0.01). Conclusion : The authors concluded that dexamethasone treatment in vivo resulted in increased levels of SP-B mRNA and SP-C mRNA. These results suggested that dexamethasone stimulates the synthesis of hydrophobic proteins associated with surfactant.
Currently, as a consequence of PACS (Picture Archiving Communication System) implementation many hospitals are replacing conventional film-type interpretations of diagnostic medical images with new digital-format interpretations that can also be saved, and retrieve However, the big limitation in PACS is considered to be the lack of mobility. The purpose of this study is to determine the optimal communication packet size. This was done by considering the terms occurred in the wireless communication. After encoding medical image using JPGE2000 image compression method, This method embodied auto-error correction technique preventing the loss of packets occurred during wireless communication. A PC class server, with capabilities to load, collect data, save images, and connect with other network, was installed. Image data were compressed using JPEG2000 algorithm which supports the capability of high energy density and compression ratio, to communicate through a wireless network. Image data were also transmitted in block units coeded by JPEG2000 to prevent the loss of the packets in a wireless network. When JPGE2000 image data were decoded in a PUA (Personal Digital Assistant), it was instantaneous for a MR (Magnetic Resonance) head image of 256${\times}$256 pixels, while it took approximately 5 seconds to decode a CR (Computed Radiography) chest image of 800${\times}$790 pixels. In the transmission of the image data using a CDMA 1X module (Code-Division Multiple Access 1st Generation), 256 byte/sec was considered a stable transmission rate, but packets were lost in the intervals at the transmission rate of 1Kbyte/sec. However, even with a transmission rate above 1 Kbyte/sec, packets were not lost in wireless LAN. Current PACS are not compatible with wireless networks. because it does not have an interface between wired and wireless. Thus, the mobile JPEG2000 image viewing system was developed in order to complement mobility-a limitation in PACS. Moreover, the weak-connections of the wireless network was enhanced by re-transmitting image data within a limitations The results of this study are expected to play an interface role between the current wired-networks PACS and the mobile devices.
Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.
Background : Surfactant protein A (SP-A) is important in the regulation of surfactant secretion, synthesis and recycling. SP-A has important roles in regulating surfactant metabolism as well as in determining surfactant's physical properties. Since systemic sepsis is one of the common causes of acute respiratory distress syndrome (ARDS) and abnormalities in surfactant function have been described in ARDS, the authors investigated the effects of endotoxemia on the accumulation of mRNA encoding SP-A and SP-A protein content. Methods : Adult rats were given various doses of intraperitoneal endotoxin from Salmonella enteritidis and sacrificed at different times. SP-A mRNA was measured by filter hybridization method. Lung SP-A protein content was determined by double sandwich ELISA assay using a polyclonal antiserum raised in rabbits against purified rat SP-A. Results : 1) The accumulation of SP-A mRNA in the endotoxin treated group 24 hours after 2mg/kg and 5mg/kg endotoxin treatments was significantly increased 50.9% and 27.3%, respectively, compared to the control group (P<0.001, P<0.025). 2) The accumulation of SP-A mRNA 24 hours in the 5mg/kg endotoxin treated group was significantly increased by 26.5% compared to the control group (P<0.01). 3) Total amount of lung SP-A was not altered at 24 hours by various doses of treatment. Total lung SP-A content 144 hours after endotoxin administration was significantly decreased by 51.4% compared to the control group (P<0.01). Conclusions : The specific regulation of SP-A by various time course in vivo is evident. The late decline in SP-A protein content was unexpected and suggests that SP-A may be differentially regulated during lung inflammation. The functional significance of these alterations remains to be clarified.
Lee, Jae Young;Kim, Mi Ok;Sohn, Jang Won;Yoon, Ho Joo;Shin, Dong Ho;Kim, Tae Wha;Park, Sung Soo
Tuberculosis and Respiratory Diseases
/
v.55
no.3
/
pp.257-266
/
2003
Background : The surfactant protein A(SP-A) is important in the regulation of surfactant secretion, synthesis and recycling. Since the acute respiratory distress syndrome(ARDS) is usually viewed as the functional and morphological expression of a similar underlying lung injury casued by a variety of insults and since abnormalities in surfactant function have been described in ARDS, the authors investigated the different effects of endotoxin and thiourea on the accumulation of mRNA encoding SP-A. Methods : Sprague-Dawley rats were given 5 mg/kg intraperitoneal endotoxin from Salmonella enteritidis and 3.5 mg/kg intraperitoneal thiourea and sacrified at different time periods. Results : 1) SP-A mRNA was significantly increased 67.0% in 6 hours and 73.4% in 24 hours after 5 mg/kg endotoxin treatment respectively(P<0.005, P<0.005). 2) SP-A mRNA significantly decreased 32.9% in 24 hours after 3.5 mg/kg thiourea treatment(P<0.05). Conclusions : These results indicate that the differential regulation of surfactant protein A in vivo is evident and suggest that surfactant protein A might be differentially regulated during different kind of insults of lung injury at different time periods without altering lung wet to dry ratios.
The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.
Sohn, Dong Hyun;Sohn, Jang Won;Yoon, Ho Joo;Shin, Dong Ho;Park, Sung Soo
Tuberculosis and Respiratory Diseases
/
v.54
no.5
/
pp.510-521
/
2003
Background : The surfactant specific proteins, SP-B and SP-C are believed to be important regulators of the surfactant function and homeostasis. Since acute respiratory distress syndrome(ARDS) is usually viewed as the functional and morphological expression of a similar underlying lung injury caused by a variety of insults, and since abnormalities in the surfactant function have been described in ARDS, the authors investigated the different effects of endotoxin and thiourea on the accumulation of mRNA encoding SP-B and SP-C. Methods : Sprague-Dawley rats were given 5 mg/kg of an intraperitoneal endotoxin from Salmonella enteritidis and 3.5 mg/kg intraperitoneal thiourea and were sacrificed at different time periods. Results : 1. The SP-B mRNA levels 6 and 24 hours after the 5 mg/kg endotoxin treatment was significantly reduced by 26.1% and 50%, respectively(P<0.01, P<0.001). 2. The SP-B mRNA levels 24 hours after the 3.5 mg/kg thiourea treatment was reduced by 9.8% and 12.5%, respectively. 3. The SP-C mRNA levels 6 and 24 hours after the 5 mg/kg endotoxin treatment was significantly reduced by 38.7% and 53.6%, respectively(P<0.01, P<0.001). 4. The SP-C mRNA level 6 hours after the 3.5 mg/kg thiourea treatment was reduced by 22.8%(P<0.05). Conclusion : These results indicate that the differential regulation of the hydrophobic surfactant proteins in vivo is evident, and suggest that the hydrophobic surfactant proteins might be differentially regulated during lung injury at different time periods without altering the lung wet to dry ratios. The mechanism of these alternations at the different time periods and the different kinds of etiology remain to be determined.
Hangeul as the Korean unique characters were invented according to some character-making principles and based on scholars' exhaustive researches. While most of the characters in the world evolved naturally, Hangeul was invented based on a precise linguistic analysis of the time, and therefore, it is most scientific and reasonable among various characters throughout the world. Nevertheless, Hangeul typeface designs do not seem to inherit the ideology of scientific and reasonable Hangeul correctly. For the square forms have been used intact due to the influences from the Chinese characters which prevailed during the time. If a single set of square characters should be designed, as much as 11,172 fonts should be designed, which suggests that advantages of Mangeul may not well be used fully; Hangeul was invented to visualize every sound with the combinations of 28 vowels and consonants. Problems of such square fonts began to be identified since 1900's when typewriters were introduced first from the West. Since a typewriter is designed with 28 characters laid out on its keyboard by using such combinations, the letters may be easily combined on it. The so-called the flexibility square-format typeface was born as such. Specially, the three-pairs fonts of these can be combined up to 67 letters including vowels and consonants. The three-pairs fonts system can help to solve the problems arising form the conventional square fonts and inherit the original ideology of Hangeul invention. This study aims to review the history of the three-pairs fonts designs facilitated by mechanic encoding of Hangeul and thereupon, suggest some desirable directions for future Hangeul fonts. Since the flexibility square-format typeface is expected to evolve more and more owing to development of the digital technology, they would serve our age of information in terms of both functions and convenience. Just as Hunminjongum tried to be literally independent from the Chinese characters, so the flexibility square-format typeface designs would serve to recover identity of our Hangeul font designs.
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