• Journal of mucopolysaccharidosis and rare diseases
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• 제4권1호
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• pp.26-36
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• 2018

Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and CNS degeneration. Characterization of animal model is the beginning point of the therapeutic clinical trial. Mouse model has a limitation in that it is not a human and does not have all of the disease phenotypes. Therefore, delineate of the phenotypic characteristics of MPS IIIA mouse model prerequisite for the enzyme replace treatment for the diseases. We designed 6-month duration of phenotypic characterization of MPS IIIA mouse biochemically, behaviorally and histologically. We compared height and weight of MPS IIIA mouse with wild type from 4 weeks to 6 months in both male and female. At 6 months, we measured GAG storage in urine kidney, heart, liver, lung and spleen. The brain GAG storage is presented with Alcian blue staining, immunohistochemistry, and electron-microscopy. The neurologic phenotype is evaluated by brain MRI and behavioral study including open field test, fear conditioning, T-maze test and Y-maze test. Especially behavioral tests were done serially at 4month and 6month. This study will show the result of the MPS IIIA mouse model phenotypic characterization. The MPS IIIA mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Asian-Australasian Journal of Animal Sciences
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• 제26권2호
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• Toxicological Research
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• 제17권
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• pp.157-166
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• 2001

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared to the well known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 추계 학술대회
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• pp.31-50
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• 2000

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene e(pression. However, we have now developed a series of reroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors the intron and exon sequences from heterologous cellular or viral genes are present, When compared to the well blown MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences

• 생명과학회지
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• 제16권2호
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• pp.274-281
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• 2006

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다.

• 미생물학회지
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• 제29권2호
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• pp.75-79
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• 1991

The yeast L-A virus (4.6 kb dsRNA genome) encodes the major coat protein and a "gag-pol" fusion minor coat protein that separately encapsidate itself and $M_{1}$, a 1.8 kb dsRNA satellite virus encoding a secreted protein toxin (the killer toxin). The teast chromosomal SKI genes prevent viral cytopathology by lowering the virus copy number. Thus, $ski^{-}$ mutants are ts and cs for growth. We transformed a ski2-2 virus-infested mutant with a yeast bank in a high copy cloning vector and selected the rare healthy transformants for analysis. One type of transformant segregated M-O L-A-O cells with high frequency. Elimination of the DNA clone from the ski2-2 strain eliminated this phinotype and introduction of the DNA clone recovered from such transformants into the parent ski2-2 strain, or into ski3 or ski6 mutants gave the same phenotype. This killer-curing phenotype was due to the curing of the helper L-A dsRNA virus. The 6.5 kb insert only had this activity when carried on a high copy vector and in $ski^{-}$ cells (not in $SKI^{+}$ cells). This 6.5 kb insert acts as a mutagen on L-A dsRNA producing a high rate of deletion mutations.mutations.

• The Plant Pathology Journal
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• 제22권4호
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• pp.369-374
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• 2006

To characterize benzimidazole-resistant and -sensitive Monilinia fructicola populations, the fungal isolates were obtained from peach plants showing brown rot and bloosom blight. Benzimidazole-sensitive isolates did not grow on potato dextrose agar(PDA) amended with $\geq1.0{\mu}g$ active ingredient(a.i.)/ml of the fungicides. However, benzimidazole-resistant isolates grew on PDA regardless of the tested concentrations of fungicides. Benzimidazole-resistant isolates did not grow on diethofencarb-PDA, but sensitive isolates grew on the same PDA. In the nucleotide sequences of $\beta$-tubulin gene, only codon 198(GAG: glutamic acid), a target site for benzimidazole, was replaced with GCG(alanine) in all of the resistant isolates, and this substitution seems to play an important role in the development of resistance. Other interesting codons such as 165(GCT), 200(TTC), and 241(GCT) were not changed among the isolates. Benzimidazole-resistant and -sensitive isolates were clustered clearly in random amplified polymerphic DNA analyses and the results revealed that low levels of genetic diversity between benzimidazole-sensitive and -resistant isolates of M. fructicola in the investigated regions.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1258-1266
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• 2005

The present study was carried out in order to assess the protective effects of calycosin-7-O-$\beta$-D-glucopyranoside, isolated from Astragali radix (AR), on hyaluronidase (HAase) and the recombinant human interleukin-$1\beta$ (IL-$1\beta$)-induced matrix degradation in human articular cartilage and chondrocytes. We isolated the active component from the n-butanol soluble fraction of AR (ARBu) as the HAase inhibitor and structurally identified as calycosin-7-O-$\beta$-D-glucopyranoside by LC-MS, IR, ${1}^H$ NMR, and ${13}^C$ NMR analyses. The $IC_{50}$ of this component on HAase was found to be 3.7 mg/ml by in vitro agarose plate assay. The protective effect of ARBu on the matrix gene expression of immortalized chondrocyte cell line C28/I2 treated with HAase was investigated using a reverse transcription polymerase chain reaction (RT-PCR), and its effect on HAase and IL-$1\beta$-induced matrix degradation in human articular cartilage was determined by a staining method and calculating the amount of degraded glycosaminoglycan (GAG) from the cultured media. Pretreatment with calycosin-7-O-$\beta$-D-glucopyranoside effectively protected human chondrocytes and articular cartilage from matrix degradation. Therefore, calycosin-7-O-$\beta$-D-glucopyranoside from AR appears to be a potential natural ant-inflammatory or antii-osteoarthritis agent and can be effectively used to protect from proteoglycan (PG) degradation.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.