• The Korean Journal of Zoology
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• v.22 no.4
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• pp.141-152
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• 1979

Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of approximately 72, 000 molecular weight as determined by both of a glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is $Mn^2+$ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The divalent cation requirement for maximum activity of RNase H is similar to those of DNA polymerase. Both DNA polymerase and RNase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. An additional RNase H without detectible polymerase activity was generated by a limited chymotrypsin digestion. This RNase H activity was inhibited equally effectively as RNase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. Neutralization and binding test showed that antibody binding to reverse transcriptase molecule did not completely inhibit the polymerase activity.

• The Korean Journal of Mycology
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• v.17 no.4
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• pp.177-183
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• 1989

Mitochondria in Pleurotus ostreatus was purified by stepped sucrose density gradient centrifugation. The mitochondrial ATP synthase was investigated by various waveof the illumination at dark room for 30 min. The mitochondrial ATP synthase activity was stimulated 2.3 fold by 480 nm illumination compared with the broad wavelength group. The mitochondrial ATP synthase activity according to various times of illumination was stimulated 4.2 fold for 15 min at 480 nm compared with the broad wavelength group. The optimum pH and optimum temperature of the mitochondrial ATP synthase were 7.5 and $56^{\circ}C$, respectively. The activity of this enzyme was stimulated by 0.5 mmol $Fe^{2+}$, 1.0 mmol $Fe^{3+}$ and 5.0 mmol $k^+$ ion, but inhibited by 0.1 mmol $Na^+$ ion.

• Korean Journal of Veterinary Research
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• v.12 no.1
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• pp.77-84
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• 1972

Throughout the studies the following experimental results were obtained and summarized. 1. Treatment of MVE virus with acetone, Tween-ether and Tween-ether-protamine sulphate caused an eight to 16 fold increase in HA activity. 2. Treatment with acetone and Tween-ether resulted in a four fold increase in CF activity. Treatment with Tween-ether-protamine sulphate decreased the activity. 3. The crude virus showed a complete loss of infectivity after treatment with Tween-ether, but three log unit was, decreased with acetone treatment. 4. The HA activity of treated and crude virus was disappeared after heating at $37^{\circ}C$ for 60 minutes but CF activity was increased. 5. Tween-ether or acetone treatment equally applicable to the preparation of haemagglutinin for HI test. 6. Zonal centrifugation of crude virus in a linear ten to 60 percent sucrose gradient showed two peaks of CF activity, and one of high buoy ant density part accompanied by HA activity and infectivity and the other of lower density part. Acetone treatment brought a decrease of the high density CF activity but not affected the second peak of low density found with crude virus, and resulted in increased HA activity and decreased infectivity. The peaks of HA, CF and infectivity after acetone treatment were not clearly separated. Tween-ether treatment caused a loss of the peak of CF activity found in the area of high density with crude virus, but the peak in the area of low density was not affected. This peak of CF activity was separated from noninfectious HA activity. The HA and CF activities were considered to be contributed by different parts of the varion.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.35-44
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• 1990

Poly (A) + RNA was isolated from mouse submaxillary gland and renin mRNA was isolated by poly (U)-sepharose chromatography and sucrose linear densiW gradient centifugation. And renin mRNA was identified by in vitro translation and immunoprecipitation. In order to construct recombinant plasmid, renin cDNA was synthesized by using reverse transcriptase and inserted into EcoRi site of PUC19. In addition, the cDNA was also synthesized using polymerase chain reaction and inserted into HindlIl site of PUC19. The recombinant plasmid was transformed into JMlO3 and the expression of the inserted renin cDNA was examined. The transformant produced renin protein having a molecular weight of 45, 000 dolton, which showed hypertensive effect upon injecting it into rabbit ear vein. A renin genomic library was prepared by inserting rabbit kidney DNA into EMBL3 phage, and was screeined for the isolation of renin gemomic DNA using renin cDNA probe.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Korean Journal of Ecology and Environment
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• v.56 no.1
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• pp.36-44
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• 2023

Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the "actual" potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.

• Journal of Periodontal and Implant Science
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• v.38 no.4
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• pp.639-644
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• 2008

Purpose: The aim of this study was to compare the number of live and dead bacteria attached to, or within, the stratified squamous epithelium lining the tissue side of the gingival sulcus. Materials and Methods: A total of 50 patients was examined and classified into healthy or diseased sites according to inflammatory status of the gingival tissue. The surface of stratified squamous epithelium was removed by gentle scraping of the gingival sulcus with curettes. The cells were processed in the laboratory by density-gradient centrifugation to separate the epithelial cells from the loose bacteria and debris. The LIVE/$DEAD^{(R)}$ $BacLight^{TM}$ Bacterial Viability Kit was applied and the specimens were observed by an epifluorescent microscope and the number of bacteria was counted. Results: Live and dead bacteria were stained to green and red, irrespectively. Generally, the number of total bacteria in the diseased sites was significantly higher than in the healthy sites. The mean number of detected bacteria in the diseased sites was $58.6{\pm}36.0$ (red bacteria $10.4{\pm}9.2$ / green bacteria $48.2{\pm}30.5$), while it was $1.5{\pm}1.7$ in the healthy sites (red bacteria $0.1{\pm}0.3$ / green bacteria $1.4{\pm}1.5$). The percentage of red bacteria was $17.5{\pm}11.2%$ in the diseased sites and $2.0{\pm}5.8%$ in the healthy sites. Conclusion: The total number of bacteria in the diseased sites was significantly higher than that of the healthy sites. The ratio and the number of red bacteria were also significantly higher in the diseased sites.

• BMB Reports
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• v.29 no.3
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• Archives of Plastic Surgery
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• v.35 no.3
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• pp.229-234
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• 2008

Purpose: There are some reports presenting that peripheral blood contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal stem cells. The purposes of this study is to isolate and characterize circulating mesenchymal progenitor cells with osteogenic potential from human peripheral blood. Methods: Human buffycoat containing mononuclear cells was harvested from peripheral blood of normal persons and isolated using a density gradient centrifugation and serially subcultured in osteogenic media for 1-4 weeks. The proliferation capability, phase-contrast microscopy, transmission electron microscopy, immunophenotype FACS analysis, Alizarin red staining and RT-PCR assays for osteogenic differentiation potential were performed. Results: The phenotype of cultured cells changed from small round or cuboidal cells at passage 1 into large spindle-shaped fibroblastic morphology cells at passage 4. Surface marker expressed CD14, but did not express CD34, CD80, CD83. Strong positive staining was observed for Alizarin reds in osteogenic medium on day 14, Using RT-PCR, the mRNA levels of bone- specific genes, such as ALP, c-bfa-1 and osteocalcin were detected. Conclusion: A new subset of peripheral blood derived progenitor cells described here has the ability to proliferate and differentiate into osteogenic cell lineages in vitro, and to be candidate for regenerative therapy.

• Journal of Korean Neurosurgical Society
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• v.56 no.5
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• pp.375-382
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• 2014

Objective : To assess the effect of bone marrow mononuclear cells (BMMNCs) transplantation in the expression of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) in spinal cord injury (SCI) in rats. Methods : BMMNCs were isolated from tibia and femur by a density gradient centrifugation. After establishment of acute transection SCI, rats were divided into experiment (BMMNCs), experiment control (0.1 M PBS infused) and sham surgery groups (laminectomy without any SCI). Locomotor function was assessed weekly for 5 weeks post-injury using BBB locomotor score and urinary bladder function daily for 4 weeks post-injury. Activity of NF-${\kappa}B$ in spinal cord was assessed by immunohistochemistry and reverse transcriptase polymerase chain reaction. Results : At each time point post-injury, sham surgery group had significantly higher Basso, Beattie, Bresnahan locomotor and urinary bladder function scores than experiment and experiment control group (p<0.05). At subsequent time interval there were gradual improvement in both experiment and experiment control group, but experiment group had higher score in comparison to experiment control group (p<0.05). Comparisons were also made for expression of activated NF-${\kappa}B$ positive cells and level of NF-${\kappa}B$ messenger RNA in spinal cord at various time points between the groups. Activated NF-${\kappa}B$ immunoreactivity and level of NF-${\kappa}B$ mRNA expression were significantly higher in control group in comparison to experiment and sham surgery group (p<0.05). Conclusion : BMMNCs transplantation attenuates the expression of NF-${\kappa}B$ in injured spinal cord tissue and thus helps in recovery of neurological function in rat models with SCI.