• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.147-151
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• 2019

Objective: The aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST). Methods: Frozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern ("a"-"g") were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed. Results: The HOST examinations showed that > 93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type "d", 98.67% in type "g", and 98.17% in type "f" spermatozoa. Conclusion: We found that the type "d" spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.

• BMB Reports
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• 제29권1호
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• pp.22-26
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• 1996

Agents that activate or inhibit the $Ca^{2+}$ release channel in cardiac sarcoplasmic reticulum (SR) were tested for their abilities to affect the activity of the SR $Ca^{2+}$-ATPase. Vesicles of junctional SR (heavy SR, HSR) from terminal cisternae were prepared from porcine cardiac muscle by density gradient centrifugation. The steady-state activity of $Ca^{2+}$-ATPases in intact HSR vesicles was/$347{\pm}5\;nmol/min{\cdot}mg$ protein (${\pm}$ SD). When the HSR vesicles were made leaky, the activity was increased to $415{\pm}5\;nmol/min{\cdot}mg$ protein. This increase is probably due to the uncoupling of HSR vesicles. Caffeine (10 mM), an agonist of the SR $Ca^{2+}$ release channel, increased $Ca^{2+}$-ATPase activity in the intact HSR vesicle preparation to $394{\pm}30\;nmol/min{\cdot}mg$ protein. However, caffeine had no significant effect in the leaky vesicle preparation and in the purified $Ca^{2+}$-ATPase preparation. The effect of caffeine on SR $Ca^{2+}$-ATPase was investigated at various concentrations of $Ca^{2+}$. Caffeine increased the pump activity over the whole range of $Ca^{2+}$ concentrations, from $1\;{\mu}M$ to $250\;{\mu}M$, in the intact HSR vesicles. When the SR $Ca^{2+}$-ATPase was inhibited by thapsigargin, no caffeine effect was observed. These results imply that the caffeine effect requires the intact vesicles and that the increase in $Ca^{2+}$-ATPase activity is not due to a direct interaction of caffeine with the enzyme. We propose that the activity of SR $Ca^{2+}$-ATPase is linked indirectly to the activity of the $Ca^{2+}$ release channel (ryanodine receptor) and may depend upon the amount of $Ca^{2+}$ released by the channels.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.3057-3062
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• 2013

Background: Selecting chemotherapy regimens guided by chemosensitivity tests can provide individualized therapies for cancer patients. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS) assay is one in vitro assay which has become widely used to evaluate the sensitivity to anticancer agents. The aim of this study was to evaluate the clinical applicability and accuracy of MTS assay for predicting chemotherapeutic response in unresectable NSCLC patients. Methods: Cancer cells were isolated from malignant pleural effusions of patients by density gradient centrifugation, and their sensitivity to eight chemotherapeutic agents was examined by MTS assay and compared with clinical response. Results: A total of 37 patients participated in this study, and MTS assay produced results successfully in 34 patients (91.9%). The sensitivity rates ranged from 8.8% to 88.2%. Twenty-four of 34 patients who received chemotherapy were evaluated for in vitro-in vivo response analysis. The correlation between in vitro chemosensitivity result and in vivo response was highly significant (P=0.003), and the total predictive accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for MTS assay were 87.5%, 94.1%, 71.4%, 88.9%, and 83.3%, respectively. The in vitro sensitivity for CDDP also showed a significant correlation with in vivo response (P=0.018, r=0.522). Conclusion: MTS assay is a preferable in vitro chemosensitivity assay that could be use to predict the response to chemotherapy and select the appropriate chemotherapy regimens for unresectable NSCLC patients, which could greatly improve therapeutic efficacy and reduce unnecessary adverse effects.

• 한국가금학회지
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• 제28권1호
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• pp.69-76
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• 2001

Avian primordial germ cells (PGCs) originate from the epiblast and appear in the germinal crescent. These PGCs enter the developing blood vessels during stage 10∼12 (H&H), circulate in the blood stream, migrate into the developing gonadal anlage and differentiate into germ cells. However, it is not clear until when the migratory ability of PGC is maintained. This study was conducted to examine whether migratory ability is present in PGCs from the gonad at later embryonic developmental stages. In the present study, gonads were dissected from 5-, 6- and 10-day old quail embryos and treated with trypsin-EDTA. Gonadal PGCs (gPGCs) were purified by Ficoll-density-gradient-centrifugation and labeled with PKH26 fluorescent dye. The PKH26-labeled gPGCs were microinjected into the blood vessel of the recipient quail embryo. Manipulated recipients were incubated for 3 days, embedded in paraffin and sdctioned. The foreign gPGCs were detected by fluorescent and confocal laser microscopy. As a result, quail gPGCs, from 10, 6 and 5 day old embryos could migrate through the recipient blood stream at early stage and settle in the gonads. Thus, results suggest that gPGCs from upto 10-day old embryos keep properties seen in circulating PGC. Therefore, the PGCs of 10-day old embryonic gonads can be used for the tools of genetic manipulation.

• 한국식물병리학회지
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• 제12권1호
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• pp.99-108
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• 1996

3종의 빙핵세균 Peudomonas syringae 8401, Pseudomonas fuorescens 8701, Erwinia herbicola 8701의 세포 외막으로부터 아무런 변성제도 사용치 않고 sucrose density gradient centrifugation, Sephacryl gel filtration chromatography, DEAE-cellulose ion exchange chromatography, non-denaturing buffer를 이용한 PAGE, electroelution, SDS-PAGE를 통해 빙핵활성 단백질을 고도로 정제할 수 있었다. P. suringae와 P. fluorescens에서는 각각 3종류(155 kD, 75 kD, 50 kD)의 빙핵활성 단백질이, E. herbicola에서는 155 kD를 제외한 2종류(75 kD, 50 kD)의 빙핵활성 단백질은 이 연구를 통해 처음 밝혀진 것으로 , 지금까지 보고된 빙핵활성 단백질(150 KD 이상)보다는 훨씬 작은 것이다. 이는 빙핵활성을 나타내는 단백질의 기본단위는 이 실험의 결과만에 의하면 최대 50 kD임을 시사한다. 이들 단백질은 그 유래된 세균의 종류나 또는 단백질 분자량의 크기에 관계없이 모두 -5.5~7.5$^{\circ}C$에서 물을 동결시키는 높은 빙핵활성을 갖고 있었다. 이는 지금까지 보고된 어느 정제단백질의 빙핵활성보다 높은 것이다. 정제된 단백질의 빙핵활성은 trypsin 처리에 의해 상실되었고, pH6~8범위에서는 안정하였으며, pH5이하, pH9이상에서는 활성을 상실하였다. 보존온도에 대한 영향은 3$0^{\circ}C$이상이 되면 점차 활성이 감소하는 경향을 보이다 37$^{\circ}C$이상에서는 활성이 완전히 상실되었다. 금속이온으로서 Hg\ulcorner이온과 SDS에 의해 활성이 상실되었으나 phosphatidylinositol의 첨가에 의해서는 활성이 약간 증가(-1$^{\circ}C$)하였다.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2000년도 제17차 정기총회 및 학술발표
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• pp.75-77
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• 2000

Retaining migratory activity is a prerequisite for the manipulation and use of PGCs. This study was conducted to examine whether migratory activity is retained in the primordial germ cells(PGCs) from gonads at the later embryonic developmental stage. In the present study, gonads were dissected from 5-, 6- and 10-day-old quail embryos and treated with trypsin-EDTA for the degradation of gonadal tissue. Gonadal PGCs (gPGCs) were purified by Ficoll density gradient centrifugation and labeled with PKH26 fluorescent dye. The PKH26-labeled gPGCs were microinjected into the blood vessels of recipient quail embryo. After further incubation of 3 days, the manipulated recipients were embedded in paraffin and sectioned. The gPGCs were detected by their fluorescence under the fluorescent microscopy and the confocal laser microscopy. As a result, 10-day-old quail gPGCs as well as 5-and 6-day-old gPGCs, could migrate to recipient embryonic gonads and settle down. These results suggest that the 10-day-old gPGCs have the properties of circulating PGCs at early stage. Therefore the PGCs from 10-day old embryonic gonads can be used for the tools of genetic manipulation.

• 한국가축번식학회지
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• 제17권4호
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• pp.271-278
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• 1994

The aim of this study was to compare the two culture systems 1) co-culture with cumulus cells and 2) chemically defined medium supplemented with amino acids (CR1aa) and fetal calf serum (FCS) of in vitro produced bovine embryos from follicular oocytes in vitro. Bovine follicular oocytes were collected from ovaries of slaughtered cows and matured in TCM199 supplemented with 10% FCS and hormones (1$\mu\textrm{g}$/ml FSH-P and 1$\mu\textrm{g}$/ml oestradiol-17$\beta$)24 hours at 39$^{\circ}C$ under 5% CO2 in air. The capacitation of spermatozoa from ejaculated or frozen bull semen was induced by centrifugation through Percoll density gradient (45%, 90%). Then capacitated spermatozoa (1$\times$106/ml) were inseminated into 50${mu}ell$ droplet containing matured follicular oocytes and incubated for 40~42 hours. Cleaved embryos of 2~4cell stage were transferred to the co-culture with cumulus cells and/or CR1aa medium supplemented with FCS. In semen source, the developmental rates to the blastocyst and the hatched blastocyst stages were higher in ejaculated semen(27.6% and 14.9%) than those of frozen-thawed semen(18.3% and 11.8%), respectively. In two culture systems, the proportions of embryonic development upto the blastocysts and the hatched blastocysts were higher of CR1aa medium (22.1% and 12.1%) than those of cumulus cell co-culture (16.8% and 5.1%), respectively. The number of cells in exapnded blastocysts was slightly higher in cumulus cells co-culture (122.6$\pm$8.5) than that in CR1aa medium (117.9$\pm$5.9). The present results indicated that the early development of in vitro produced bovine embryos can be maintained efficiently in CR1aa medium as well as in co-culture with cumulus cells.

• 한국균학회지
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• 제15권4호
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• pp.217-223
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• 1987

1. 표고버섯의 미토콘드리아는 설탕 농도 $44{\sim}50%$ 사이에서 분리정제 되었다. 2. 파장 변화에 따른 미토콘드리아성 ATPase의 비활성도는 680 nm파장에서 가장 증가하였다. 3. 680nm에서 빛 조사 시간 변화에 따른 활성도는 5분 동안 조사하였을 때 가장 증가하였다. 4. 최적 광조건(680nm, 5분)에서 조사한 이 효소의 최적 pH 및 최적 온도는 7.5와 $59^{\circ}C$였다. 5. 최적 광조건에서 얻은 이 효소는 $Fe^{3+},\;Fe^{2+},\;Mg^{2+},\;K^+$ 및 $Ca^{2+}$ 이온에 의하여 활성화되었고, $Na^+$ 이온에 의하여 억제되었다.

• 한국균학회지
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• 제15권4호
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• pp.224-230
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• 1987

표고버섯의 미토콘드리아는 설탕밀도 선형기울기 원심분리법으로 정제하여, 광감응성 mitochondrial ATPase의 유기물 효과, 광증감제 효과 및 파장 변화에 따른 $K^+$ 이온의 유입 효과를 실험하였다. 1. 이 효소는 10m mol dithiothreitol 및 0.1m mol quinacrine에 의하여 각각 139% 및 128%의 활성도를 증가시켰다. 2. $100\;{\mu}g$의 oligomycin과 1m mol phlorizin은 이 효소의 활성을 각각 48% 및 45% 억제시켰다. 3. 광증감제인 0.1m mol phenazine methosulfate는 이 효소의 활성도를 36% 촉진시켰다. 4. $K^+$ 이온 유입 효과의 최적 파장은 690nm였고, 이때의 최적 pH 및 최적 온도는 각각 7.2 및 $55^{\circ}C$였다.

• 한국균학회지
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• 제17권2호
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• pp.99-104
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• 1989

1. 최적광조사 조건, 470 nm에서 15초 동안 빛을 조사한 미토콘드리아성 ATP synthase는 1mmole DCPI에 의하여 그 활성이 85% 증가되었다. 2. 1mmole DNP, $10\;{\mu}mole$ HHQNO 및 $100\;{\mu}g/ml$의 oligomycin은 이 효소의 활성을 각각 61, 41% 및 12% 억제시켰다. 3. 최적 조건의 빛에 의한 $Fe^{3+}$ 및 $Fe^{2+}$ 이온의 유입효과에서, 5mmole $Fe^{3+}$ 및 0.5mmole $Fe^{2+}$ 이온에 의하여 이 효소 활성이 각각 14% 및 12% 증가되었다. 4. 광증감제인 phenazine methosulfate의 존재하에서 470nm의 빛을 조사한 후의 효소의 활성도가 크게 증가되는 것으로 보아, 미토콘드리아성 ATP synthase내에 미지의 흡광물질이 존재함을 추정할 수 있었다.