• Title/Summary/Keyword: injection rate

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Electrical properties of metal-oxide-semiconductor structures containing Si nanocrystals fabricated by rapid thermal oxidation process (급속열처리산화법으로 형성시킨 $SiO_2$/나노결정 Si의 전기적 특성 연구)

  • Kim, Yong;Park, Kyung-Hwa;Jung, Tae-Hoon;Park, Hong-Jun;Lee, Jae-Yeol;Choi, Won-Chul;Kim, Eun-Kyu
    • Journal of the Korean Vacuum Society
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    • v.10 no.1
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    • pp.44-50
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    • 2001
  • Metal oxide semiconductor (MOS) structures containing nanocrystals are fabricated by using rapid thermal oxidations of amorphous silicon films. The amorphous films are deposited either by electron beam deposition method or by electron beam deposition assisted by Ar ion beam during deposition. Post oxidation of e-beam deposited film results in relatively small hysteresis of capacitance-voltage (C-V) and the flat band voltage shift, $\DeltaV_{FB}$ is less than 1V indicative of the formation of low density nanocrystals in $SiO_2$ near $SiO_2$/Si interface. By contrast, we observe very large hysteresis in C-V characteristics for oxidized ion-beam assisted e-beam deposited sample. The flat band voltage shift is larger than 22V and the hysteresis becomes even broader as increasing injection times of holes at accumulation condition and electrons at inversion condition. The result indicates the formation of slow traps in $SiO_2$ near $SiO_2$/Si interface which might be related to large density nanocrystals. Roughly estimated trap density is $1{\times}10^{13}cm^{-2}$. Such a large hysteresis may be explained in terms of the activation of adatom migration by Ar ion during deposition. The activated migration may increase nucleation rate of Si nuclei in amorphous Si matrix. During post oxidation process, nuclei grow into nanocrystals. Therefore, ion beam assistance during deposition may be very feasible for MOS structure containing nanocrystals with large density which is a basic building block for single electron memory device.

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Radioprotective Effect of Red Ginseng in Irradiated Mice with ${\gamma}$-ray (생쥐에서 홍삼의 감마선조사에 의한 방어효과)

  • Seung, Ka-Yeon;Lee, Heung-Man;Kim, Jong-Sang;Lee, Jun-Haeng
    • Journal of the Korean Society of Radiology
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    • v.4 no.1
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    • pp.31-35
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    • 2010
  • Recently, the incidents of direct or indirect radiation exposure due to increase of use of radiation or radioisotope are on the increase in medical and industrial circles. If cells are irradiated, free radicals are created through biological process, and cells are directly or indirectly damaged. This research intends to explore into the effect of saponin at the level of cell (in vitro) and entity (in vivo), using red ginseng extract "saponin", as radioprotective agent. In the experiment implemented at the level of cell (in vitro), degree of cell activity was measures by adding mouse mesenchymal stem cells "C3H/10T1/2 cells" into red ginseng extract "saponin(0, 0.05, 0.2, and 0.4 g/L)", and then the optimal concentration of saponin influencing cells was calculated, in 24, 48, 72, and 96 hours after gamma irradiation at the optimal concentration of saponin, each cell survival rate was observed through XTT assay. The best time period of cultivation for the optimal activity of C3H/10T1/2 cells was as 48 hours, and the degree of optimal activity was shown at 0.05 g/L. In 48 hours after irradiation of 5 Gy to C3H/10T1/2 cells at 0.05 g/L, the degree of activity of cells increased by 10%. In the experiment implemented at the level of entity (in vivo), red ginseng extract "saponin" at a dose of 100 mg/kg/day was injected into the abdominal cavity of six-week immature mouse for two weeks. Right after the last abdominal injection, total body irradiation of gamma rays was carried out at a dose of 5 Gy and 10 Gy. And after irradiation, the blood sample was taken, and then the number of red corpuscles was counted. In result, the decrement of experimental group treated with red ginseng extract "saponin" was 2.3 times larger than that of control group. In view of the results so far achieved, it was revealed that red ginseng extract "saponin" has a radiation exposure protection effect in the experiment implemented at the level of cell (in vitro). In case of animal experiment, the decrement of number of red corpuscles decreased. Finally, it is necessary to carry out more various researches continuously.

Determination and Survey of Fluoroquinolones Residue in Chicken Muscle by HPLC with Fluorescence Detector (액체크로마토그래피-형광검출기를 이용한 닭고기 중 플루오로퀴놀론계 항균물질 정량분석 및 잔류조사)

  • 박은정;임지흔;이성모
    • Journal of Food Hygiene and Safety
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    • v.19 no.1
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    • pp.12-18
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    • 2004
  • Ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin in chicken muscle were seperated by liquid extraction and determined with high performance liquid chromatography (HPLC) with fluorescence detector. Analysis was carried out using following conditions; Cl8 column (250${\times}$4.6 mm i.d. 5 ${\mu}{\textrm}{m}$ particle size), mobile phase composed of D.W. (containing 0.4% triethylamine and phospholic acid): methanol : acetonitrile (800:100:100, v/v/v), isocratic pump at a flow rate of 1.0 $m\ell$/min and 50 ${mu}ell$ of injection volume, fluorescence detector with EX278 nm/EM.456 nm. The calibration curves of four fluoroquinolones showed linearity (${\gamma}$$^2$$\geq$0.999) at concenration range of 0.025-0.6 $\mu\textrm{g}$/ml. The recoveries in fortified chicken muscle represented more than 80% with low coefficient of variation (〈10%) for concentration range of four fluoroquinolones. The detection limits for ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin were 23.5, 3.4, 3.0 and 2.5 ng/g in chicken muscle, respectively. We also monitored fluoroquinolones residue in muscle of chickens (broiler 1:227, Korean native chicken 219, laying chicken 77) using EEC-4-plate screening and HPLC conformation methods. Ten(broiler 5, Korean native chicken 5) out of the fifteen samples which were positively detected by EEC-plate screening method from 1,523 chicken meat were confirmed with ciprofloxacin and enrofloxacin by HPLC. The ranges of residual concentration were 0-0.12 ppm for ciprofloxacin and 0.01-6.79 ppm for enrofloxacin. In conclusion, our method could be applied effectively to determine four fluoroquinolones residues in chicken meat, and further survey for fluoroquinolones residue in chicken meat are needed for more effective control of fluoroquinolones used in livestock.

Biogenesis of Lysosome-related Organelle Mutant Silkworms by Direct Injection of a Cas9 Protein-guided RNA Complex into Bombyx mori Embryos (Cas9 단백질/ 가이드 RNA 복합체를 이용한 누에 BmBLOS 유전자 편집)

  • Kim, Kee Young;Yu, Jeong Hee;Kim, Su-Bae;Kim, Seong-Wan;Kim, Seong-Ryul;Choi, Kwang-Ho;Kim, Jong Gil;Park, Jong Woo
    • Journal of Life Science
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    • v.29 no.5
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    • pp.537-544
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    • 2019
  • Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

Ultrasound-Guided Axillary Brachial Plexus Block, Performed by Orthopedic Surgeons (정형외과 의사가 시행한 초음파 유도 액와 상완 신경총 차단술)

  • Kim, Cheol-U;Lee, Chul-Hyung;Yoon, Ja-Yeong;Rhee, Seung-Koo
    • Journal of the Korean Orthopaedic Association
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    • v.53 no.6
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    • pp.513-521
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    • 2018
  • Purpose: The purpose of this study was to assess the effectiveness and complications of an ultrasound-guided axillary brachial plexus block performed by orthopedic surgeons. Materials and Methods: From March to May 2017, an ultrasound-guided axillary brachial plexus block was performed on a total of 103 cases of surgery. A VF13-5 transducer from Siemens Acuson X300 was used. The surgical site was included in the range of the anatomic sensory distribution of the blocked nerve, except for the case where an operation time of more than 2 hours was expected due to multiple injuries and the operation of the upper arm. The procedure was performed by 2 orthopedic surgeons in the same method using 50 ml of solution (20 ml of lidocaine HCl in 2%, 20 ml of ropivacaine in 0.75%, 10 ml of normal saline in 0.9%). The success rate of anesthesia induction during surgery, anesthetic induction time, anatomical range of operation, duration of postoperative analgesia and complications were investigated. Results: The results from the 2 practices were similar. The anesthesia was successful in 100 out of 103 patients (97.1%). In these patients, the average needling time was 5.5 minutes (2.5-13.2 minutes), the average induction time to complete anesthesia was 18.4 minutes (5-40 minutes), and the average duration of postoperative analgesia was 402.8 minutes (141-540 minutes). The post-anesthesia immediate complications were dizziness in 1 case, nausea and vomiting in 4 cases, and peri-oral numbness in 2 cases, but surgery was performed without problems. All these 7 cases with complications recovered on the same day. A total of 3 cases failed with anesthesia, and they were treated by an injection with local anesthesia in the operation room in 2 cases and switched to general anesthesia in 1 case. Conclusion: An ultrasound-guided axillary brachial plexus block, which was performed by orthopedic surgeons allows anesthesia in a brief period and the high success rates of anesthesia for certain surgeries of the elbow and surgeries on forearm, wrist and hand. Therefore, it can reduce the waiting time to the operating room. This technique is a relatively safe procedure and dose selective anesthesia is possible.

Factors Affecting Physicians who will be Vaccinated Every Year after Receiving the COVID-19 Vaccine in Healthcare Workers (의료종사자의 COVID-19 예방 백신 접종받은 후 향후 매년 예방접종 의향에 미치는 요인)

  • Hyeun-Woo Choi;Sung-Hwa Park;Eun-Kyung Cho;Chang-hyun Han;Jong-Min Lee
    • Journal of the Korean Society of Radiology
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    • v.17 no.2
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    • pp.257-265
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    • 2023
  • The purpose of this study was to vaccinate every year according to the general characteristics of COVID-19, whether to vaccinate every year according to the vaccination experience, whether to vaccinate every year according to knowledge/attitude about vaccination, and negative responses to the vaccinate every year In order to understand the factors affecting the vaccination physician every year by identifying the factors of Statistical analysis is based on general characteristics, variables based on vaccination experience, and knowledge/attitudes related to vaccination. The doctor calculates the frequency and percentage, A square test (-test) was performed, and if the chi-square test was significant but the expected frequency was less than 5 for 25% or more, a ratio difference test was performed with Fisher's exact test. Through multiple logistic regression analysis using variables that were significant in simple analysis, a predictive model for future vaccination and the effect size of each independent variable were estimated. As statistical analysis software, SAS 9.4 (SAS Institute Inc., Cary, NC, USA) was used, and because the sample size was not large, the significance level was set at 10%, and when the p-value was less than 0.10, it was interpreted as statistically significant. In the simple logistic regression analysis, the reason why they answered that they would not be vaccinated every year was that they answered 'to prevent infection of family and hospital guests' rather than 'to prevent my infection' as the reason for the vaccination. It was 11.0 times higher and 3.67 times higher in the case of 'for the formation of collective immunity of the local community and the country'. The adverse reactions experienced after the 1st and 2nd vaccination were 8.42 times higher in those who did not experience pain at the injection site than those who did not, 4.00 times higher in those who experienced swelling or redness, and 5.69 times higher in those who experienced joint pain. There was a 5.57 times higher rate of absenteeism annually than those who did not. In addition, the more anxious they felt about vaccination, the more likely they were to not get the vaccine every year by 2.94 times.

Development of High Intensity Focused Ultrasound (HIFU) Mediated AuNP-liposomal Nanomedicine and Evaluation with PET Imaging

  • Ji Yoon Kim;Un Chul Shin;Ji Yong Park;Ran Ji Yoo;Soeku Bae;Tae Hyeon Choi;Kyuwan Kim;Young Chan Ann;Jin Sil Kim;Yu Jin Shin;Hokyu Lee;Yong Jin Lee;Kyo Chul Lee;Suhng Wook Kim;Yun-Sang Lee
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.9 no.1
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    • pp.9-16
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    • 2023
  • Liposomes as drug delivery system have proved useful carrier for various disease, including cancer. In addition, perfluorocarbon cored microbubbles are utilized in conjunction with high-intensity focused-ultrasound (HIFU) to enable simultaneous diagnosis and treatment. However, microbubbles generally exhibit lower drug loading efficiency, so the need for the development of a novel liposome-based drug delivery material that can efficiently load and deliver drugs to targeted areas via HIFU. This study aims to develop a liposome-based drug delivery material by introducing a substance that can burst liposomes using ultrasound energy and confirm the ability to target tumors using PET imaging. Liposomes (Lipo-DOX, Lipo-DOX-Au, Lipo-DOX-Au-RGD) were synthesized with gold nanoparticles using an avidin-biotin bond, and doxorubicin was mounted inside by pH gradient method. The size distribution was measured by DLS, and encapsulation efficiency of doxorubicin was analyzed by UV-vis spectrometer. The target specificity and cytotoxicity of liposomes were assessed in vitro by glioblastoma U87mg cells to HIFU treatment and analyzed using CCK-8 assay, and fluorescence microscopy at 6-hour intervals for up to 24 hours. For the in vivo study, U87mg model mouse were injected intravenously with 1.48 MBq of 64Cu-labeled Lipo-DOX-Au and Lipo-DOX-Au-RGD, and PET images were taken at 0, 2, 4, 8, and 24 hours. As a result, the size of liposomes was 108.3 ± 5.0 nm at Lipo-DOX-Au and 94.1 ± 12.2 nm at Lipo-DOX-Au-RGD, and it was observed that doxorubicin was mounted inside the liposome up to 52%. After 6 hours of HIFU treatment, the viability of U87mg cells treated with Lipo-DOX-Au decreased by around 20% compared to Lipo-DOX, and Lipo-DOX-Au-RGD had a higher uptake rate than Lipo-DOX. In vivo study using PET images, it was confirmed that 64Cu-Lipo-DOX-Au-RGD was taken up into the tumor immediately after injection and maintained for up to 4 hours. In this study, drugs released from liposomes-gold nanoparticles via ultrasound and RGD targeting were confirmed by non-invasive imaging. In cell-level experiments, HIFU treatment of gold nanoparticle-coupled liposomes significantly decreased tumor survival, while RGD-liposomes exhibited high tumor targeting and rapid release in vivo imaging. It is expected that the combination of these models with ultrasound is served as an effective drug delivery material with therapeutic outcomes.

Studies on the Utilization of Woodland for Livestock Farming II. Problem and Its Improvement Followed by the Join Cattle Grazing in king Won Do (임지의 축산적 이용에 관한 연구 제2보. 강원도의 새마을 "소" 임간공동방목사업의 문제점과 개선책)

  • 맹원재;윤익석;유제창;정승헌
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.3 no.2
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    • pp.100-111
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    • 1983
  • The research results reported herein had the objectives to understand and analyze the present problems of saemaeul woodland joint cattle grazing system in Kang Won Do and to take steps of improvement. The study results on actual management conditions, problems analyzed and improvement plan of total 208 joint cattle grazing area which was established 105 area in 1981 and 103 area in 1982 were summarized as follows: 1. the effectiveness of joint cattle grazing projects 1) Average daily weight gain of cattle during joint cattle grazing period was 0.4kg, showing higher daily than the conventional feeding of 0.33kg. 2) Increase of total farm income over the conventional feeding system were \1,031,357,320 during the grazing period from May to October in 1982 by adapting the 208 joint cattle grazing system, of which effectiveness of weight gain was \293,075,300 and labor saving was \543,838,750. 3) According to the results of questionaire investigation from 208 joint cattle grazing area, effectiveness of joint cattle grazing system over the conventional system were (1) labor saving, (2) feed cost saving, (3) reduced diseases, (4) increase of number of feeding, (5) inspiration of joint endeavor, (6) effect of more gain, (7) easiness of feeding and feed cost savings. 2. Problems of joint cattle grazing system. 1) Shortages of grass were a problem at second year of joint cattle grazing period due to the low regrowth rate of wild grass. 2) Proper land for woodland joint cattle grazing is belonging to land of Government ownership and it is very hard to get the permission from office of forestry for cattle grazing purpose. 3) It is also difficult to find a proper time of breeding in grazing area by the difficulty of estrus detection. 4) There are a difficulty to give a proper vaccination and medical examination for the grazing cattle. 3. Improvement plans for woodland joint cattle grazing projects. 1) Obtain sufficient roughages by hoof cultivation and oversowing pasture from the second year of joint cattle grazing period. 2) In order to increase the beef production and to use for a calf production area, Government should arrange that all proper grazing land of Government owned in Kang Won Do convert into woodland joint cattle grazing area. 3) Make a good reproductive record by mixed grazing with a excellent breeding cow in a remote area. And carry out the collective artificial insemination with synchronous puberty induced by injection of puberty stimulation hormone. 4) Make a preventive injection for blackleg, twice medication of fasciola hepatica in a year, and spray and medication of tick insecticide. 4. A policy towards upbringing of woodland joint cattle grazing area. 1) Government should thoroughly investigate about a proper land for woodland joint cattle grazing from all forests. 2) When the area is suitable for the woodland joint cattle grazing, though it is national forest or restricted area, government should make it possible to establish a grazing area. 3) On the proper land foe joint cattle grazing in the remote place, Government should support for the road construction and electric fence equipments by using of national funds. 4) There should be an administrative consideration for well promotion of the project that make woodland joint cattle grazing suitable to the characteristics of Kang Won Do. 5) In order to improve the reproduction record, Government should reform the insufficiency of artificial insemination in the joint cattle grazing area. 6) In order to maintain a proper price of cow, Government should carry out the price plan. 7) When there is any request for grassland formation in the woodland joint cattle grazing area, Government should permit it with preference.

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Studies on Ancylostomiasis I. An Experimental Study on Hookworm Infection and Anemia (구충증(鉤蟲症)에 관(關)한 연구(硏究) 제1편(第1篇) 구충(鉤蟲)의 감염(感染) 및 구충성빈혈(鉤蟲性貧血)에 관(關)한 고찰(考奈))

  • Lee, Mun-Ho;Kim, Dong-Jip;Lee, Jang-Kyu;Seo, Byong-Sul;Lee, Soon-Hyung
    • The Korean Journal of Nuclear Medicine
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    • v.1 no.1
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    • pp.55-66
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    • 1967
  • In view of its prevalence in the Far East area, a more detailed knowledge on the hookworm infection is one of the very important medical problems. The present study was aimed to; determine the infectivity of the artificially hatched ancylostoma duodenale larvae in man after its oral administration, evaluate the clinical symptomatology of such infection, determine the date of first appearance of the ova in the stool, calculate the blood loss per worm per day, assess the relation-ships between the ova count, infectivity(worm load), blood loss and severity of anemia. An erythrokinetic study was also done to analyse the characteristics of hookworm anemia by means of $^{59}Fe\;and\;^{51}Cr$. Materials and Methods Ten healthy male volunteers(doctors, medical students and laboratory technicians) with the ages ranging from 21 to 40 years were selected as the experimental materials. They had no history of hookworm infection for preceding several years, and care was taken not to be exposed to reinfection. A baseline study including a through physical examinations and laboratory investigations such as complete blood counts, stool examination and estimation of the serum iron levels was done, and a vermifuge, bephenium hydroxynaphoate, was given 10 days prior to the main experiment. The ancylostoma duodenale filariform larvae were obtained in the following manner; The pure ancylostoma duodenale ova were obtained from the hookworm anemia patients and a modified filter paper method was adopted to harvest larger number of infective larvae, which were washed several times with saline. The actively moving mature larvae were put into the gelatine capsules, 150 in each, and were given to the volunteers in the fasting state with 300ml. of water. The volunteers were previously treated with intramuscular injection of 15mg. of chlorpromazine in order to prevent the eventual nausea and vomiting after the larvae intake. The clinical symptoms and signs mainly of the respiratory and gastrointestinal tracts, appearance of the ova and occult blood in the stool etc. were checked every day for the first 20 days and then twice weekly until the end of the experiment, which usually lasted for about 3 months. Roentgenological survey of the lungs was also done. The hematological changes such as the red blood cell, white blood cell and eosinophil cell counts, hemoglobin content and serum iron levels were studied. The appearance of the ova in the stool was examined by the formalin ether method and the ova were counted in triplicate on two successive days using the Stoll's dilution method. The ferrokinetic data were calculated by the modified Huff's method and the apparent half survival time of the red blood cells by the modified Gray's method. The isotopes were simultaneously tagged and injected intravenously, and then the stool and blood samples were collected as was described by Roche et al., namely, three separate 4-day stool samples with the blood sample drawing before each 4-day stool collection. The radio-activities of the stools ashfied and the blood were separately measured by the pulse-height analyser. The daily blood loss was calculated with the following formula; daily blood loss in $ml.=\frac{cpm/g\;stool{\times}weight\;in\;g\;of\;4-day\;stool}{cpm/ml\;blood{\times}4}$ The average of these three 4-day periods was given as the daily blood loss in each patient. The blood loss per day per worm was calculated by simply dividing the daily blood loss by the number of the hookworm recovered after the vermifuge given twice a week at the termination of the experiment. The iron loss in mg. through the gastrointestinal tract was estimated with the daily iron loss in $mg=\frac{g\;Hgb/100ml{\times}ml\;daily\;blood\;loss{\times}3.40}{100}$ 3.40=mg of iron per g Hgb following formula; Results 1. The respiratory symptoms such as cough and sputum were noted in almost all cases within a week after the infection, which lasted about 2 weeks. The roentgenological findings of the chest were essentially normal. A moderate degree of febril reaction appeared within 2 weeks with a duration of 3 or 4 days. 2. The gastrointestinal symptoms such as nausea, epigastric fullness, abdominal pain and loose bowel appeared in all cases immediately after the larvae intake. 3. The reduction of the red blood cell count was not remarkable, however, the hemoglobin content and especially the serum iron level showed the steady decreases until the end of the experiment. 4. The white blood cells and eosinophil cells, on the contrary, showed increases in parallel and reached peaks in 20 to 30 days after the infection. A small secondary rise was noted in 2 months. 5. The ova first appeared in the stool in 40. 1 days after the infection, ranging from 29 to 51 days, during which the occult blood reaction of the stool became also positive in almost cases. 6. The number of ova recovered per day was 164, 320 on the average, ranging from 89,500 to 253,800. The number of the worm evacuated by vermifuge was in rough correlation with the number of ova recovered. 7. The infectivity of ancylostoma duodenale was 14% on the average, ranging from 7.3 to 20.0%, which is relatively lower than those reported by other workers. 8. The mean fecal blood loss was 5.78ml. per day, with a range of from 2.6 to 11.7ml., and the mean blood loss per worm per day was 0.30ml., with a range of from 0.13 to 0.73ml., which is in rough coincidence with those reported by other authors. There appeared to exist, however, no correlation between the blood loss and the number of ova recovered. 9. The mean fecal iron loss was 2.02mg. per day, with a range of from 1.20 to 3.89mg., which is less than those appeared in the literature. 10. The mean plasma iron disappearance rate was 0.80hr., with a range of from 0.62 to 0.95hr., namely, a slight accerelation. 11. The hookworm anemia appeared to be iron deficiency in origin caused by continuous intestinal blood loss.

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Preselection of Bovine Blastocysts Expressing Exogeneous Gene Following Microinjection (외래유전자를 주입한 소 수정란에서 형질전환가능 수정란의 선발)

  • 공일근
    • Korean Journal of Animal Reproduction
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    • v.21 no.2
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    • pp.167-176
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    • 1997
  • This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

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