• 분석과학
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• 제22권4호
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• pp.326-335
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• 2009

계란 중에 잔류하는 6 종의 글루코코티코이드 약물을 극미량으로 분석하는 방법이 개발되었다. 동시분석을 위한 시료 추출방법과 정제방법을 확립하였으며 액체 크로마토그래프와 질량분석기의 최적화 조건을 확립하였으며, 글루코코티코이드를 분석하기 위해서는 5 g의 계란에 초산 완충용액을 사용하여 시료의 pH를 5.2로 조절한 후 효소 가수분해를 위해 Helilx pomatia를 사용하였다. 혼합물을 원심분리하여 20 mL n-헥산으로 두 번 추출하였다. 다시 HLB 카트리지에서 메탄올에 의한 추출이 이루어진 후 연속해서 실리카 카트리지에서 메탄올/에틸아세테이트를 사용하여 정제하였다. 분석물질은 역상 HPLC/ESI-MS/MS로 분석하였으며 ESI는 음이온 모드를 사용하였다. 검정곡선의 상관계수는 0.99 이상을 나타내었고 검출한계는 $0.09-0.17{\mu}g/kg$ 이었으며 회수율은 55.7-69.8%이었다. 바탕 계란 중에서 실험된 유효성 검증방법에 근거하여 확립된 분석법은 계란 중 글루코코티코이드를 수 ${\mu}g/kg$ 농도까지 검출하는데 사용될 수 있을 것이다.

• 한국동물학회지
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• 제22권3호
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• pp.103-114
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• 1979

양서류 피부과립선의 미세구조를 관찰하기 위하여 무미양서류인 무당개구리(Bombina orientalis), 두꺼비 (Bufo bufo gargarizans), 개구리 (Rana nigromaculata) 및 옴개구리 (Rana rugosa)의 피부조직은 2.5% glutaraldehyde-paraformaldehyde (pH 7.2)와 1%osmium tetroxide에 전후 고정한 후 ethanol과 acetone으로 탈수, Epon 812 mixture에 포매하여 LKB ultramicrotome으로 초박절표본을 만들어 uranyl acetate와 lead citrate으로 염색하여 JEOL-100B형 전자현미경으로 관찰하여 다음과 같은 결과를 얻었다. 1. 양서류 피부과립선은 선상피세포와 근상피세포로 이루어졌다. 2. 양서류 피부과립선의 선상피세포는 암세포들로 구성되었으나 무당개구리에서는 명세포도 관찰되었다. 3. 양서류 피부과립선은 전분지를 하였다. 4. 양서류 피부과립선 상피세포의 핵은 원형 내지 타원형으로 크고 작은 핵경함요를 보였고, heterochromatin은 주로 핵경 인접부에 많았다. 세포질에는 mitochondria가 핵주변부에 비교적 많이 산재하였고, rough-surfaced endoplasmic reticulum은 핵 주변부에 발달하였지만 smooth-surfaced endoplasmic reticulum은 미약하였다. 5. 분비과립들은 구형 또는 난형으로 높은 전자밀도를 보였으며 다소 약한 전자밀도를 보이는 과립도 관찰되었다. 6. 양서류 과립선내 분비과립들이 같은 세포내에서 다소 전자밀도의 차이를 보이는 것은 분비과립의 형성단계에 따른 농도 차이에 기인하며 그 화학적 조성은 유사하다고 생각된다.

• 한국동물학회지
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• 제21권1호
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• pp.29-39
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• 1978

양서류 피부 점액선의 선상피세포의 미세구조를 관찰하기 위하여 양서류 피부조직을 2.5% glutaraldehyde-paraformaldehyde(pH7.2) 1% osmium tetroxide에 전후 고정 한후 ethanol과 aceton으로 탈수, Epon 812에 포매 Sorvall MT-2B ultramicrotome으로 ultrathin section을 만들어 uranyl acetate와 lead citrate으로 염색하여 JEOL-100B형 전자현미경으로 관찰하여 다음과 같은 결과를 얻었다. 1. 양서류 피부 점액선은 선상피세포와 근상피세포로 이루어졌다. 2. 양서류 피부 점액선의 선상피세포는 미세구조적인 면에서 종에 따라 여러형의 세포들이 관찰되었다. a. 도롱뇽에서는 암세포와 명세포가 관찰되었다. b. 무당개구리에서는 mitochondria가 많은 세포와 원형의 분비과립을 갖는 세포들이 관찰되었다. c. 맹꽁이에서는 구형의 분비과립을 갖는 세포와 거품모양의 과립괴를 갖는 세포들이 관찰되었다. d. 개구리 피부 점액선은 A형과 B형점액선으로 구분되었고, A형점액선 상피에서는 mitochondria가 많은 세포와 원형의 분비과립을 갖는 세포, B형점액선 상피에서는 mitochondria가 많은 세포와 많은 분비과립을 갖는 세포 및 거의 전 세포질이 endoplasmic reticulum으로 채워진 세포들이 관찰되었다. 3. 위의 사실들로 미루어 양서류 피부점액선의 선상피는 mitochondria가 많은 미분화세포와 점액분비과립을 많이 함유한 성숙세포 및 전 세포질이 거의 rough endoplasmic reticulum으로 채워진 분비후기세포로 구성된다고 생각된다.

• KSBB Journal
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• 제10권1호
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• pp.63-70
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• 1995

실험실에서 자체 제작한 자유유통 전기이동 장치 에서 등전집속법을 이용한 대두 단백질의 분리를 통 해 운전 조건들이 분리에 미치는 영향을 조사하였 다. 매 실험마다 pH, 전기전도도, uv 흉광도 (280nm) 등을 측정하였고 시료의 순도는 SDS­P PAGE 분석을 통해 점검하였다. Tris와 boric acid로 처리한 대두단백질 추출액에 g glutamic acid, histidine, argmme, glycine 등 아 미노산 각 ImM과 dipeptide로 glycyl-glycine 2mM, 배경 전해액으로서 KCI ImM로 구성된 시료 의 완충액을 혼합하여 시료로 사용하였다. 분리막을 셀룰로오스 아세테이트를 사용할 경우 pH는 양극쪽에서 3, 음극쪽에서 8 정도의 값을 보였으며 2개의 변곡점을 나타내었다. 가해준 전압은 3 300V에서 lOOOV의 범위였으며 전압이 높을수록 더 나은 분리도를 얻었으나 전압을 더 높일 경우 과도한 Joule열의 발생으로 인해 한계가 있었다. 시간이 지남에 따라 단백질들은 분리조 중앙 부근에서 집속이 일어났으며 pH와 전기전도도의 변화로부터 분리 조내의 이온들이 막을 통해 전극쪽으로 이통해 가고 있음을 알 수 있었다. 완충용액의 농도를 5배로 증 가시킬 경우 300V에서 좋은 집속을 얻었으냐 10배 이상으로 농도를 높일 경우에는 분리조 입구와 출구 의 유체 온도차가 $25^{\circ}C$ 이상이 되어 단백질의 변성 이 일어날 수 있어 더 높일 수 없었다. 이온교환막을 사용할 경우 이온의 분극화현상을 일으켜 U자 형태 의 전기전도도 분포를 나타내었다. 아미노산 혼합물 대신 상용의 ampholyte를 사용하더라도 분리도에 있어 큰 차이가 없었다.

• Asian Journal of Atmospheric Environment
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• 제5권3호
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• pp.146-151
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• 2011

1-Nitropyrene (1-NP), 2-NP and 2-nitrofluoranthene (2-NFR) are useful markers for studying the atmospheric behaviors of polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs). However, present methods for measuring trace levels of these compounds are lesssensitive and laborious. Here we describe several improvements to a previously reported high-performance liquid chromatography-chemiluminescence detection system that allows it to determine trace levels of 1-, 2-NPs and 2-NFR. The proposed system was equipped with a reducer column packed with Pt/Rh instead of zinc whose life-time was limited. The combination of Cosmosil MS-II (monomeric ODS) and AR-II (polymeric ODS) columns was used instead of polymeric ODS columns as the separator column to improve the separation. An ethanol mixture with acetate buffer (pH 5.5) was used in place of an acetonitrile mixture with the same buffer to activate the reducer column. The same ethanol mixture was used as the mobile phase for the clean-up column. The switching time of the column switching valve was optimized to concentrate the amino-derivatives of above NPAHs quantitatively on the concentrator column. The concentrations of bis(2,4,6-trichlorophenly) oxalate and hydrogen peroxide in the chemiluminescence reagent solution were optimized to 0.4 mM and 30 mM, respectively, to increase the sensitivity. Under the above conditions, the detection limits (S/N=3) of 1-, 2-NPs and 2-NFR were 1 fmol (0.25 pg), 10 fmol (2.5 pg) and 4 fmol (1 pg), respectively. The proposed system was effectively used to determine trace levels of 1-, 2-NPs and 2-NFR in airborne particulates collected at Noto Peninsula. The atmospheric concentrations of 1-, 2-NPs and 2-NFR were not more than sub pg $m^{-3}$ levels. They were higher in winter (January) than in summer (July). In both seasons, the concentrations were in decreasing order, [2-NFR]>[1-NP]>[2-NP].

• Applied Microscopy
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• 제17권1호
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• pp.47-64
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• 1987

Morphological difference of the renal glomerulus at different age groups have been studied in young (three month-old), adult (twelve month-old) and old (thirty month-old) Fisher strain 344 rats. Pieces of the tissues were taken from renal corticies prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), following by post-fixation with 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3) and embedded within Araldite. The ultrathin sections contrasted with uranyl acetate and lead citrate were observed under a JEM 100CX electron microscope. The mean thickness of glomerular basal lamina and Bowman's capsule were determined by measuring the thinnest portion of basal lamina, and by taking the average of 50 readings from electron micrographs at different ages. The numerical changes of the slit pores were compared based upon the numbers over the length of 10um of glomerular basal lamina. The results were as follow: 1. The thickness of glomerular basal lamina is increased during aging; 140.4 nm in young rats, 270.0 nm in adult ones, and 437.8 nm in old ones. 2. The thickness of basal lamina of parietal cells of Bowman's capsule is 187.5 nm in young rats, 914.0 nm in adult ones, and 2850.0 nm in old ones. 3. The numbers of the slit pores of basal lamina are reduced during aging, 30.3 slit pores/$10{\mu}m$ in adult ones, and 24.2 slit pores/$10{\mu}m$ in old ones. 4. Accumulation of dense intracytoplasmic filamentous material in the parietal cells of Bowman's capsule is increased in the vicinity of the basal lamina during aging. The proximal tubule-like epithelial cell in Bowman's capsule is observed at one glomerulus in a young rat. 5. The endothelial cells are edematous and form balloon-like structure protruding into capillary lumen in young and old rats. 6. Cytoplasm of the podocyte shows a variety of alteration during aging, such as swelling of mitochondria and of endoplasmic reticulum, and increase of microtubules, microfilaments, lysosomes and lamellated myelin structures, etc. Accumulation of dense intracytoplasmic material in the foot processes is increased in the vicinity of the basal lamina during aging. The podocytic membrane-like structures are seen in young and o]d rats. 7. The mesangial matrices and mesangial cells are increased during aging, and slight swelling of endoplasmic reticulum and Golgi cisternae in young and old rats.

• Applied Microscopy
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• 제17권1호
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• pp.83-97
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• 1987

In order to discriminate the enteroendocrine cell types in the mucosal epithelium of the normal duodenum of the Korean hedgehog (Erinaceus koreanus). The tissues were fixed in the mixture of 1% paraformaldehyde and 1% glutaraldehyde in phosphate buffer (pH 7.2), and postfixed in 2% osmium tetroxide (phosphate buffer, pH 7.2). They were embedded in Araldite, and the ultrathin sections were made by LKB-V ultratome following the inspection of semithin sections stained with toluidine blue-borax solutions. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100B electron microscope. At least six types of enteroendocrine cells distributed in the mucosal epithelium of the duodenum were identified according to their morphological characteristics mainly based on the size, shape, number and electron density of the secretory granules. Type I cells had moderately developed organelles. The secretory granules were pleomorphic ($370X510nm$), and the granule cores with high electron density were enveloped in limiting membrane and characterized by a narrow halo. Type II cells contained an indented nucleus and well-developed organelles. The secretory granules were round (350 nm) and classified in two kinds by electron density, moderate and high. Both granules were surrounded by limiting membrane and those with high electron density showed often a wide halo. Type III cells had an indented nucleus. The secretory granules with various electron density were round (220 nm) in shape. The granules with high electron density were enveloped in limiting membrane and characterized by a narrow halo, but those with low or moderate electron density had not been observed the limiting membrane. Type IV cells contained an indented nucleus and moderately developed organelles. The secretory granules were round (180 nm) in shape, and the granule cores with high electron density were enveloped in limiting membrane and showed often a wide halo. Type V cells had a large amount of rough endoplasmic reticulum. Secretory granules with low or moderate electron density were round (230 nm) in shape, and surrounded by limiting membrane and showed a narrow halo. Type VI cells contained an oval nucleus and well-developed organelles, especially Golgi complex. The secretory granules with high electron density were round (210 nm) in shape. The granules were enveloped in limiting membrane and showed often a wide halo.

• Applied Microscopy
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• 제27권4호
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• pp.357-372
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• 1997

To study the age-related morphological differences of the retinal pigment cells and Bruch's membrane of mouse, retinae of one week-old, five weeks-old, eight weeks-old, six months-old, twelve months-old, eighteen months-old, twenty-four months-old and thirty months-old ICR mice were dissected out under anesthesia. Pieces of the tissue taken from the posterior region of the retina were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), and 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3), and embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed under a JEM 100 CX-II electron microscope. Observed results were as follows: 1. Retinae of one week old mouse exhibit that some parts of the pigment cell provided with basal foldings, whereas other parts of the one contain without basal foldings. After (ive weeks-old, all retinal pigment cells have the basal infoldings. 2. In the one week-old, stage 1 and stage 2 melanosomes were observed in the retinal pigments cells, but after five weeks-old, most of the retinal pigment cells contain some matured stage melanosomes (stage III and stage IV). 3. The phagosomes in the retinal pigment cells were increased during aging. 4. After eighteen months-old, electron dense materials are observed within the basal infoldings. 5. After eighteen months-old, the thickness of the Bruch's membrane is prominently increased. The thickness of the basal laminae of the pigment cell and the choriocapillary endothelium is more prominently increased as compared with that of the other components of the Bruch's membrane. 6. The thickness of the basal lamina of the pigment cell is more prominently increased as compared with that of the choriocapillary endothelium on aging. From the above results, it was suggested that the pigment cell and Bruch's membrane matures structurally In five weeks, and the function of the pigment cell is prominently suppressed around eighteen months-old, and thereafter the functional suppression is continued on aging.

• Applied Microscopy
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• 제27권4호
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• pp.373-389
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• 1997

Morphological difference of the renal glomerulus at different age groups have been studies in one week-old, five weeks-old, eight weeks-old, six months-old, twelve months-old, eighteen months-old, twenty-four months-old, and thirty months-old ICR mice. Pieces of the tissue taken from the renal corticies were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution (0.1 Millonig's phosphate buffer pH 7.3), and 1% osmium tetroxide solution (0.1 M Millonig's phosphate buffer, pH 7.3), and were embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate solution, and were observed under a JEM 100CX-II electron microscope. The results were as follows: 1 In the one week-old mouse, thicknesses of the three layers of the glomeluar basal lamina (lamina densa, lamina rara interna and lamina rara externa) are similar, but in the five weeks-old mouse, thick lamina densa becomes a greater portion of the thickness of whole glomerular basal lamina. 2. No difference was noticed between thickness of the renal glomerular basal lamina of the five weeks-old mouse compare with that of the one week-old one, but basal lamina of the eight weeks-old one is thickened considerably and thicknesses were maintained through twelve months-old one. After eighteen months, the thickness of the glomerular basal lamina is increased remarkably. 3. After eighteen months, electron dense deposits within the basal lamina of the renal glomeruli are observed frequently. 4. Amount of the microfilaments in the mesangial cells and the mesangial matrices are increasing during aging. 5. The thicknesses of the basal laminae of the Bowman's capsule are increasing during aging. 6. After twenty four months, the proximal tubular cell-like parietal cells with well developed microvilli are observed frequently. From the above results, it was suggested that the renal glomerulus matures structurally in five weeks, and the function of the glomerulus is suppressed after eighteen months.

• Applied Microscopy
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• 제22권2호
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• pp.1-14
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• 1992

This experiment was performed to study the morphological responses of the parafollicular cells of rat following X-ray irradiation. Male rats were divided into normal and experimental groups. The head and neck region of the rat, under sodium thiopental anesthesia, was exposed to 3,000 rads or 6,000 rads of radiation in a single dose, respectively. The source was a Mitsubishi Linear Accelerator ML-4MV. The target to skin distance was 80 cm, and the dose rate was 200 rads/min. The rate of experimental groups were sacrificed on the 6th hour, 2nd and 6th day after X-ray irradiation. Pieces of the tissue taken from the thyroid gland were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1M Millonig's phosphate buffer, pH 7.3), and in 1% osmium tetroxide (0.1M Millonig's phosphate buffer, pH 7.3), and embedded in araldite mixture. The ultrathin sections stained with uranyl acetate and lead citrate were observed with JEM 100 CX-II electron microscope. The results were as follow; 1. Two types of the parafollicular cells, according to their electron densities, were found, i. e., light cells and dark cells. 2. Three types of the parafollicular cells, according to their sizes of secretory granules were found, i.e., small granule cells ($85nm{\pm}20.1;64{\sim}102nm$), medium granule cells ($120nm{\pm}26.5;77{\sim}179nm$), and large granule cells ($165nm{\pm}25.7;128{\sim}189nm$). 3. The differential ultrastructural changes of the cells according to their cell types, i.e., dark and light cell, or small, medium and large granule cells, were hardly observed in the time and dose range covered by this study. 4. The morphological changes of the parafollicular cells were not pronounced after exposure to 3,000 rads of X-ray. 5. Swollen cisternae of the granular endoplasmic reticulum and partial cytolysis were observed after exposure to 6,000 rads of X-ray. 6. Above results suggest that the parafollicular cells showed the alterations of mitochondrial and granular endoplasmic reticular swelling, and partial cytolysis, but only in doses of 6,000 rads.