• Applied Microscopy
• /
• v.27 no.4
• /
• pp.461-472
• /
• 1997

The substantia nigra of the Mongolian gerbil was studies by tyrosine hydroxylase immunohistochemistry and immunoelectron microscopy with preembedding method. The purpose was to obtain information on the distribution and ultrastructure of the Tyrosine hydroxylase immunoreactive and dopaminergic neurons in the substantia nigra, in order to provide the necessary background for the gerbil. Large number of tyrosine hydroxylase immunoreactive neurons were located in the compact part of substantia nigra. Findings in the gerbil, compared to observations in the other species, included the presence of prominent bundles of tyrosine hydroxylase immunoreactive cytoplasmic processes passing in the dorsoventral direction from pars compacta into pars reticulata at middle and caudal levels of the substantia nigra, and the presence of a distinct tyrosine hydroxylase immunoreactive substantia nigra pars lateralis. Tyrosine hydroxylase immunoreactive neurons had well-developed cell organelles, especially rough endoplasmic reticulum, free ribosome and poly-ribosome, and showed the infoldings of the nuclear envelope. We anticipate that the present description of the cellular organization of the tyrosine hydroxylase immunoreactive dopaminergic area in the substantia nigra of gerbil will be useful for the animal experimental model of Parkinson's disease.

• Applied Microscopy
• /
• v.35 no.2
• /
• pp.73-80
• /
• 2005

To observe the morphological changes and the expression of tenascin in synovium of human knee joint, between normal condition and degenerative arthritis, were processed by immunoelectron microscopic method. The results were summarized as follows. 1. In degenerative arthritis, the hyperplasia of synovial membrane was characterized by the increase of cell number in secretory synovial cells. 2. In normal condition, there was no marking of the immuno-gold for tenascin in synovial membrane. 3. In degenerative arthritis, the immuno-gold for tenascin were observed in endoplasmic reticulum of secretory synovial cells and extracellular matrix of synovial layer. On the basis of above findings, the hyperplasia of synovial membrane and the pathologic processes may be concerned with the increase of number of secretory synovial cells and of expression of tenascin, in degenerative arthritis.

• The Korean Journal of Zoology
• /
• v.35 no.3
• /
• pp.308-321
• /
• 1992

심근세포는 골격근 세포와 더불어 세포의 분화기작을 이해하기 위한 좋은 모델이라 할 수 있다. 본 연구에서는 심근세포의 배양중에 일어나는 심근 근원섬유형성과정에 대한 이해를 위하여 SDS- 및 two-dimensional gel electrophoresis와 immunofluorescent 및 immunoelectron microscopy를 수단으로 이를 검토하였다. 배양과정에서 나타나는 주요한 단백질 중에서 SDS-PAGE에 의해 209 kDa와 53 kDa 및 46 kDa band가 각각 막분획 및 세포질 분획 단백질에서 배양과정동안에 가장 현저한 양상을 보였다. 특히 배양 3일과 4일의 양상에서 뚜렷한 변화를 나타내어 이들의 OD 격은 배양 96시간에서의 막분획에서 0.38(209 kDa)와 0.52(53 kDa) 및 배양 72시간에서의 세포질 분획에서 0.34(46 kDa)로 각각 최고치를 나타내었다. 또한 이차원 전기영동상의 양상에서도 세포질 및 막분획 단백질의 전반적인 전개양상이 배양 96시간에서 가장 두드러짐으로써 결국 배양 72시간 및 96시간대에서 심근 모세포의 분화와 관련된 일련의 단백질 합성과정이 가장 활발하다는 것을 알 수 있었다. 배양중에 발현되는 actin에 대한 형광현미경적 분석에 의해, actin은 세포내에서 불균등하게 분포하였고 근원섬유가 형성되는 시기인 배양 96시간에 세포외곽부에서 나타나는 다소 강한 염색성을 관찰할 수 있었다. 심근세포의 근원섬유는 전자현미경 관찰에 의해 primitive forms로 배양 96시간에 출현함을 알 수 있었으며 이 시기의 근원섬유의 형태는 여러 구성대(A, H and M bands)가 미약하나 1대는 비교적 뚜렷하였다. 따라서 심근세포의 T근원섬유형성과정에서 관찰되는 뚜렷한 단백질양상의 변화와 근원섬유의 출현시기 및 구성과정간에는 중요한 관련성이 있으며 이러한 과정에서 actin은 세포질내에서 불균등한 분포를 나타낸다는 것을 알 수 있었다.

• The Plant Pathology Journal
• /
• v.15 no.5
• /
• pp.270-279
• /
• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• Applied Microscopy
• /
• v.38 no.3
• /
• pp.221-233
• /
• 2008

Endogenous nitric oxide (NO) has been known to regulate many physiological and pathological processes, especially the glandular secretion and blood flow. However, nitric oxide synthase (NOS) responsible for NO synthesis has not been well studied ultrastructurally in rat salivary gland. The present study was performed to investigate the distribution of nitric Oxide synthase isoforms (endothelial. neuronal, and inducible NOS). Immunoelectron microscopic study, using monoclonal mouse anti-endothelial NOS, anti-neuronal NOS, and anti-inducible NOS, was performed in the salivary gland of rat. Endothelial NOS (eNOS)-positive immunoreactivities were most prominent in the secretory granules of serous cells of the salivary gland of the rat. Immunoreactivities were well concentrated on serous secretory granules in the serous cells. However, weak eNOS-positive immunoreactivity was observed in the mucous secretory granules of the mucous cells. Positive endothelial NOS (eNOS) immunoreactivities were most prominent in the secretory granules of intralobular ducts. Ductal secretory granules and acinar serous secretory granules have a similar pattern of labeling as eNOS suggestings. Neural NOS (nNOS)-positive immunoreactivity was not detected in duct systems or in acinar cells. Inducible NOS (iNOS)-positive immunoreactivity was not seen in acinar and ductal cells. These results reveal the presence of eNOS in the salivary gland of the rat, which may be related with regulation of the glandular secretion and blood flow through the gland.

• Applied Microscopy
• /
• v.24 no.2
• /
• pp.78-92
• /
• 1994

Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.

• Applied Microscopy
• /
• v.17 no.2
• /
• pp.1-22
• /
• 1987

Pemphigus vulgar교의 본태(本態)를 알아보기 위한 일환으로 본질병(本疾病)의 병변조직(病變組織)을 전자현미경적(電子顯微鏡的)으로 관찰(觀察)하고 본질병(本疾病)에 관여(關與)하는 세포(細胞)들에 대(對)해 면역전자현미경적(免疫電子顯微鏡的)으로 추구(追究)하였던 바, 그 결과(結果)를 보고(報告)하는 바이다. 견(犬)의 pemphigus vulgaris의 구강(口腔) 및 식도(食道)의 점막층(粘膜層)을 전자현미경적(電子顯微鏡的)으로 관찰(觀察)하였던 바 acantholysis를 일으켜도 desmosome과 기저막(基底膜)은 큰 변화(變化)가 없었으며 세포간(細胞間)은 세포간물질(細胞間物質)의 집적(集積)에 의(依)해 확장(擴張)되고 이들 세포간강물질(細胞間腔物質)은 집괴(集塊) 또는 무구조(無構造)한 물질(物質)로 나타났다. 그리고 기저세포(基底細胞)는 기저막(基底膜)에 단단히 부착(附着)되어 있었고 dendritic cell이 기저세포층(基底細胞層)위로 분포(分布)되어 있었으며, 이들 dendritic cell 중(中)에서는 가끔 여러 형태(形態)의 퇴행성변화(退行性變化)를 볼 수 있었다. Mouse 피부유리상피세포(皮膚遊離上皮細胞)에 있어서 immunogold-labeling 방법(方法)에 의해 dendritic cell을 동정(同定)하는 데에는 post-fixation, pre-embedding immunogold-labeling technique가 좋았으며 15nm와 40nm 크기의 colloid-fold 입자(粒子)로 Langerhans cell과 Thy-1양성(陽性) dendritic cell이 표식(標識)될 수 있었다. 이들 세포(細胞)들은 세포막항원(細胞膜抗元)에 따라 monoclonal antibody의 반응(反應)에 이어 치밀한 colloid-gold 입자(粒子)가 세포막표면(細胞膜表面)을 따라 일정(一定)하게 표식(標識)되었다. 또한 이들 상피세포(上皮細胞)들을 투과전자현미경적(透過電子顯微鏡的)으로 관찰(觀察)하였을 때 초미세구조(超微細構造)가 잘 보존(保存)되었으나 Langer-hans cell내(內)의 Birbeck granule은 유리전(遊離前) 피부상피조직내(皮膚上皮組織內)의 Langerhans cell내(內)의 Birbeck granule에 비(比)해 수적(數的)으로 현저히 감소(減少)되어 있었다. 그러나 Thy-1 양성(陽性) dendritic cell에서 볼 수 있는 dense-core 과립(顆粒)은 별변화(別變化)없이 쉽게 관찰(觀察)될 수 있었다. 조직배양(組織培養)을 한 견(犬)의 keratinocyte에 대(對)해 사람 pemphigus vulgaris의 항체(抗體)로 반응(反應)시킨 후 protein-A gold(15 nm)로 표식(標識)시킨 바 제일 바깥 상층(上層)의 keratinocyte에 있어서 세포막표면(細胞膜表面)을 따라 표식(標識)되어 세포막항원(細胞膜抗元)을 나타내었으며, 이와 같은 소견(所見)으로 미루어 정상피부(正常皮膚) 중층편평상피세포(重層扁平上皮細胞)에서도 동일(同一)한 소견(所見)을 관찰(觀察)할 수 있다고 본다.

• The Korean Journal of Physiology and Pharmacology
• /
• v.2 no.5
• /
• pp.617-628
• /
• 1998

In order to understand the pathogenetic mechanism of adult respiratory distress syndrome (ARDS), the role of phospholipase A2 (PLA2) in association with oxidative stress was investigated in rats. $Interleukin-1{\alpha}\;(IL-1,\;50\;{\mu}g/rat)$ was used to induce acute lung injury by neutrophilic respiratory burst. Five hours after IL-1 insufflation into trachea, microvascular integrity was disrupted, and protein leakage into the alveolar lumen was followed. An infiltration of neutrophils was clearly observed after IL-1 treatment. It was the origin of the generation of oxygen radicals causing oxidative stress in the lung. IL-1 increased tumor necrosis factor (TNF) and cytokine-induced neutrophil chemoattractant (CINC) in the bronchoalveolar lavage fluid, but mepacrine, a PLA2 inhibitor, did not change the levels of these cytokines. Although IL-1 increased PLA2 activity time-dependently, mepacrine inhibited the activity almost completely. Activation of PLA2 elevated leukotriene C4 and B4 (LTC4 and LTB4), and 6-keto-prostaglandin $F2{\alpha}\;(6-keto-PGF2{\alpha})$ was consumed completely by respiratory burst induced by IL-1. Mepacrine did not alter these changes in the contents of lipid mediators. To estimate the functional changes of alveolar barrier during the oxidative stress, quantitative changes of pulmonary surfactant, activity of gamma glutamyltransferase (GGT), and ultrastructural changes were examined. IL-1 increased the level of phospholipid in the bronchoalveolar lavage (BAL) fluid, which seemed to be caused by abnormal, pathological release of lamellar bodies into the alveolar lumen. Mepacrine recovered the amount of surfactant up to control level. IL-1 decreased GGT activity, while mepacrine restored it. In ultrastructural study, when treated with IL-1, marked necroses of endothelial cells and type II pneumocytes were observed, while mepacrine inhibited these pathological changes. In histochemical electron microscopy, increased generation of oxidants was identified around neutrophils and in the cytoplasm of type II pneumocytes. Mepacrine reduced the generation of oxidants in the tissue produced by neutrophilic respiratory burst. In immunoelectron microscopic study, PLA2 was identified in the cytoplasm of the type II pneumocytes after IL-1 treatment, but mepacrine diminished PLA2 particles in the cytoplasm of the type II pneumocyte. Based on these experimental results, it is suggested that PLA2 plays a pivotal role in inducing acute lung injury mediated by IL-1 through the oxidative stress by neutrophils. By causing endothelial damage, functional changes of pulmonary surfactant and alveolar type I pneumocyte, oxidative stress disrupts microvascular integrity and alveolar barrier.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.11
• /
• pp.1971-1982
• /
• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.