• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.495-501
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• 2009

A new spore display method is presented that enables recombinant proteins to be displayed on the surface of Bacillus spores via fusion with InhA, an exosporium component of Bacillus thuringiensis. The green fluorescent protein and $\beta$-galactosidase as model proteins were fused to the C-terminal region of InhA, respectively. The surface expression of the proteins on the spores was confirmed by flow cytometry, confocal laser scanning microscopy, measurement of the enzyme activity, and an immunogold electron microscopy analysis. InhA-mediated anchoring of foreign proteins in the exosporium of Bacillus spores can provide a new method of microbial display, thereby broadening the potential for novel applications of microbial display.

• Parasites, Hosts and Diseases
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• v.39 no.2
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• pp.119-132
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• 2001

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of Dl fraction (DIA) among 5 antigenic protein fractions partially purified by DEAE- anion exchange chromatography from water- soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. DlA showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that DlA was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immune-reactivity of two immunoglobulins (PI-Ig, Dl-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with Dl-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in Dl antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue .

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

• Parasites, Hosts and Diseases
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• v.42 no.4
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• pp.185-193
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• 2004

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of $Ca^{2+}$ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and $Ca^{2+}$ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the $Ca^{2+}$ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular $Ca^{2+}$ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.

• Biomedical Science Letters
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• v.2 no.1
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• pp.1-12
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• 1996

Antigenic components reacting with IgE and IgG antibodies were localized in muscular layer of adult and of larva, sparganum. But the antigenic components inducing IgG were localized at tegument and parenchyma in addition to muscular layer in adult and sparganum. Also in sparganum, the surface of calcareous corpuscles of parenchyma showed immunoreactivity to IgG antibody. However antigenic components inducing IgE antibody were not localized in tegument and parenchyma, but in adult worm, we observed the immunopositive reaction at the lining of vitelline follicles in mature proglottis and on surface of egg shell within uterus of graved proglottis. By the method of immunogold-labelling, we observed the location of antigenic particles in tegument of sparganum. The density of antigenic particles inducing IgG was higher than that of antigen particles inducing IgE in syncytial tegument, tegument cells. A total of 43 and 36 protein bands were resolved from crude extracts of adult and sparganum, respectively, by SDS-PAGE. 34 bands from crude extracts of adult and larva were migrated to same positions. By EITB, 21 bands of 44 bands in adult were recognized with IgG antibody, and also 21 bands of 36 bands in sparganum. 13 bands of them were common antigenic components both in the adult worm and sparganum. Because 19 bands of 44 bands in adult worm were reacted with IgE antibody, they were IgE antigenic component. In sparganum, 13 bands were IgE antigenic components. 9 bands of them were common antigenic component inducing IgE antibody in both a-dult and sparganum. 3 bands of antigenic component recognized by IgE and IgG antibody were nonspecific antigen in both adult and sparganum of Spirometra erinacei.

• Applied Microscopy
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• v.40 no.4
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• pp.261-266
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• 2010

ATP is the energy source synthesized at the electron transferase that consist of complex I, II, III, IV and V in mitochondrial cristae. The complex V functions as ATPase which composed of sub-complex $F_0$ and $F_1$. Porin or VDAC (voltagedependent anion-selective channel), is a family of small pore-forming proteins of the mitochondrial outer membrane, and play important roles in the regulated flux of anion, proton and metabolites between the cytosolic and mitochondrial compartments. The channel allows the diffusion of negatively charged solutes such as succinate, malate, and ATP in the fully open state, but of positively charged ions in subconducting state. In this study, in order to investigate the relationship of the function and localization between porin and ATPase we observed the distribution of porin and ATPase in the mitochondria of the bovine heart. Monoclonal antibodies against porin and ATPase ${\beta}$-subunit were used to detect porin and ATPase using light microscope with immunohistochemistry and immunofluorescence, and using electron microscope with immunogold-labeling. ATPase were stained in longitudinal section region in cardiac muscle, porin were stained in longitudinal section region in cardiac muscle. We viewed more specific pattern of localization and distribution of these proteins using immunofluorescence method. There were some region which were labeled with porin or ATPase respectively, and others which were labeled both proteins in cardiac muscle. The electron microscope results showed that immunogold labeled porin were labeled locally at mitochondrial outer membrane and ATPase were labeled evenly at mitochondrial cristae. But ATPase was not labeled at mitochondria cristae. These results confirmed the subcellular localizations of porin and ATPase in mitochondrial outer membrane and cristae. Also, we assumed that ATP synthesis always does not activation in all mitochondria exist in the bovine cardiac muscle.

• KSBB Journal
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• v.21 no.5
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• pp.325-330
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• 2006

This study presents the characterization of an integrated portable microfluidic electrical detection system for fast and low volume immunoassay using polystyrene microbead, which are used as immobilization surfaces. In our chip, a filtration method using the microbead was adopted for sample immobilization and immunogold silver staining(IGSS) was used to increase the electrical signal. The chip is composed of an inexpensive and biocompatible Polydimethylsiloxane(PDMS) layer and Pyrex glass substrate. Platinum microelectrodes for electric signal detection were fabricated on the substrate and microchannel and pillar-type microfilters were formed in the PDMS layer. With a fabricated chip, we reacted antigen and antibody according to the procedures. Then, silver enhancer was injected to increase the size of nanogold particles tagged with the second antibody. As a result, microbeads were connected to each other and formed an electrical bridge between microelectrodes. Resistance measured through the electrodes showed a difference of two orders of magnitude between specific and nonspecific immuno-reactions. The detection limit was 10 ng/ml. The developed immunoassay chip reduced the total analysis time from 3 hours to 50 min. Fast and low-volume biochemical analysis has been successfully achieved with the developed microfilter and immuno-sensor chip, which is integrated to the microfluidic system.

• Applied Microscopy
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• v.32 no.2
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• pp.91-105
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• 2002

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). The cDNA of five isoforms of rat UT-A, UTA1, UT-A2, UT-A3, UT-A4, and UT-A5 have been cloned. The purpose of this study was to examine the expression of UT-A (L194), which marked UT-A1, UT-A2 and UT-A4. Male Sprague-Dawley rats, weighing approximately 200 g, were divided into three group: control rats had free access to water, dehydrated rats were deprived of water for 3 d, and water loaded rats had free access to 3% sucrose water for 3 d before being killed. The kidneys were preserved by in vivo perfusion through the abdominal aorta with the 2% paraformaldehyde-lysine- periodate (PLP) or 8% paraformaldehyde solution for 10 min. The sections were processed for immunohistochemical studies using pre-embedding immunoperoxidase method and immunogold method. In the normal rat kidney, UT-A1 was expressed intensely in the cytoplasm of the inner medullary collecting duct (IMCD) cell and UT-A2 was expressed on the plasma membrane of the terminal portion of the shortloop descending thin limb (DTL) cells (type I epithelium) and of the long-loop DTL cells (type II epithelium) in the initial part of the inner medulla. Immunoreactivity for UT-A1 in the IMCD cells, was decreased in dehydrated animals whereas strongly increased in water loaded animals compared with control animals. In the short-loop DTL, immunoreactivity for UT-A2 was increased in intensity in both dehydrated and water loaded groups. However, in the long-loop DTL of the outer part of the inner medulla, immunoreactivity for UT-A2 was markedly increase in intensity in dehydrated group, but not in water loaded group. In conclusion, in the rat kidney, UT-A1 is located in the cytoplasm of IMCD cells, whereas UT-A2 is located in the plasma membrane of both the short-and long-loop DTL cells. Immunohistochemistry studies revealed that UT-A1 and UT-A2 may have a different role in urea transport and are regulated by different mechanisms.

• Applied Microscopy
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• v.31 no.1
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• pp.71-83
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• 2001

Laminin, a kind of multidomain glycoproteins, is mainly localized in the basement membranes of various tissues. It is known that laminin plays an important part in mammalian lung morphogenesis. The authors have undertaken this study to investigate the changes in the distribution of laminin, and to find out cells which synthesize laminin during the organogenesis and differentiation of the lung. The fetal and neoantal rats (Sprague-Dawley strain) were used as experimental animals. The immunohisto-chemical methods were employed for detection of laminin within the developing lung tissue and the immunegold cytochemical methods were performed for detection of cells which synthesize laminin according to each stage of development. The results are as follows; 1. During fetal life, strong immunoreactivity for laminin is maintained in the basement membranes of the blood vessels and the bronchioles, the extracellular matrix of the mesenchyme, and basal lamina of the alveolar septum in the fetal rat lung. 2. After birth, laminin immunoreactivity at the alveolar septum is gradually reduced. 3. During fetal life, laminin is mainly detected within the cytoplasm of the mesenchymal cells, the endothelial cells of blood vessels and the fibroblasts in fetal rat lung. 4. According to the differentiation of type I and type II pneumocyte after birth, laminin is detected within cytoplasm of the type I pneumocytes, type II pneumocytes and fibroblasts. It is consequently suggested that laminin is largely expressed in the developing lung and laminin may be also synthesized by the type II pneumonocytes at early newborn stages.