• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.253-257
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• 2013

This study examined the mechanism of action of a local anesthetic, lidocaine HCl. Energy transfer between the surface fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, and the hydrophobic fluorescent probe, 1,3-di(1-pyrenyl) propane, was used to determine the effect of lidocaine HCl on the thickness (D) of the synaptosomal plasma membrane vesicles (SPMV) isolated from the bovine cerebral cortex, and liposomes of the total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. The thickness (D) of the intact SPMV, SPMVTL and SPMVPL were $1.044{\pm}0.008$, $0.914{\pm}0.005$ and $0.890{\pm}0.003$ (arbitrary units, n=5) at $37^{\circ}C$ (pH 7.4), respectively. Lidocaine HCl decreased the thickness of the neuronal and model membrane lipid bilayers in a dose-dependent manner with a significant decrease in the thickness, even at 0.1 mM. The decreasing effect of lidocaine HCl on the membrane thickness might be responsible for some, but not all of its anesthetic action.

• Membrane Journal
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• v.15 no.4
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• pp.297-309
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• 2005

As a number of potential endocrine disrupting compounds (EDCs) are released into the environment, recently growing attention has been drawn to them. Therefore sensitive and reliable analytical methods are essential to monitor those compounds. In this study, complementary CC-MS and LC-MS were employed to analyze the endocrine disrupters, and the results of two methods were compared for di(2-ethylhexyl)phthalate (DEHP), benzylbutylphthalate (BBP), pentachlorophenol (PCP), and 4,4'-Isopropylidenediphenol (Bisphenol-A, or BPA). The results indicate that it was possible to lower the detection limits of EDCs by LC-MS. Also, LC-MS enabled to identify the EDCs as almost intact molecules. Furthermore, this study presented a nanofiltration membrane (MWCO 250) and a ultrafiltration membrane (MWCO 1,000) filtration system as methods far removing EDCs from drinking water containing $\gamma$-BHC, p,p'-DDE, BBP, p,p'-DDT, DEHP, PCP, and BPA. Cross-flow type nanofiltrations showed $100\%$ removal of EDCs, and the result implies that MWCO 250 nanofilter was sufficient for treatment of EDCs. The ratio of permeate flux to mass transfer coefficient of nanofiltration, high flux ultrafiltration, and low flux ultrafiltration with ultrapure water were 0.67, 3.4, and 0.44, respectively. It was found that nanofiltration and low flux ultrafiltration were operated at a diffusion dominant condition, and the high flux ultrafiltration was operated at a convection dominant condition. Furthermore, a diffusion dominant process attained reasonable rejection of EDCs. The removal in the ultrafiltration was depending on the molecular weight of an EDC, and the filtration was governed by diffusion-dominant hydrodynamic conditions.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.339-347
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• 2001

Sperm-mediated DNA transfer has a potential to markedly simplify techniques for the generation of transgenic animals. The exogenous DNA transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently introduced in the production of transgenic animals. In this study, the developmental competence and tile expression rates of transgene were investigated after injection of spermatozoon or sperm head with enhanced green fluorescent protein (EGFP) gene into the mature porcine oocytes. The porcine oocytes were injected with intact sperm, membrane-disrupted sperm or sperm head. After injection. embryos were cultured in NCSU23 medium up to the blastocyst stage, and the developmental competence and expression rates were studied. The developmental rate (67.0%) of sperm injection group was higher than that (59.7%) of sperm head injection group, and the rates of EGFP expression were also significantly different between sperm injection and sperm head injection groups (42.1 vs 20.0%) (F＜0.05). In the porcine oocytes injected with sperm treated with different methods of membrane disruption, the removal of sperm membrane did not alter the developmental competence of embryos. The rate of blastocysts at 7 days after injection with intact and membrane disrupted sperm were 15.0 and 14.2%, respectively. The EGFP expression rates, 38.4% in embryos injected with frozen-thawed sperm was higher than that, 22.4% of embryos injected with the Triton X-100 treated sperm. Prior to injection, sperm were cultured in different EGFP gene concentrations from 0.Ol to 1ng/u${mu}ell$. However, no significant difference in developmental rates of embryos among different concentrations of EGFP gene were observed. The highest expression rate of EGFP gene, 37.4% was obtained from the embryos injected with spermatozoa treated with 0.1 ng/${mu}ell$ EGFP gene. These results suggested that exogenous DNA could be attached to the membrane disrupted sperm, and that these sperm could be used as a vector carrying foreign DNA into embryos.

• Development and Reproduction
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• v.14 no.4
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• pp.281-286
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• 2010

Recent evidence has revealed that the intratesticular injection of hypertonic saline(20%) resulted in a chemically castrated state such as nadir testosterone levels in rats. To confirm the efficacy of this simple saline-injection method further, we investigated the changes in the gross and microscopic anatomy of testis. Our study comprised three groups; intact(control) group, orchidectomy group and saline-injection (experimental) group. Single dose of hypertonic saline (sterilized, $750{\mu}{\ell}/testis$) were directly administered into both testis of adult rats (about 300 g BW). Bilateral orchidectomy was performed at the same day of saline injection. Following 30 days post-injection, reproductive tissues were surgically removed, weighed and fixed for histological examination. The body weights were not changed in both orchidectomy group and saline-injection group when compared to those in intact group. The wet weights of testis were significantly decreased in saline-injection group when compared to those in intact group. The wet weights of epididymis and seminal vesicle and prostate were significantly decreased in orchidectomy group and saline-injection group when compared to those in intact group. Macroscopically, the testes exerted slight atrophy and the tunica albuginea seemed to be intact in saline injection group. Histologically, however, larger parts of testicular tissue underwent necrosis and were barely recognizable after hematoxylin-eosin staining. In the same section, only the opposite part of the injection site was stained showing abnormal state of cell layers mostly fibrosis and infiltrated leukocytes. Sloughing of immature germ cells from the basement membrane along with shedding cells in the intraluminal space was notable in most seminiferous tubules from the saline injected testis. The present study confirmed that the direct injection of hypertonic saline into testis can induce a castration-like, testosterone-depriving effects on accessory sex organs. Our findings suggest that the efficacy of this less expensive and minimally invasive method seems to be almost even with that of conventional orchidectomy and chemical castration, though more in-depth evaluation should be supported.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.309-314
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• 1994

To develop simple and efficient transformation methods of monocotyledonous plane, electroporation-mediated delivery of DNA into intact embryogenic cell clumps was investigated in zoysiagrass and rice. Calli of zoysiagrass, induced from 3-week-old immature embryos, were suspension-cultured in MS basic medium supplemented with 1.0 mg/t 2.4-D and used for elechuporation. Calli, derived from immature inflorescences of 20 mm lenth of rice, were also suspension-cultured on N6 basic medium supplemented with 1.0 mg/L 2.4-D. Suspension-cultured embryogenic cell clumps were electroporated in liqid MS medium added with a Plasmid DNA (30 $\mu\textrm{m}$/ml), pGA1074, encoding ${\beta}$-glucuronidiase (GUS). DNA delivery into the cells through cell walls and cell membrane was confirmed by the transient expression of the GUS gene. Cell clumps of zoysiagrass and rice, electroporated with 400 volt at 800 pF capacitance, expressed GUS gene activity at a mean frequency of 25 units (one unit = one clony of blue cells) per 200 ${\mu}\ell$ of packed cell volume. Untreated cells and healed non-embryogenic cells did not exhibit GUS activity These results indicate that electroporation-mediated transformation can use intact embryogenic cells (thus avoiding the use protoplasts) in zoysiagrass and rice.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• Archives of Craniofacial Surgery
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• v.11 no.1
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• pp.41-44
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• 2010

Purpose: The epicanthus is a specific feature in Asian. Many techniques have been described to eliminate the epicanthal fold: resection of glabellar skin, resection of medial canthal skin, V-Y advancement, V-W technique, modified Z-plasty, multiple Z-plasties, and others. The authors observed postoperative epiphora after correction of epicanthal fold by periciliary skin flap without damaging lacrimal duct. Methods: A 19-year-old woman underwent non-incisional blepharoplasty, septorhinoplasty, and periciliary epicanthoplasty. On her history, she didn't have any symptom of epiphora preoperatively. And there was no specific complaint of epiphora during the postoperative two weeks. However epiphora got worse from one month after the surgery. She was out of this country, so the patient re-visited the clinic on the postoperative six months for this on-going symptom. On an ophthalmologic examination, patient's lacrimal duct and sac was intact but both lacrimal puncta of the patient were covered with a thin membrane. This membrane was punctuated by a 25 gauge needle and dilated with a standard dilator. Results: After ophthalmologic treatment, no recurrence was observed during five weeks of follow-up periods. Conclusion: Both lacrimal puncta of the patient were only covered with membranes. And we could not confirm the direct relationship between periciliary epicanthoplasty and postoperative epiphora. The probable factors will be a predisposing narrowed punctum, post operative peri-punctal edema and decrease in muscular function of orbicularis oculi.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1047-1054
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• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.529-543
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• 2020

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca²⁺ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

• Journal of Sericultural and Entomological Science
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• v.32 no.1
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• pp.49-57
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• 1990

To investigate the pathway of Nuclear Polyhedrosis Virus(NPV) of Bombyx mori in early stage infection of 2nd instar larva, the larval tissues were observed under electron microscope at interval of 6, 12, 24, and 48 hours after virus inoculation. The results are as follows. 1. The intact and enveloped nucleocapsids released from the polyhedra protein in the gut lumen apparently entered with the microvilli. 2. Virus progenies were observed in columnar cell nuclei 24 hours after inoculation, but polyhedra was not seen. The enveloped virus was observed in some of the intercellular spaces between mid-gut cells. 3. Many enveloped virus particles appeared in the basement membrane. These enveloped virus particles passed the basement membrane and gathered at blood cells in heomocoeal. 4. The NPV muliplicates in nuclei of the blood cells and the tracheal cells normally.