• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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• pp.201-206
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• 2004

Mitochondrial ATP-sensitive potassium $(mitoK_{ATP})$ channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of $mitoK_{ATP}$ channels remains unclear. In this study, we investigated the subunit composition of $mitoK_{ATP}$ channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while $mitoK_{ATP}$ channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of $mitoK_{ATP}$ channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands $({\sim}44,\;{\sim}46,\;and\;{\sim}30\;kDa)$ were detected, and a specific ${\sim}46-kDa$ immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of $mitoK_{ATP}$ channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

• Journal of Yeungnam Medical Science
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• 제15권2호
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• pp.286-296
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• 1998

비중격 만곡증 환자의 비내수술시 채취한 정상 하비갑개 점막조직으로 부터 상피세포만을 분리세포의 단층배양법(monolayer culture of dissociated cells)으로 배양하여 비점막 상피세포 배양법을 정립하고 또한 배양된 세포가 상피세포임을 동정하기 위하여 간접 면역형광항체법으로 상피세포 특유의 cytokeratin을 확인하였고 투과전자현미경으로 상피세포만이 가지고 있는 교소체(desmosome) 및 장세사(tonofilament) 등을 확인하였으며 6회까지 계대배양을 할 수 있었다. 향후 본 연구에서 배양된 비점막 상피세포를 이용하여 알레르기성 비염 등과 같은 비점막 염증성 질환의 병태생리에서 상피세포의 역할에 대한 연구와 호산구등 염증 세포의 침윤과 상피세포의 손상에 접착분자 및 cytokine의 상호작용에 관한 연구가 계속될 수 있을 것으로 기대한다.

• Parasites, Hosts and Diseases
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• 제48권4호
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• pp.319-324
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• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

• Asian-Australasian Journal of Animal Sciences
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• 제26권12호
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• pp.1680-1688
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• 2013

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly ($88.9{\rightarrow}114.4$). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1971-1982
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• 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.

• 생명과학회지
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• 제17권2호통권82호
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• pp.241-247
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• 2007

Paenibacillus macerans 유래의 cycloinulooligosaccharide fructanotransferase (CFTase) 유전자(cft, 2832 bp, 103.8 kDa)를 효모 표층발현 vector인 pCTcon (GAL1 promoter)에 subcloning 하였다. 구축된 pCTECFTN (9.0 kb)를 숙주세포인 s. cerevisiae EBY100에 형질전환한 후, uracil이 결핍된 배지와 inulin 함유배지에서 선별하였다. cft 유전자는 GAL1 promoter에 의해 효모 형질전환체에서 성공적으로 발현되었다. inulin으로부터 cyclofructans (CFs)로 생산하는 효소적 능력으로부터 표층발현 유무를 확인하였다. YPDG배지에서 48시간 배양 후 분획한 균체는 5.52 unit/ l 의 활성을 보였다. CF 생산을 위한 효소의 최적 반응 조건으로 pH 8.0, 반응온도 $50^{\circ}C$, 기질농도 5%, 기질은 Jerusalem artichoke 등의 inulin과 표층 발현 CFTase 효소반응 결과, cycloinulohexaose (CF6), cycloinuloheptaose (CF7), 그리고 cycloinulooctaose (CF8)이 생성되었고, 이 중에서 CF6가 주 생성물이었다.

• Journal of Periodontal and Implant Science
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• 제45권3호
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• pp.120-126
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• 2015

Purpose: With the significance of stable adhesion of alveolar bone and peri-implant soft tissue on the surface of titanium for successful dental implantation procedure, the purpose of this study was to apply microgrooves on the titanium surface and investigate their effects on peri-implant cells and tissues. Methods: Three types of commercially pure titanium discs were prepared; machined-surface discs (A), sandblasted, large-grit, acid-etched (SLA)-treated discs (B), SLA and microgroove-formed discs (C). After surface topography of the discs was examined by confocal laser scanning electron microscopy, water contact angle and surface energy were measured. Human gingival fibroblasts (hGFs) and murine osteoblastic cells (MC3T3-E1) were seeded onto the titanium discs for immunofluorescence assay of adhesion proteins. Commercially pure titanium implants with microgrooves on the coronal microthreads design were inserted into the edentulous mandible of beagle dogs. After 2 weeks and 6 weeks of implant insertion, the animal subjects were euthanized to confirm peri-implant tissue healing pattern in histologic specimens. Results: Group C presented the lowest water contact angle ($62.89{\pm}5.66{\theta}$), highest surface energy ($45{\pm}1.2mN/m$), and highest surface roughness ($Ra=22.351{\pm}2.766{\mu}m$). The expression of adhesion molecules of hGFs and MC3T30E1 cells was prominent in group C. Titanium implants with microgrooves on the coronal portion showed firm adhesion to peri-implant soft tissue. Conclusions: Microgrooves on the titanium surface promoted the adhesion of gingival fibroblasts and osteoblastic cells, as well as favorable peri-implant soft tissue sealing.

• Archives of Pharmacal Research
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• 제29권8호
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• pp.666-676
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• 2006

Gangliosides are widely distributed in mammalian cells and play important roles in various functions such as cell differentiation and growth control. In addition, diabetes and obesity cause abnormal development of reproductive processes in a variety of species. However, the mechanisms underlying these effects, and how they are related, are not fully understood. This study examined whether the differential expression of gangliosides is implicated in the abnormal follicular development and uterine architecture of streptozotocin (STZ)-induced and db/db diabetic mice. Based upon the mobility on high-performance thin-layer chromatography, mouse ovary consisted of at least five different ganglioside components, mainly gangliosides GM3, GM1, GD1a and GT1b, and diabetic ovary exhibited a significant reduction in ganglioside expression with apparent changes in the major gangliosides. A prominent immunofluorescence microscopy showed a dramatic loss of ganglioside GD1a expression in the primary, secondary and Graafian follicles of STZ-induced and db/db diabetic mice. A significant decrease in ganglioside GD3 expression was also observed in the ovary of db/db mice. In the uterus of STZ-induced diabetic mice, expression of gangliosides GD1a and GT1b was obviously reduced, but gangliosides GM1, GM2 and GD3 expression was increased. In contrast, the uterus of db/db mice showed a significant increase in gangliosides GM1, GD1a and GD3 expression. Taken together, a complex pattern of ganglioside expression was seen in the ovary and uterus of normoglycemic ICR and $db/^+$ mice, and the correspoding tissues in diabetic mice are characterized by appreciable changes of the major ganglioside expression. These results suggest that alterations in ganglioside expression caused by diabetes mellitus may be implicated in abnormal ovarian development and uterine structure.

• Journal of Periodontal and Implant Science
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• 제30권1호
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• pp.111-120
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• 2000

Purpose We designed this study for the purpose of determining the relationship between periodontal disease activity and PLBW, using the evaluation of probing pocket depth, loss of attachment, gingival index, gingival crevicular fluid amount and subgingival microflora. Methods A total of 100 volunteer mothers(mean age 30.44) at the Department of Obstetrics and Gynecology Seoul National University Hospital were selected for this study.Pregnancy outcomes were categorized into cases and controls in two ways. our definition was based on the following; Group 1 : Any PLBW cases Vs. All NBW controls Group 2 : PLBW cases Vs. NBW controls A periodontal exam was performed on the Ramfjord( #16, 21, 24, 36, 41, 44) teeth and Clinical evaluation consisted of probing pocket depth, loss of attachment, gingival index and gingival crevicular fluid amount. Subgingival plaque samples were collected by three sterile #35 paper points. The total number of anaerobic colonies and aerobic bacteria were enumerated after incubation. Antisera to P. gingivalis, P. intermedia, A. actinomycetemcomitans were produced in white rabbits with live whole cells suspensions. The specific fluorescent bacteria obtained by immunofluorescence and total cell counts obtained by dark-field microscopy were counted on four fields. The percent of each specific microorganism in the total cell count was determined. Results Any PLBW and PLBW cases showed significantly greater probing depth and attachment loss than all NBW and NBW controls. Cases group had significantly increased anaerobic bacterial counts compared with control group and no differences in the other microbes. This study confirmed that periodontal disease is a statistically significant risk factor for PLBW by investigating clinical parameters and subgingival plaque analysis.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.297-303
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• 2019

Corticosteroids are commonly used for pain control in rotator cuff tear. Deregulated $NF-{\kappa}B$ activation is a hallmark of chronic inflammatory diseases and has been responsible for the pathogenesis of rotator cuff tear. The Dexamethasone(DEXA) is a synthetic corticosteroid. The purpose of this study was to examine the exact effect of dexamethasone on $NF-{\kappa}B$ signaling in rotator cuff tear. We measured $NF-{\kappa}B$ expression in four groups: control, $TNF-{\alpha}$-treated, DEXA-treated, and combined treatment with $TNF-{\alpha}$ and DEXA. Tenocytes were isolated from patients with rotator cuff tears and pre-incubated with $TNF-{\alpha}$ (10 ng/ml), DEXA ($1{\mu}M$), or both of them for 10 min, 1 h, and 2 h. Expression of p65, p50, and p52 in the nuclei and cytosol was analyzed by western blotting and immunofluorescence imaging using confocal microscopy. We also evaluated nucleus/cytosol (N/C) ratios of p65, p50, and p52. In our study, the combined treatment with DEXA and $TNF-{\alpha}$ showed increased N/C ratios of p65, p50, and p52 compared with those in the $TNF-{\alpha}$ group at all time points. Additionally, in the DEXA group, N/C ratios of p65, p50, and p52 gradually increased from 10 min to 2 h. In conclusion, DEXA promoted the nuclear localization of p65, p50, and p52, but was not effective in inhibiting the inflammatory response of $TNF-{\alpha}$-stimulated rotator cuff tear.