• Archives of Pharmacal Research
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• v.18 no.2
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• pp.84-89
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• 1995

The complexation and physical characteristics of egg phosphatidylcholine (PC) liposome containing amphotericin B(AmB) were investigated through circular dichrosim(CD) spectra, the size distribution, the turbidity change, and the calcein release. CD spectra of AmB-containing egg PC mxture exhibited a positive peak around 330 nm indicative of complexation of AmB and four negative peaks. The positive peak increased up to $2.2{\;}millidegree/{\mu}g$ AmB as AmB contents increased up to 12% (w/w), suggesting that AmB-phospholipid complexation was promoted by the antibiotics. The effective diameter of liposomesa by dynamic light scattering decreased from 450 nm to 220 nm as the amount of AmB in liposomes increased from o to 30% (w/w). The complexation may be responsible for the reduction in size. On the other hand, at around 1 mN deoxycholate (DOC), the reltive turbidities of 5 and 10% (w/w) AmB-containing liposome suspension were less than 1 probably due to the soblubilization of the complex, while those of pure PC liposome suspension were larger than 1 at the same concentration. Deoxycholate-induced release of liposomes, indicating the intercalation of the drug into the bilayers. Therefore, it is concluded that in AmB/eggPC/water system, AmB-phospholipid complexcoexists with AmB-containing liposomes.

• Herbal Formula Science
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• v.32 no.1
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• pp.99-109
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• 2024

Objectives : In this study, we investigated the cytoprotective effect of Eriobotrya japonica L. (EJ) extract against Arachidonic acid (AA)+iron-induced oxidative stress. Methods : To confirm the cytoprotective effect of EJ against AA+iron-induced oxidative stress in HepG2 cells, it was evaluated by MTT assay, immunoblot anaylsis, and Calcein-AM/propidium iodide (PI) staining. Additionally, the mechanism of action of the cytoprotective effect was evaluated through molecular mechanisms. Results : EJ (100 ㎍/mL) inhibited Arachidonic acid (AA)+iron-induced cell death in a concentration-dependent manner. It also inhibited AA+iron-induced mitochondrial dysfunction and ROS production. EJ activated the LKB1-AMPK signaling pathway. Conclusions : In conclusion, EJ has the ability to protect liver cells from oxidative stress, indicating that it is related to AMPK-LKB1 signaling pathways.

• Clinical and Experimental Pediatrics
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• v.46 no.7
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• pp.702-709
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• 2003

Purpose : $IFN{\gamma}$ sentitizes many tumor cells to $TNF{\alpha}$ and FASL-mediated apoptosis by enhancing the expression of TNF or FAS/CD95 receptor and modulating the activation of caspase and Bcl-2 family. It has been reported that $IFN{\gamma}$ and $TNF{\alpha}$ synergistically caused differentiation and growth inhibition of neuroblastoma cells. Even though some neuroblastoma cell express FASR/FASL on the cell surface, they could not induce apoptosis by ligation of the FAS/CD95 receptor. But the treatment of $IFN{\gamma}$ is reported to induce apoptosis in some neuroblastoma cell lines through the CD95/CD95L autocrine circuit. In this study, we examined whether $IFN{\gamma}$ could affect $TNF{\alpha}$ and agonistic FAS/CD95 antibody(CH-11)-induced apoptosis against neuroblastoma cell lines that had shown diverse drug sensitivity and resistance. Methods : CHLA-15, CHLA-90 and LA-N-2 neuroblastoma cells were cultured in IMDM and treated with recombinant $IFN{\gamma}$, $TNF{\alpha}$ and CH-11 antibody. Cell viability was measured by DIMSCAN with a fluorescent calcein-AM. Apoptosis was analyzed through flow cytometry using Annexin V-PE and 7-ADD staining and confirmed by pancaspase and caspase-8 blocking experiments. The expression of TNF RI and FAS/CD95 receptor was evaluated by flow cytometry using the corresponding antibody and PE-conjugated secondary antibody. Results : $IFN{\gamma}$ or $TNF{\alpha}$ alone had no demonstrable cytotoxic effects, whereas both cytokines in combination induced apoptosis synergistically in CHLA-15 and CHLA-90 cells. Although there was no cytotoxicity with the ligation of CH-11 alone in CHLA-90 cells, pretreatment of $IFN{\gamma}$ increased the sensitivity of CH-11-mediated apoptosis. The expression of TNFRI and FAS/CD95R were non-specifically enhanced after treatment of $IFN{\gamma}$ without relation to sensitivity to $TNF{\alpha}$ and CH-11. This finding suggest up-regulation of both receptors may contribute to sensitization of $TNF{\alpha}$ and CH-11-mediated apoptosis by $IFN{\gamma}$ in only sensitive cell lines. Conclusion : $IFN{\gamma}$ induced sensitization of $TNF{\alpha}$ and agonistic FAS/CD95 antibody-mediated apoptosis on some neuroblastoma cells through up-regulation of TNFRI and FAS/CD95 receptor.

• Clinical and Experimental Pediatrics
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• v.49 no.4
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• pp.439-445
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• 2006

Purpose : ${\alpha}$-Galactosylceramide (${\alpha}$-GalCer)-stimulated human $V{\alpha}24$ natural killer T (NKT) cells exert antitumor activity against some leukemia in a CD1d dependent and TCR-mediated manner, but could not kill CD1d - negative neuroblastoma (NB) cells. There are few reports about the direct antitumor effect of highly secreted cytokines by these cells on activation. In this study, using a cell-free supernatant (SPN) collected from plate bound hCD1d/${\alpha}$ GalCer tetramers-stimulated NKT cells, we examined whether they could be helpful in the immunotherapeutic treatment of NB. Methods : Cells were cultured in IMDM. The cytokines produced by NKT cells were measured with Cytometric Bead Array (CBA) analysis. Cell viability was evaluated by calcein-AM fluorescence with digital image microscopy scanning (DIMSCAN). The percentage of specific apoptosis was calculated by flow cytometric detection of apoptosis using annexin V and 7-AAD. Results : The activated NKT cells secreted high levels of IL-2, INF-${\gamma}$, TNF-${\alpha}$. The SPN was significantly cytotoxic against four out of eight tested NB cell lines, through mainly apoptosis as evidenced by annexin-V staining and inhibition with the pretreatment of pancaspase blocker. This apoptosis was significantly inhibited when anti-TNF-${\alpha}$ and anti-IFN-${\gamma}$ neutralizing mAbs were used separately and it was completely abolished when the two mAbs were combined. Conclusion : IFN-${\gamma}$ and TNF-${\alpha}$ produced by NKT cells could exert synergistically direct antitumor activity through apoptosis on some NB cell lines.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.207-215
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• 2009

Purpose: The aim of the present study is to evaluate the long term bone healing after horizontal ridge augmentation using auto block bone graft for implant installation timing. Materials and Methods: Five Beagle dogs(which were 14 months old and weighted approximately 10kg). In surgery 1(extraction & bone defect), premolars(P2, P3,P4) were extracted and the buccal bone plate was removed to create a horizontally defected ridge. After three months healing, in surgery 2(ridge augmentation). Auto block bone grafts from the mandibular ramus were used in filling the bone defects were fixed with stabilizing screws. The following fluorochrome labels were given intravenously to the beagle dogs: oxytetracycline 1week after the surgery, alizarin red 4 weeks after the surgery, calcein blue 8 weeks after the surgery. The tissue samples were obtained from the sacrificed dogs of 1, 4, 8, 12, 16 weeks after the surgery. Non-decalcified sections were prepared by resin embedding and microsection to find thickness of $10{\mu}m$ for the histologic examination and analysis. Results: 1. We could achieve the successful reconstruction of the horizontal bone defect by auto block bone graft. The grafted bone block remained stable morohologically after 16 weeks of the surgery. 2. In the histologic view. We observed osteoid tissue from the sample $4^{th}$ week sample and active capillary reconstruction in the grafted bone from the $12^{th}$ week sample. Healing procedures of auto bone grafts were compared to that of the host bone. 3. Bone mineralization could be detected from the $8^{th}$ week sample. 4. Fluorochrome labeling showed active bony changes and formation at the interface of the host bone and the block graft mainly. Bony activation in the grafted bone could be seen from the $4^{th}$ week samples. Conclusions: Active bone formation and remodeling between the grafted bone and host bone can be seen through the revascularization. After the perfect adhesion to host bone, Timing of successful implant installation can be detected through the ideal ridge formation by horizontal ridge augmentation.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4597-4606
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• 2012

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median $IC_{50}$ (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and $27.86{\mu}g/ml$, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.

• Journal of Periodontal and Implant Science
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• v.29 no.1
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• pp.233-249
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• 1999

Primary fixation is one of the most important factor in establishing adequate osseointegration between implant and bone. To evaluate the initial healing response of bone around implants without primary bone contact, this study was designed to create considerable space between implant and bone in 5 mongrel dogs, about 1-year old. After 3 holes of 6.0mm in diameter were prepared at the femur neck of the dogs, commercially pure titanium thread type implants(STERI-$OSS^{(R)}$), 8mm in length and 3.8mm, 5.0mm and 6.0mm in diameter, were inserted. Implants were supported by only nonresorbable membrane($Teflon^{(R)}$), and the penetration of upper soft tissue into the gap was inhibited by it. The each implant was positioned in the center of the drilled hole. 9 implants with different diameters were inserted in 3 dogs for histologic observation, and 12 were inserted in 2 dogs for mobility test and removal torque test.Fluorescent dyes were injected in order of Doxycycline, Alizarin Red S, and Calcein at intervals of 2 weeks. At 4-, 8-, and 12-week after placement, 3 dogs were sacrificed for histologic observation, and at 8- and 12-week after placement, 2 dogs were sacrificed for mobility test using $Periotest^{(R)}$ (Simens AG, Bensheim, Germany) and torque test using Autograph AGS-1000D $series^{(R)}$(Japan). The result were as follows: 1. The wider the gap between bone and implant was, the less bone maturity was, and the later osseointegration was occurred. Trabecular direction of new bone around implant was changed from parallel to perpendicular to the implant, and the gap was filled with new bone, over time. 2. There was a decreasing tendency over time in the mobility of all implants, but the wider gap between bone and implant was, the smaller decrease of the mobility was. 3. There was a increasing tendency over time in the removal torque gauge of all implants, and the wider gap was, the smaller increase of the removal torque gauge was. The results suggest that osseointegration in case of implant without primary bone contact may be obtained by guided bone regeneration technique with prolonged healing period, but the time of second surgery should be considered carefully.

• Journal of Periodontal and Implant Science
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• v.29 no.2
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• pp.415-433
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• 1999

Implant stability is the key to long-term successful outcome for osseointegrated implants. To evaluate the initial healing response of bone around HA-coated implants without primary bone contact. 21 HA-coated thread type implants(STERI-OSS?) were placed in the femurs of 5 mongrel dogs, about 1-year old. Implants, 8 mm in length and 3.8mm(experimental 1group), 5.0mm(experimental 2group) and 6.0mm(control group) in diameter, were inserted after 3 holes of 6.0mm in diameter and 10mm in depth were prepared in the surgical sites each dog. Implants were supported by only nonresorbable membrane($Teflon^{(R)}$), in order to prevent the ingrowth of upper soft tissue into the gap between bone and implant, and to maintain each implant to be positioned in the center of the drilled hole. 9 implants with different diameters were inserted in 3 dogs for histologic observation, and 12 implants were inserted in 2 dogs for mobility test and removal torque test. Fluorescent dyes were injected for the observation of new bone formation in order of $Terramycin^{(R)}$, Arizarin $Red^{(R)}$, and $Calcein^{(R)}$ at an interval of 2 weeks. 3 dogs were sacrificed for histologic observation at 4, 8, and 12-week after placement. Light microscopy and confocal laser scanning microscopy were used to qualitatively characterize the bone around HA-coated implant. 2 dogs were sacrificed for mobility test($Periotest^{(R)}$, Simens AG, Bensheim, Germany) and removal torque test($Autograph^{(R)}$ AGS-1000D series, Japan) at 8 and 12-week after placement The results were as follows: 1. Histologic observation showed that osseointegration occurred to both control and experimental groups as time lapse, but delayed bone healing was revealed in 3.8mm group (experimental 1group), compared to contrtol group and 5.0mm group (experimental 2group). 2. The mobility test showed that the experimental groups had no distinguishable movement during experimental periods of 8 and 12-week, and there was no difference in mobility depending on the gap between bone and implant, and time lapse. 3. The removal torque forces were increased depended on the gaps decreasing between bone and implant, and time lapse. The results suggest that HA-coated implant without primary bone contact, based on guided bone regeneration could obtain its stability in all experimental groups as time lapse, but bone healing was delayed in experimental group of 3.8mm. And the results suggested that studies on correlationship between mobility test and removal torque test for implant stability would be necessary.

• Journal of Life Science
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• v.26 no.5
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• pp.620-631
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• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.41-57
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• 2001

It is well known that the apposition of bone at implant surface would be influenced by the microstructure of titanium implants. The purpose of this study was to compare bone healing around the screw-shaped titanium implant with three different surface topographies in the canine mandibles by histological and biomechanical evaluation. All mandibular premolars of six mongrel dogs were extracted and implants were placed one month later. The pure titanium implants had different surface topographies: smooth and machined ($Steri-OSS^{(R)}$: Group II); sandblasted and acid-etched ($ITI^{(R)}$, SLA: Group III) surface. The fluorescent dyes were injected on the 2nd (calcein), 4th (oxytetracycline HCI) and 12th (alizarin red) weeks of healing. Dogs were sacrificed at 4 and 12 weeks after implantation. The decalcified and undecalcified specimens were prepared for histological and histo-metrical evaluation of implant-bone contact. Some specimens at 12 weeks after implantation were used for removal torque testing. Histologically, direct bone apposition to implant surface was found in all of the treated groups. More mature and dense bone was observed at the implant-bone interface at 12 weeks than that at 4 weeks after implantation. Under the fluorescent microscope, thick regular green fluorescent lines which mean early bone apposition were observed at the implant-bone interface in Group III, while yellow and red fluorescent areas were found at the implant-bone interface in Group I and II. The average implant-bone contact ratios at 4 weeks of healing were 54.3% in Group I, 57.7% in Group II and 66.2% in Group III. In Group I, implant-bone contact ratio was significantly lower than Group II and III(p<0.05). The average implant-to-bone contact ratios at 12 weeks after implantation were 64.3% in Group I, 66.7% in Group II and 71.2% in Group III. There was no significant difference among the three groups. In Group I and II, the implant-bone contact ratio at 12 weeks increased significantly in comparison to ratio at 4 weeks(p<0.05). The removal torque values at 12 weeks after implantation were 90.9 Ncm in Group I, 81.6 Ncm in Group II and 77.1 Ncm in Group III, which were significantly different(p<0.05). These results suggest that bone healing begin earlier and be better around the surface-treated implants compared to the smooth surface implants. The sandblasted and acid-etched implants showed the most favorable bone response among the three groups during the early healing stage and could reduce the waiting period prior to implant loading.