Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.
Background: Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about Isolation of Korean CDV since 1980 in Korea. Objectives: To investigate the biological properties and the genetic characterization of Korean CDV. Methods: Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. Results: A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (105.5 50% tissue culture infectious dose [TCID50]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. Conclusions: We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.
HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.
TheraCal LC, a new light-cured, resin-modified calcium silicate-filled base/liner material, has been introduced as a pulpotomy agent. The aim of this study was to evaluate the capacity of hard tissue formation and pulpal response after pulpotomy with TheraCal LC. Twenty-two 9-week-old male rats were anesthetized, cavities were prepared in maxillary first molars and pulps were capped with formocresol (FC), mineral trioxide aggregate (MTA), and TheraCal LC. Specimens obtained from rats were scanned using a high-resolution micro CT system. The specimens were prepared and evaluated histologically, and immunofluorescence assay was performed to assess the dentin matrix protein-1 (DMP-1) expression. On micro CT analysis, the MTA and TheraCal LC groups showed thicker hard tissue formation than the FC group. On hematoxylin and eosin (H&E) staining, MTA and TheraCal LC groups showed dentine bridge formation with vital pulp beneath the materials. On immunofluorescence analysis, DMP-1 was highly expressed in the TheraCal LC group compared to the FC group. TheraCal LC showed similar capacity to form hard tissue as MTA when it was used as a pulpotomy agent. Because of its good manipulation and faster setting time compared to MTA, TheraCal LC could be considered as a good alternative to MTA.
Thirty-day-old piglets were intranasally or subcutaneously inoculated with 2ml of Aujeszky's disease virus, NYJ-1 strain, at the titer of $10^{6.75}$$TCID_{50}/0.1ml$, that was isolated from the diseased piglets in Korea, and histopathological studies were performed to elucidate the pathognomonic characters of the isolate. Results obtained through the experiments were as follows: 1. Major clinical signs on the 2nd and 3rd days post inoculation (p.i.) were fever, anorexia and dyspnea. On the 6th and 7th days p.i., nervous signs, severe dyspnea and salivation were observed in the group of intranasal inoculation, and one out of 3 piglets in this group died on the 7th day p.i.. General signs were more severe in the group of intranasal inoculation than the group of subcntaneous injection. Between the 8th and l0th days p.i., the signs subsided and the piglets were completely recovered from the illness. 2. Hematologically, most of the inoculated pigs showed a mild lymphocytopenia on the 5th and 6th days p.i.. 3. By necropsy, swelling and hemorrhagic lesions were observed in tonsil, central nervous system and lung. No specific changes were grossly found in other parenchymatous organs. 4. In histopathological study, degeneration and necrosis of nervous cells, non-suppurative meningoencephalitis, diffuse or focal gliosis, perivascular cutting and degeneration of ganglion cells were observed in central nervous system, and swelling and hemorrhagic changes were shown in the tissues of liver, lung and lymph nodes. 5. By indirect immunofluorescence antibody assay using ADV-monoclonal antibody, specific ADV antigens were detected in the tissues of tonsil, brain and spleen of the succumbed piglet. However, in the experimentally slaughtered piglets, the specific reactions were noted only in the tonsils.
Background: The inhibition of tumor cell growth without toxicity to normal cells is an important target in cancer therapy. One possible way to increase the efficacy of anticancer drugs and to decrease toxicity or side effects is to develop traditional natural products, especially from medicinal plants. Paris polyphylla Smith has shown anti-tumour effects by inhibition of tumor promotion and inducement of tumor cell apoptosis, but mechanisms are still not well understood. The present study was to explore the effect of Paris polyphylla Smith extract (PPSE) on connexin26 and growth control in human esophageal cancer ECA109 cells. Methods: The effects of PPSE on Connexin26 were examined by RT-PCR, western blot and immunofluorescence; cell growth and proliferation were examined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Results: PPSE inhibited the growth and proliferation on esophageal cancer ECA109 cells, while increasing the expression of connexin26 mRNA and protein; conversely, PPSE decreased Bcl-2 and increased Bad. Conclusion: This study firstly shows that PPSE can increase connexin26 expression at mRNA and protein level, exerting anti-tumour effects on esophageal cacner ECA109 cells via inhibiting cell proliferation and inducing cell apoptosis.
Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis ${\alpha}$-actinin 2 ($Tv{\alpha}$-actinin 2) has been used to diagnose trichomoniasis. $Tv{\alpha}$-actinin 2 was dissected into three parts; the N-terminal, central, and C-terminal portions of the protein (#1, #2, and #3, respectively). Western blot of these $Tv{\alpha}$-actinin 2 proteins with pooled patients' sera indicated that #2 and #3, but not #1, reacted with those sera. Immunofluorescence assays of two different forms of T. vaginalis (trophozoites and amoeboid forms), using anti-$Tv{\alpha}$- actinin 2 antibodies, showed localization of $Tv{\alpha}$-actinin 2 close to the plasma membranes of the amoeboid form. Fractionation experiments indicated the presence of $Tv{\alpha}$-actinin 2 in cytoplasmic, membrane, and secreted proteins of T. vaginalis. Binding of fluorescence-labeled Trichomonas to vaginal epithelial cells and prostate cells was decreased in the antibody blocking experiment using anti-$Tv{\alpha}$-actinin 2 antibodies. Pretreatment of T. vaginalis with anti-$rTv{\alpha}$-actinin 2 antibodies also resulted in reduction in its cytotoxicity. Flow cytometry, ligand-binding immunoblotting assay, and observation by fluorescence microscopy were used to detect the binding of recombinant $Tv{\alpha}$-actinin 2 to human epithelial cell lines. Specifically, the truncated N-terminal portion of $Tv{\alpha}$-actinin 2, $Tv{\alpha}$-actinin 2 #1, was shown to bind directly to vaginal epithelial cells. These data suggest that ${\alpha}$-actinin 2 is one of the virulence factors responsible for the pathogenesis of T. vaginalis by serving as an adhesin to the host cells.
Curcumin is a widely used flavoring agent in food, and it has been reported to inhibit cell growth, to induce apoptosis, and to have antitumor activity in many cancers. Cisplatin is one of the most potent known anticancer agents and shows significant clinical activity against a variety of solid tumors. This study was undertaken to investigate the synergistic apoptotic effects of co-treatment with curcumin and cisplatin on human tongue SCC25 cells. To investigate whether the co-treatment efficiently reduced the viability of the SCC25 cells compared with the two treatments separately, an MTT assay was conducted. The induction and the augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and an analysis of DNA hypoploidy. Western blot, MMP and immunofluorescence tests were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following the co-treatment. In this study, following the co-treatment with curcumin and cisplatin, the SCC25 cells showed several forms of apoptotic manifestation, such as nuclear condensation, DNA fragmentation, reduction of MMP, increased levels of Bax, decreased levels of Bcl-2, and decreased DNA content. In addition, they showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40 (CAD) to the nuclei, and activation of caspase-7, caspase-3, PARP, and DFF45 (ICAD). In contrast, separate treatments of $5{\mu}M$ of curcumin or $4{\mu}g/ml$ of cisplatin, for 24 hours, did not induce apoptosis. Therefore, our data suggest that combination therapy with curcumin and cisplatin could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.
The standard procedure outlined by the United States Environmental Protection Agency (US EPA) in Method 1623 for analyzing Giardia lamblia cysts and Cryptosporidium parvum oocysts in water samples consists of filtration, elution, centrifugal concentration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) followed by microscopic examination. In this study, the extent of (oo)cyst loss in each step of this procedure was evaluated by comparing recovery yields in segmented analyses: (i) IMS + IFA, (ii) concentration + IMS + IFA, and (iii) filtration/elution + concentration + IMS + IFA. The complete (oo)cyst recovery by the full procedure was $52{\sim}57%$. The (oo) cyst loss in the IMS step was only $0{\sim}6%$, implying that IMS is a fairly reliable method for (oo)cyst purification. Centrifugal concentration of the eluted sample and pellet collection before IMS resulted in a loss of $8{\sim}14%$ of the (oo)cysts. The largest (oo)cyst loss occurred in the elution step, with $68{\sim}71%$ of the total loss. The permeated loss of (oo)cysts was negligible during filtration of the water sample with a $1.0-{\mu}m$ pore polyethersulfone (PES) capsule. These results demonstrated that the largest fraction of (oo)cyst loss in this procedure occurred due to poor elution from the filter matrix. Improvements in the elution methodology are therefore required to enhance the overall recovery yield and the reliability of the detection of these parasitic protozoa.
Objectives:eurotoxicity is mediated by nitric oxide(NO) in the rat primary neuronal cultures and assess the effect of $Mn^{2+}$ on the N-methyl-D aspartate(NMDA) receptors. Methods: We have used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay to examine the effect of cytotoxicity of $MnCl_2$ in neuronal cells , NO production was determined by measuring nirites, a stable oxidation product of NO. The neurons in the rat that contains neuronal nitric oxide synthase(nNOS) were examined by immunofluorescence and confocal microscopy. The effects of $Mn^{2+}$ on the NMDA receptors was assesed by the whole cell voltage clamp technique. Results: We showed that the NO release and NOS expression was increased with 500uM $MnCl_2$ treatment and an NOS inhibitors, $N^G-nitro-L-arginine$, prevented neurotoxicity elicited by manganese. In the electrophysiological study, $Mn^{2+}$ does not block or activate the NMDA receptors and not pass through the NMDA receptors in a neurons of basal ganglia. Conclusions: It is concluded that manganese neurotoxicity in basal ganglia was partially mediated by nitric oxide in the cell culture model.
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