• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1591-1595
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• 2012

We previously isolated a novel compound, HY251, with the molecular structure of 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol from the roots of Aralia continentalis. The current study was designed to evaluate the detailed molecular mechanisms underlying the apoptotic induction by HY251 in human ovarian cancer PA-1 cells. TUNEL assay and Western blot analyses revealed an appreciable apoptotic induction in PA-1 cells treated with $60{\mu}M$ of HY251 for 24 h. This apoptotic induction was associated with caspase-8-dependent Bid cleavage, which in turn resulted in the formation of pro-apoptotic truncated Bid (tBid), and activation of caspase-9 and -3, as well as the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, we found that this death event was also associated with the significant up-regulation and activation of the p53 tumor-suppressor protein through phosphorylation at Ser15. Therefore, we suggest that HY251 may be a potent cancer chemotherapeutic candidate for the treatment of ovarian cancer.

• BMB Reports
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• v.40 no.3
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• pp.426-431
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• 2007

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of a117 GluAsp and $\alpha$132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.

• Molecules and Cells
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• v.41 no.3
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1343-1348
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• 2015

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression and was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.5
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• pp.286-290
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• 2005

Oxytocin (OT)-related peptide, isotocin was purified from the brain extract of conger eel (Conger myriaster) using reverse-phase, ion-exchange and size exclusion high performance liquid chromatography (HPLC). The sequence of the peptide, with a molecular weight of 967.30 Da, was determined as Cys-Tyr-Ile-Ser-Asn­Cys-Pro-Ile-Gly-$NH_2$, where the Cys between 1st and 6th residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis and MALDI-TOF mass spectrometry. The sequence was confirmed by identical elution with the purified and synthetic peptide using the HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of OT -superfamily member, isotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-7}M$ on the intestinal smooth muscle of goldfish (Carassius auratus).

• Journal of Animal Science and Technology
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• v.45 no.3
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• pp.491-498
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• 2003

The purpose of this study is to observe purification and properties of osteopontin(OPN) from bovine milk. The purification of osteopontin from bovine milk was performed by using ion-exchange and hydrophobic chromatography. SDS-PAGE analysis revealed that the protein migrated at Mw. 60,000. NH2-terminal sequence analysis of the first seven amio acids revealed the protein to be identical to that previously reported for bovine OPN. 35-wk-old chickens, including 3 Single Comb White Leghorn (SCWL), were used to produce egg yolk antibody(IgY) against OPNas a antigen. However, the anti-OPN antibody activities determined by ELISA. Immunological assy of OPN in milk was performed using radial immunodiffusion test based on the standard curve of pure OPN. The radial precipitation lines of four different milk samples indicated that the concentrations of OPN in the milk samples were within the range of 31.7 to 39.7${\mu}g$/ml. On inhibition with OPN on precipitation of calcium phosphate, OPN was slightly higher than casein phosphopeptide(CPP) and poly-glutamic acid.

• Food Science of Animal Resources
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• v.30 no.6
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• pp.923-929
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• 2010

Colostral whey prepared from colostrum (pooled from first six post-partum milkings) was heated for 10 min at $100^{\circ}C$ Heated colostral whey was incubated with 1% enzymes (protein equivalent basis) for 15, 30, 60, 90, and 120 min at $50^{\circ}C$. Papain, pepsin, trypsin, and alcalase produced different degrees of hydrolysis (DH), 10.66%, 12.42%, 10.83%, and 25.31%, respectively, at an incubation time of 120 min. The SDS-PAGE reveals that significant amounts of bovine serum albumin (BSA), ${\beta}$-lactoglobulin (${\beta}$-LG), and ${\alpha}$-lactalbumin (${\alpha}$-LA) survived papain digestion. In contrast, pepsin completely removed BSA but not ${\beta}$-LG present in heated colostral whey. Alcalase completely eliminated BSA, ${\beta}$-LG, and ${\alpha}$-LA. This differential hydrolysis was confirmed by reversed-phase HPLC analysis. Using ion-exchange chromatography, fraction-1 (F-1) was obtained from alcalase hydrolysate at a NaCl gradient concentration of 0.25 M. Reversed-phase HPLC chromatograms of alcalase F-1 showed numerous small peaks, which probably indicate that a variety of new peptides were produced. Iron content of alcalase F-1 was 28.94 ppm, which was the highest among all enzyme fractions, whereas iron content of colostral whey was 36.56 ppm. Main amino acids contained in alcalase F-1 were Thr (15.45%), Glu (14.12%), and Ser (10.39%). Therefore, alcalase can be used to generate good iron-binding peptides in heated colostral whey, and the resulting iron-binding peptides could be suitable as a value-added food ingredient for food supplements.

• Journal of Life Science
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• v.9 no.1
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• pp.26-34
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• 1999

A thermostable pullulanase has been isolated and purified from Thermus caldophilus GK-24 to a homogeneity by gel-filtration and ion-exchange chromatography. The specific activity of the purified enzyme was 431-fold increase from the crude culture broth with a recovery of 11.4%. The purified enzyme showed $M_{r}$ of 65 kDa on denaturated and natural conditions. The pI of the enzyme was 6.1 and Schiff staining was negative, suggesting that the enzyme is not a glycoprotein. The enzyme was most active at pH 5.5. The activity was maximal at $75^{\cire}C$ and stable up to $95^{\cire}C$ for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at $4^{\cire}C$ for 24hr. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was simulated by $Mn^{2+}$ ion, }$Ni^{2+}$, $Ca^{2+}$, $Co^{2+}$ ions. The enzyme hydrolyzed the ${\alpha}$-1,6-linkages of amylopectin, glycogens, ${\alpha}$, ${\beta}$-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose and the activity was inhibited by $\alpha$, $\beta$, or $\gamma$-cyclodextrins. The $NH_{2}$-terminal amino acid sequence [(Ala-Pro-Gln-(Asp of Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] was compared with known sequences of various sources and that was compared with known sequences of various sources and that was different from those of bacterial and plant enzymes, suggesting that the enzymes are structurally different.

• Genomics & Informatics
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• v.12 no.4
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• pp.268-275
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• 2014

The harshness of legionellosis differs from mild Pontiac fever to potentially fatal Legionnaire's disease. The increasing development of drug resistance against legionellosis has led to explore new novel drug targets. It has been found that phosphoglucosamine mutase, phosphomannomutase, and phosphoglyceromutase enzymes can be used as the most probable therapeutic drug targets through extensive data mining. Phosphoglucosamine mutase is involved in amino sugar and nucleotide sugar metabolism. The purpose of this study was to predict the potential target of that specific drug. For this, the 3D structure of phosphoglucosamine mutase of Legionella pneumophila (strain Paris) was determined by means of homology modeling through Phyre2 and refined by ModRefiner. Then, the designed model was evaluated with a structure validation program, for instance, PROCHECK, ERRAT, Verify3D, and QMEAN, for further structural analysis. Secondary structural features were determined through self-optimized prediction method with alignment (SOPMA) and interacting networks by STRING. Consequently, we performed molecular docking studies. The analytical result of PROCHECK showed that 95.0% of the residues are in the most favored region, 4.50% are in the additional allowed region and 0.50% are in the generously allowed region of the Ramachandran plot. Verify3D graph value indicates a score of 0.71 and 89.791, 1.11 for ERRAT and QMEAN respectively. Arg419, Thr414, Ser412, and Thr9 were found to dock the substrate for the most favorable binding of S-mercaptocysteine. However, these findings from this current study will pave the way for further extensive investigation of this enzyme in wet lab experiments and in that way assist drug design against legionellosis.

• Journal of Microbiology
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• v.39 no.2
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• pp.95-101
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• 2001

In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergilius nidulans, the PCR-amplified coding sequence for alkaline pretense (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans aicA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the host's own pretense, the alcA prumoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence or the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.