• Asian Pacific Journal of Cancer Prevention
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• v.15 no.13
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• pp.5437-5442
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• 2014

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase $C{\varepsilon}$ gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigat the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5825-5831
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• 2013

Background/Objectives: Maintenance of cellular function in culture is vital for transfer and development following adoptive immunotherapy. Dual properties of IL-21 in activating T cells and reducing activation induced cell death led us to explore the mechanism of action of IL-21 enhanced proliferation and cytotoxic potential of CIK cells. Method: CIK cells cultured from PBMCs of healthy subjects were stimulated with IL-21 and cellular viability and cytotoxicity to K562 cells were measured. To elucidate the mechanism of action of IL-21, mRNA expression of cytotoxic factors was assessed by RT-PCR and protein expression of significantly important cytotoxic factors and cytokine secretion were determined through flow cytometry and ELISA. Western blotting was performed to check the involvement of the JAK/STAT pathway following stimulation. Results: We found that IL-21 did not enhance in vitro proliferation of CIK cells, but did increase the number of cells expressing the CD3+/CD56+ phenotype. Cytotoxic potential was increased with corresponding increase in perforin ($0.9831{\pm}0.1265$ to $0.7592{\pm}0.1457$), granzyme B ($0.4084{\pm}0.1589$ to $0.7319{\pm}0.1639$) and FasL ($0.4015{\pm}0.2842$ to $0.7381{\pm}0.2568$). Interferon gamma and TNF-alpha were noted to increase ($25.8{\pm}6.1ng/L$ to $56.0{\pm}2.3ng/L$; and $5.64{\pm}0.61{\mu}g/L$ to $15.14{\pm}0.93{\mu}g/L$, respectively) while no significant differences were observed in the expression of granzyme A, TNF-alpha and NKG2D, and NKG2D. We further affirmed that IL-21 signals through the STAT-3 and STAT-5b signaling pathway in the CIK cell pool. Conclusion: IL-21 enhances cytotoxic potential of CIK cells through increasing expression of perforin, granzyme B, IFN-gamma and TNF-alpha. The effect is brought about by the activation of STAT-3 and STAT-5b proteins.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1544-1550
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• 2008

The retinoids (vitamin A and its derivatives) play a critical role in vision, growth, reproduction, cell differentiation and embryonic development. Using the IMpRH panel, porcine cellular retinol binding protein genes 5 and 7 (RBP5 and RBP7) were assigned to porcine chromosomes 5 and 6, respectively. The complete coding sequences (CDS) of the RBP5 and RBP7 genes were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) method, and the deduced amino acid sequences of both genes were compared to human corresponding proteins. The mRNA distributions of the two genes in adult Wuzhishan pig tissues (lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph node, small intestine, liver, brain, kidney and fat) were examined. A total of nine single nucleotide polymorphisms (SNPs) were identified in two genes. Three of these SNPs were analyzed using the polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) method in Laiwu, Wuzhishan, Guizhou, Bama, Tongcheng, Yorkshire and Landrace pig breeds. Association analysis of genotypes of these SNP loci with economic traits was done in our experimental populations. Significant associations of different genotypes of $RBP5-A/G^{63}$, $RBP5-A/G^{517}$ and $RPB5-T/C^{intron1-90}$ loci with traits including maximum carcass length (LM), minimum carcass length (LN), marbling score (MS), back fat thickness at shoulder (SBF), meat color score (MCS) and hematocrit (HCT) were detected. These SNPs may be useful as genetic markers in genetic improvement for porcine production.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.943-954
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• 2009

Diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the final and rate-limiting step of triglyceride synthesis. Both DGAT1 and DGAT2 genes code proteins with DGAT activity. Studies have shown DGAT1 polymorphisms associate with intramuscular fat deposition in beef cattle, but fewer associations between DGAT2 and beef cattle economic traits have been reported. The objective of this study was to investigate single nucleotide polymorphism (SNP) in intron3 of bovine DGAT2 and evaluate the associations of that with carcass, meat quality, and fat yield traits. Test animals were 157 commercial feedlot steers belonging to 3 Chinese native breeds (22 for Luxi, 24 for Jinnan, and 23 for Qinchuan), 3 cross populations (20 for Charolais${\times}$Fuzhou, 18 for Limousin ${\times}$Luxi, and 17 for Simmental${\times}$Jinan) and 1 Taurus pure breed population (16 Angus steers). In the current study, 15 SNP were discovered in intron3 and exon4 of DGAT2 at positions 65, 128, 178, 210, 241, 255, 270, 312, 328, 334, 365, 366, 371, 415, and 437 (named as their positions in PCR amplified fragments). Only 7 of them (128, 178, 241, 270, 312, 328, and 371) were analyzed, because SNP in three groups (65-128-255, 178-210-365 and 241-334-366) were in complete linkage disequilibrium within the group, and SNP 415 was a deletion and 437 was a null mutation. Frequencies for rare alleles in the 3 native breed populations were higher than in the 3 cross populations for 178 (p = 0.04), 270 (p = 0.001), 312 (p = 0.03) and 371 (p = 0.002). A general linear model was used to evaluate the associations between either SNP genotypes or allele substitutions and the measured traits. Results showed that SNP 270 had a significant association with the fat yield associated with kidney, pelvic cavity, heart, intestine, and stomach (KPHISY). Animals with genotype CC and CT for 270 had less (CC: -7.71${\pm}$3.3 kg and CT: -5.34${\pm}$2.5 kg) KPHISY than animals with genotype TT (p = 0.02). Allele C for 270 was associated with an increase of -4.26${\pm}$1.52 kg KPHISY (p = 0.006) and $-0.92{\pm}0.45%$ of retail cuts weight percentage (NMP, Retail cuts weight/slaughter body weight) (p = 0.045); allele G for 312 was associated with an increase of -5.45${\pm}$2.41 kg KPHISY (p = 0.026). An initial conclusion was that associations do exist between DGAT2 gene and carcass fat traits. Because of the small sample size of this study, it is proposed that further effort is required to validate these findings in larger populations.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.10
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• pp.1446-1458
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• 2013

The study assessed the effect of dietary supplementation of leaf meal mixture (LMM) containing condensed tannins (CT) on feed intake, nutrient utilization and performance of sheep infected with Haemonchus contortus. Eighteen adult sheep of similar age and body weight ($25.03{\pm}1.52$) were included in this study and out of these, 12 sheep were infected with single dose of infective third stage larvae of H. contortus at 2,000 larvae per sheep. The experimental sheep were allocated in three different groups' i.e. negative control (NC; no infection), control (C; H. contortus infected) and treatment (T; H. contortus infected+CT at 1.5% of the DM through LMM) and the experiment was conducted for a period of 90 d. The intake of dry matter (DM), organic matter (OM) and digestibility of DM, OM, neutral detergent fibre (NDF) and acid detergent fibre (ADF) were comparable among three animal groups. However, digestibility of crude protein (CP) and ether extract (EE) were significantly (p<0.05) higher in NC group as compared to both C and T groups. Nitrogen (N) retention (g/d or % of N intake) was significantly (p = 0.038) lower in C group as compared to T and NC groups. Daily intake (g/kg $W^{0.75}$) of digestible crude protein (DCP), digestible organic matter (DOM) and total digestible nutrient (TDN) did not differ significantly (p<0.05) in the three groups. Haemoglobin (Hb) and packed cell volume (PCV) were significantly (p<0.001) higher in treatment group as compared to control. The level of Hb and PCV reduced (p<0.001) after 30 days of experimental feeding. CT significantly (p<0.001) reduced serum urea in T group as compared to NC and C groups. Serum proteins differed significantly (p<0.01) among the three groups. The activity of serum enzymes AST, ALT, ALP and LDH were also statistically non significant (p<0.05) among treatments. The weight of abomasal lymph nodes (ALN) in T group was higher (p<0.05) than in C group. Treatment group had lower (p<0.05) total worms and fecal egg count compared to control group. It may be concluded that dietary supplementation of CT through LMM significantly improved the N retention, and inhibited the different developmental stages of Haemonchus contortus in experimental sheep.

• KSBB Journal
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• v.11 no.1
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• pp.8-14
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• 1996

Bacillus subtillis strains with transition state regulator mutations and a spore mutation were developed for the overexpression of apsE and for the enhancement of expression level. Among the many regulator genes, degU and hpr were chosen as a representative positive and negative regulator for the aprE, respectively. Spo II G was used for the construction of asporogeneous strains. All the mutants were constructed from two protease-deleted strain DB104 and the apsE gene was transformed with an integration vector pMK101. DB104(deg$U^h$(32) $his^+$)::pMK101(Cm) and DB104($\Delta$her(Em))::pMKl01(Cm) show 7-fold and about 2-fold increase in aprE expression level, respectively. But the effect of transition state regulator mutation on the aprE expression was diminished when the integrated aprE gene was amplified by the high concentration of chloramphenicol, i. e. 30 $\mu\textrm{g}$/ml. DB104($\Delta$spoIIG(Pm) degUh(32) his+)::pMK101(Cm) and DB104($\Delta$spoIIG(Pm) $\Delta$hpr(Em))::pMK101 double mutant show 10-fold and 3-fold increase in aprE expression level, respectively. The results suggest that sporulation mutation and transition state regulator mutation have independent and additive effect on the aprE expression, and the same gene dosage effect on the transition state regulator mutation was also identified.

• Microbiology and Biotechnology Letters
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• v.41 no.3
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• pp.341-349
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• 2013

In this study, the anti-oxidative and anti-obesity activities of two medicinal herb extracts, Tetrapanax papyriferus (TP) and Siegesbeckia pubescens (SP), were evaluated using DPPH radical scavenging activity assay, lipase enzyme inhibition assay, and the cell culture model system. Both methanol extracts of TP and SP showed DPPH radical scavenging activities dose-dependently, and the $IC_{50}$ of DPPH radical scavenging activities of the two medicinal herbs were 65.23 and 47.79 ${\mu}g/ml$, respectively. Furthermore, both extracts suppressed effectively lipase enzyme activity dose-dependently. Moreover, TP and SP extracts significantly suppressed adipocyte differentiation, lipid accumulation, triglyceride (TG) contents on 3T3-L1 preadipocytes in a dose-dependent manner without cytotoxicity. Their anti-obesity effect was modulated by cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding proteins ${\alpha}$ ($C/EBP{\alpha}$), $C/EBP{\beta}$ and peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) gene and protein expressions. Furthermore, TP and SP possessed a synergistic effect on anti-obesity activity. The identification of the active compounds that confer the anti-obesity activity of TP and SP might be needed.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.6
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• pp.445-453
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• 2000

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of $Ca^{2+}-activated$ potassium channel $(K_{Ca}\;channel)$ and is involved in the activation of $K_{Ca}$ channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide $(SIN-1,\;50\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate $(8-pCPT-cGMP,\;100\;{\mu}M),$ a membrane-permeable analogue of cGMP, increased the $K_{Ca}$ channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP $(100\;{\mu}M),$ cGMP $(100\;{\mu}M),$ ATP+cGMP $(100\;{\mu}M\;each),$ PKG $(5\;U/{\mu}l),$ ATP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l),$ or cGMP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ did not increase the channel activity. ATP $(100\;{\mu}M)+cGMP\;(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer $(Rp-pCPT-cGMP,\;100\;{\mu}M),$ a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The $K_{Ca}$ channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of $K_{Ca}$ channels, or some associated proteins in the membrane patch increase the activity of the $K_{Ca}$ channel, and the activation may be associated with the vasodilating action.

• Korean Journal of Food Science and Technology
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• v.30 no.5
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• pp.1070-1076
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• 1998

The processing conditions of low salt mackerel (Scomber japonicus) fillet was investigated, in which fresh mackerel was filleted, salted in brine until the expected salt concentration reached, dried with cool air (3 m/sec, $10{\sim}20^{\circ}C$), and finally packed individually in polyvinyl chloride film. Salting time and salt concentration of brine decided the final salt level penetrated into the fillet. As the final salt level was fixed to $0.8{\sim}1.0%, salting for $15{\sim}20 hours with 5% or 10% brine at $5^{\circ}C$ was enough to get that level of salt. Formation of histamine during salting was negligible. Changes in VBN, salt soluble proteins, and histamine formation of salted mackerel fillet during the storage occurred more rapidly in cases of storage at $5^{\circ}C than af $-2^{\circ}C and $-20^{\circ}C. Oxidation of lipid during the storage progressed, however it was delayed longer then 100 days in case of storage at $-20^{\circ}C. Addition of sodium erythrobate or ginger extracts could provide some extent of browning retardation. The shelf-life of the salted mackerel fillet based on panel scores of brown color and rancidity appealed to be 14 days when stored at $5^{\circ}C, and more than 28 days in case of storage at $-2^{\circ}C and about 3 months stored at $-20^{\circ}C.

• The Korean Journal of Mycology
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• v.19 no.2
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• pp.101-108
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• 1991

To classify fungal species employed for pharmacological effects, mycelial proteins of six isolates of Ganoderma lucidum, five isolates of Schizophyllum commune and five isolates of Cordyceps spp. were separated on polyacrylamide gel to compare them by esterase, acid phosphatase, leucine aminopeptidase and peroxidase patterns. Similarity of isozyme patterns among the isolates of G. lucidum IY003, IY004, IY005 and IY008 was indicated over 70%, but that among the isolates of G. lucidum IY009, IY010 and others was indicated from 48% to 9%. Highest similarity of isozymes of S. commune was observed to be between IY803 and IY805, and similarity between these two isolates was 57%. Similarity among other isolates was shown to be from 40% to 56%. Isozyme patterns of Cordyceps spp. were comparatively different, even though they were originated from the same kind of insect as their isolate. Similarity between Cordyceps spp. IY901 and IY904, which was isolated from moths, was 67% and that of IY905 and IY909, which was originated from the larvae, was 42%. Similarity among other isolates was shown to be from 12% to 67%.