5-Azacytidine (5-aza) Induces p53-associated Cell Death Through Inhibition of DNA Methyltransferase Activity in Hep3B and HT-29 Cells.

BACKGROUND/AIM: DNA methylation regulates the expression of genes that control mechanisms of cell death. TP53 gene expression inhibits tumorigenesis, and its action is closely associated with cell death. 5-Azacytidine (5-aza), increases the expression of the TP53 gene by inhibiting DNA methyltransferase. MATERIALS AND METHODS: Using 5-aza, we induced DNA hypomethylation in p53-null and p53-expressing cancer cell lines and investigated potential mechanisms of cancer cell death. RESULTS: TP53 expression promoted cell death. Notably, methylation-specific PCR (MSP) and bisulfite sequencing revealed more methylation sites at the TP53 promoter region in p53-null cells than in p53-expressing cells. CONCLUSION: This study suggests a novel mechanism of tumorigenesis regulated by p53 expression.

Effect of 5-azacytidine (5-aza) on UCP2 expression in human liver and colon cancer cells.

The function of the uncoupling protein 2 (UCP2) is different for each cancer cell. However, the mechanism of expression is still unclear. DNA methylation affects protein expression and is one factor that transforms normal cells into cancer cells. In this study, the hepatocellular carcinoma Hep3B and HepG2 cells and colorectal cancer HT-29 cells were treated with 5-azacytidine (5-aza), a DNA demethylation agent, to observe the modification of UCP2 expression and the methylation degree in the UCP2 promoter region. Promoter basal activity and degree of UCP2 expression were measured in Hep3B, HepG2, and HT-29 cells. In addition, methylation-specific PCR (MSP) was performed to investigate the degree of methylation in the UCP2 promoter region. The methylation region in the UCP2 promoter was confirmed based on bisulfite sequencing. In Hep3B cells in which UCP2 mRNA was not transcribed, the promoter basal activity was significantly higher than in HT-29 or HepG2 cells in which UCP2 mRNA was transcribed. Treatment with 5-aza increased UCP2 expression in Hep3B and HT-29 cells; however, the expression in HepG2 cells was unchanged. The UCP2 promoter in Hep3B cells has numerous methylated regions compared with HT-29 and HepG2 cells. The results of the present study revealed that inhibition of UCP2 expression in Hep3B cells was due to methylation of the promoter region. Investigating the mechanism that induces UCP2 expression in cancer cells is important to understand the function of UCP2, which could aid in cancer treatment.

Microencapsulation of Caramel Flavor and Properties of Ready-to-drink Milk Beverages Supplemented with Coffee Containing These Microcapsules.

This study aimed to extend the retention of flavor in coffee-containing milk beverage by microencapsulation. The core material was caramel flavor, and the primary and secondary coating materials were medium-chain triglyceride and maltodextrin, respectively. Polyglycerol polyricinoleate was used as the primary emulsifier, and the secondary emulsifier was polyoxyethylene sorbitan monolaurate. Response surface methodology was employed to determine optimum microencapsulation conditions, and headspace solid-phase microextraction was used to detect the caramel flavor during storage. The microencapsulation yield of the caramel flavor increased as the ratio of primary to secondary coating material increased. The optimum ratio of core to primary coating material for the water-in-oil (W/O) phase was 1:9, and that of the W/O phase to the secondary coating material was also 1:9. Microencapsulation yield was observed to be approximately 93.43%. In case of in vitro release behavior, the release rate of the capsules in the simulated gastric environment was feeble; however, the release rate in the simulated intestinal environment rapidly increased within 30 min, and nearly 70% of the core material was released within 120 min. The caramel flavor-supplemented beverage sample exhibited an exponential degradation in its flavor components. However, microcapsules containing flavor samples showed sustained flavor release compared to caramel flavor-filled samples under higher storage temperatures. In conclusion, the addition of coffee flavor microcapsules to coffee-containing milk beverages effectively extended the retention of the coffee flavor during the storage period.

Effect of Antioxidant Addition on Milk Beverage Supplemented with Coffee and Shelf-life Prediction.

This study aimed to extend the shelf-life of coffee-containing milk beverage by adding Theobroma cacao (cacao nibs) extract. To prepare the beverage sample containing cacao nibs extract, 0.8% cacao nibs hydrothermal extract was aseptically injected. Qualitative changes in the beverage samples, including antioxidant effect, peroxide value (POV), caffeine content, and sensory parameters were monitored regularly during storage at 10°C, 20°C, and 30°C for 4 wk. The inclusion of cacao nibs extract produced higher antioxidant activity compared to the control. As the storage temperature increased, the POV of all samples increased. Samples with cacao nibs extract generally displayed lower POV than the control. The caffeine content of all samples tended to decrease during storage, with the decrease accentuated by higher storage temperatures. In the shelf-life prediction using the Arrhenius model, the kinetic regressions of the cacao nibs extract-added sample and control were Y POV=1.2212X-2.1141 (r2=0.9713) and Y POV=1.8075X-2.0189 (r2=0.9883), respectively. Finally, the predicted shelf-life of cacao nibs-added group and control to reach the quality limit (20 meq/kg POV) were approximately 18.11 and 12.18 wk, respectively. The results collectively indicate that the addition of cacao nibs extract extends the shelf-life of the coffee-containing milk beverage and heightened the antioxidant effect.

Optimization of electrochemical reaction for nitrogen removal from biological secondary-treated milking centre wastewater.

In order to remove the residual nitrogen from the secondary-treated milking centre wastewater, the electrochemical reaction including NH4-N oxidation and NOx-N reduction has been known as a relatively simple technique. Through the present study, the electrochemical reactor using the Ti-coated IrO2 anode and stainless steel cathode was optimized for practical use on farm. The key operational parameters [electrode area (EA) (cm(2)/L), current density (CD) (A/cm(2)), electrolyte concentration (EC) (mg/L as NaCl), and reaction time (RT) (min)] were selected and their effects were evaluated using response surface methodology for the responses of nitrogen and colour removal efficiencies, and power consumption. The experimental design was followed for the central composite design as a fractional factorial design. As a result of the analysis of variance, the p-values of the second-order polynomial models for three responses were significantly fit to the empirical values. The nitrogen removal was significantly influenced by CD, EC, and RT (p < .05), whereas colour removal was significantly governed by EA, CD, RT, the interaction of EA and EC (p < .05). For higher efficiency of nitrogen removal over 90%, the combination of [EA, 20 cm(2)/L; CD, 0.044 A/cm(2); EC, 3.87 g/L as NaCl; RT, 240 min] was revealed as an optimal operational condition. The investigation on cathodic reduction of NOx-N may be required with respect to nitrite and nitrate separately as a future work.

Nitrogen Removal from Milking Center Wastewater via Simultaneous Nitrification and Denitrification Using a Biofilm Filtration Reactor.

Milking center wastewater (MCW) has a relatively low ratio of carbon to nitrogen (C/N ratio), which should be separately managed from livestock manure due to the negative impacts of manure nutrients and harmful effects on down-stream in the livestock manure process with respect to the microbial growth. Simultaneous nitrification and denitrification (SND) is linked to inhibition of the second nitrification and reduces around 40% of the carbonaceous energy available for denitrification. Thus, this study was conducted to find the optimal operational conditions for the treatment of MCW using an attached-growth biofilm reactor; i.e., nitrogen loading rate (NLR) of 0.14, 0.28, 0.43, and 0.58 kg m(-3) d(-1) and aeration rate of 0.06, 0.12, and 0.24 m(3) h(-1) were evaluated and the comparison of air-diffuser position between one-third and bottom of the reactor was conducted. Four sand packed-bed reactors with the effective volume of 2.5 L were prepared and initially an air-diffuser was placed at one third from the bottom of the reactor. After the adaptation period of 2 weeks, SND was observed at all four reactors and the optimal NLR of 0.45 kg m(-3) d(-1) was found as a threshold value to obtain higher nitrogen removal efficiency. Dissolved oxygen (DO) as one of key operational conditions was measured during the experiment and the reactor with an aeration rate of 0.12 m(3) h(-1) showed the best performance of NH4-N removal and the higher total nitrogen removal efficiency through SND with appropriate DO level of ~0.5 mg DO L(-1). The air-diffuser position at one third from the bottom of the reactor resulted in better nitrogen removal than at the bottom position. Consequently, nitrogen in MCW with a low C/N ratio of 2.15 was successfully removed without the addition of external carbon sources.

Optimal Incorporation Level of Dietary Alternative Phosphate (MgHPO4) and Requirement for Phosphorus in Juvenile Far Eastern Catfish (Silurus asotus).

A growth trial was conducted to determine the optimal incorporation level of dietary magnesium hydrogen phosphate (MHP, MgHPO4), which was manufactured from swine manure and phosphorus (P), required by juvenile far eastern catfish (Silurus asotus). Graded MHP of 0.5%, 1.0%, 1.5%, and 2.0%, and 2.0% monocalcium phosphate (MCP) each was added to the basal diet (control) in lieu of cellulose to become the range of available P (AP) from 0.4% to 0.8% of which diets were designated as control, MHP0.5, MHP1.0, MHP1.5, MHP2.0, and MCP, respectively. Control diet contained fish meal (20%), soybean meal (40%), wheat flour (27%), corn gluten meal (5%), fish oil (2%) and soy oil (2%) as main ingredients. Following a 24 h fasting, 540 fish with a mean body weight of 11.8 g were randomly allotted to 6 groups in triplicate, whereby 18 tanks (0.4×0.6×0.36 cm, water volume of 66 L) were prepared. The feeding experiment lasted for 8 weeks. Fish group fed the control diet showed the lowest weight gain (WG) and feed efficiency (FE) among treatments. The WG was, however, not significantly different (p>0.05) from that of fish group fed MHP0.5. Fish group fed MHP2.0 showed the highest WG and FE of which values were not significantly different from those of fish groups fed diets MHP1.0 and MHP1.5 as well as MCP (p>0.05) except fish groups fed control and MHP0.5. Aspartate aminotransferase was significantly decreased with an increase in available P, while alanine aminotransferase did not show a significant difference among treatment. The highest inorganic P in plasma was observed in fish fed MHP2.0. From the present results, a second-order regression analysis revealed that the optimal dietary MHP level and the AP requirement were found to be 1.62% and 0.7%, respectively.

Effects of Dietary Garlic Powder on Growth, Feed Utilization and Whole Body Composition Changes in Fingerling Sterlet Sturgeon, Acipenser ruthenus.

A 12 week growth study was carried out to investigate the supplemental effects of dietary garlic powder (GP) on growth, feed utilization and whole body composition changes of fingerling sterlet sturgeon Acipenser ruthenus (averaging weight, 5.5 g). Following a 24-h fasting, 540 fish were randomly distributed to each of 18 tanks (30 fish/tank) under a semi-recirculation freshwater system. The GP of 0.5% (GP0.5), 1% (GP1), 1.5% (GP1.5), 2% (GP2) and 3% (GP3) was added to the control diet (GP0) containing 43% protein and 16% lipid. After the feeding trial, weight gain (WG) of fish fed GP1.5, GP2 and GP3 were significantly higher (p<0.05) than those of fish fed GP0, GP0.5 and GP1. Feed efficiency and specific growth rate (SGR) showed a similar trend to WG. Protein efficiency ratio of fish fed GP1.5, GP2, and GP3 were significantly higher (p<0.05) than those of fish groups fed the other diets. A significant difference (p<0.05) was found in whole body composition (moisture, crude protein, crude lipid, ash, and fiber) of fish at the end of the experiment. Significantly higher (p<0.05) protein and lipid retention efficiencies (PRE and LRE) were also found in GP1.5, GP2, and GP3 groups. Broken-line regression model analysis and second order polynomial regression model analysis relation on the basis of SGR and WG indicated that the dietary optimal GP level could be greater than 1.77% and 1.79%, but less than 2.95% and 3.18% in fingerling sterlet sturgeon. The present study suggested that dietary GP for fingerling sterlet sturgeon could positively affect growth performance and protein retention.

Effects of Dietary Supplementation of Magnesium Hydrogen Phosphate (MgHPO4) as an Alternative Phosphorus Source on Growth and Feed Utilization of Juvenile Far Eastern Catfish (Silurus asotus).

The present study was conducted to investigate a supplemental effect of magnesium hydrogen phosphate (MHP, MgHPO4) as an alternative phosphorus (P) source on growth and feed utilization of juvenile far eastern catfish (Silurus asotus) in comparison with three conventional P additives (monocalcium phosphate (MCP), dicalcium phosphate (DCP) and tricalcium phosphate [TCP]) as positive controls. A basal diet as a negative control was prepared without P supplementation and four supplemental P sources were added at the level of 2%. Five groups of 450 fish having mean body weight of 11.3 g following 24 h fasting after three week adaptation period were randomly distributed into each of 15 tanks (30 fish/tank). Fish were hand-fed to apparent satiety twice a day for 8 weeks. Fish fed MHP had weight gain (WG), protein efficiency ratio and specific growth rate comparable to those fed MCP. Fish fed MHP and MCP had feed efficiency (FE) significantly higher (p<0.05) than those fed DCP. Fish groups fed control and TCP showed the lower FE than the other groups which was significantly different (p<0.05) from those of fish fed the other diets. Survival rate was not significantly different (p>0.05) among treatments. Fish fed control had the lowest hematocrit, which was significantly different (p<0.05) from that of fish fed MHP. Fish fed MCP and MHP had plasma P higher (p<0.05) than fish fed the other diets. Relative efficiencies of MCP, DCP and TCP to MHP were found to be 100.5 and 101.3%, 92.0 and 91.6%, and 79.1 and 80.9% for WG and FE, respectively. P availability was determined to be 88.1%, 75.2%, 8.7%, and 90.9% for MCP, DCP, TCP, and MHP, respectively. Consequently, MHP recovered from wastewater stream showed that as an alternative P source its performance was comparative with MCP on growth and feed utilization of juvenile far eastern catfish.

Effects of Dietary Garlic Extracts on Whole Body Amino Acid and Fatty Acid Composition, Muscle Free Amino Acid Profiles and Blood Plasma Changes in Juvenile Sterlet Sturgeon, Acipenser ruthenus.

A series of studies were carried out to investigate the supplemental effects of dietary garlic extracts (GE) on whole body amino acids, whole body and muscle free amino acids, fatty acid composition and blood plasma changes in 6 month old juvenile sterlet sturgeon (Acipenser ruthenus). In the first experiment, fish with an average body weight of 59.6 g were randomly allotted to each of 10 tanks (two groups of five replicates, 20 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the level of 2% of fish body weight per day for 5 wks. Whole body amino acid composition between the GE and control groups were not different (p>0.05). Among free amino acids in muscle, L-glutamic acid, L-alanine, L-valine, L-leucine and L-phenylalanine were significantly (p<0.05) higher in GE than in control. However, total whole body free amino acids were significantly lower in GE than in control (p<0.05). GE group showed higher EPA (C22:6n3) and DHA (C22:5n3) in their whole body than the other group (p<0.05). In the second experiment, the effects of dietary garlic extracts on blood plasma changes were investigated using 6 month old juvenile sterlet sturgeon averaging 56.5 g. Fish were randomly allotted to each of 2 tanks (300 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the rate of 2% of body weight per day for 23 d. At the end of the feeding trial, blood was taken from the tail vein (n = 5, per group) at 1, 12, and 24 h after feeding, respectively. Blood plasma glucose, insulin and the other serological characteristics were also measured to assess postprandial status of the fish. Plasma glucose concentrations (mg/dl) between two groups (GE vs control) were significantly (p< 0.05) different at 1 (50.8 vs 62.4) and 24 h (57.6 vs 73.6) after feeding, respectively, while no significant difference (p>0.05) were noticed at 12 h (74.6 vs 73.0). Plasma insulin concentrations (µIU/ml) between the two groups were significantly (p<0.05) different at 1 (10.56 vs 5.06) and 24 h (32.56 vs 2.96) after feeding. The present results suggested that dietary garlic extracts could increase dietary glucose utilization through the insulin secretion, which result in improved fish body quality and feed utilization by juvenile sterlet sturgeon.

Effects of Dietary Garlic Extract on Growth, Feed Utilization and Whole Body Composition of Juvenile Sterlet Sturgeon (Acipenser ruthenus).

This study was carried out to investigate the supplemental effects of dietary garlic extract (GE) on growth performance of juvenile sterlet sturgeon (Acipenser ruthenus). The first experiment was designed to determine the optimum levels of garlic extract as growth promoter during 10 weeks. Three groups (two replicates/group) of 240 fish with mean body weight of 85 g were fed with diets containing 0 (control), 0.5 and 1.0% of GE. The highest weight gain (%) and feed efficiency (%) were found in fish groups fed with diet containing 0.5% GE. Subsequently, the supplemental effects of dietary GE was studied on growth of juvenile sterlet sturgeon (Acipenser ruthenus) with an average body weight of 59.6 g. Fish cultured in freshwater were randomly allotted to each of 10 tanks (two groups of five replicates, 20 fish/tank) and fed diets with 0.5% GE or without GE (control), respectively, at the level of 2.0% of fish body weight per day for 5 weeks. Weight gain (51.1%), feed efficiency (79.1%), specific growth rate (1.18%) and protein efficiency ratio (1.50) of fish fed 0.5% GE were significantly (p<0.05) higher than those fish fed the control diet. Significantly higher protein (PRE 20.4%) and lipid retention efficiencies (LRE, 74.5%) were also found in 0.5% GE group (p<0.05). The present results suggested that dietary GE could improve growth and feed utilization of juvenile sterlet sturgeons.