B. G. Kim

Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways

Nano-scale materials are noted for unique properties, distinct from those of their bulk material equivalents. In this study, we prepared spherical silver nanoparticles (AgNPs) with an average size of about 30 nm and tested their potency to induce neuronal differentiation of SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells are considered an ideal in vitro model for studying neurogenesis, as they can be maintained in an undifferentiated state or be induced to differentiate into neuron-like phenotypes in vitro by several differentiation-inducing agents. Treatment of SH-SY5Y cells by biologically synthesized AgNPs led to cell morphological changes and significant increase in neurite length and enhanced the expression of neuronal differentiation markers such as Map-2, β-tubulin III, synaptophysin, neurogenin-1, Gap-43, and Drd-2. Furthermore, we observed an increase in generation of intracellular reactive oxygen species (ROS), activation of several kinases such as ERK and AKT, and downregulation of expression of dual-specificity phosphatases (DUSPs) in AgNPs-exposed SH-SY5Y cells. Our results suggest that AgNPs modulate the intracellular signaling pathways, leading to neuronal differentiation, and could be applied as promising nanomaterials for stem cell research and therapy.

Withaferin-A Inhibits Colon Cancer Cell Growth by Blocking STAT3 Transcriptional Activity.

Background: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. Methods: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. Results: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. Conclusions: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116.

Regulation of apoptosis by modified naringenin derivatives in human colorectal carcinoma RKO cells

Flavonoids are micronutrients that are widely detected in foods of plant origin and have been ascribed pharmacological properties. Several biological functions of flavonoids have been thus far identified, whereas there currently exists a lack of evidence to support the relationship between the structure‐activity relationship and apoptosis‐inducing activity. In an attempt to determine the importance of the OH group or substitution of the 5‐ or 7‐carbon in the diphenylpropane skeleton of flavonoids, we selected 14 different flavonoids with different structures, particularly with regard to the 5‐ or 7‐carbon, and found that naringenin treatment caused a slight decrease in the cell viability of the human colorectal carcinoma RKO cells. Next, in order to characterize the effects of specific substitutions of the 7‐carbon of naringenin on apoptosis‐regulatory activities, and in an attempt to develop anti‐proliferative flavonoid derivatives that would be more effective against colon cancer, we originally synthesized several modified naringenin derivatives (MNDs) including 7‐O‐benzyl naringenin (KUF‐1) and 7‐O‐(m‐metoxybenzyl) naringenin (KUF‐2). Treatment with KUF‐1 or KUF‐2 resulted in significant apoptosis‐inducing effects concomitant with losses in mitochondrial membrane potential, caspase activation, intracellular ROS production, and sustained ERK activation. Our data show that KUF‐1 or KUF‐2 regulate the apoptosis of RKO cells via intracellular ROS production coupled with the concomitant activation of the ERK signaling pathway, thereby implying that hydroxylation or substitution at C7 is critical for the apoptosis‐inducing activity of flavonoids. J. Cell. Biochem. 104: 259–273, 2008.

Effects of Feeding Barley Naturally Contaminated with Fusarium Mycotoxins on Growth Performance, Nutrient Digestibility, and Blood Chemistry of Gilts and Growth Recoveries by Feeding a Non-contaminated Diet

The objectives of this study were to investigate the effects of feeding barley naturally contaminated with Fusarium mycotoxins on growth performance, vulva swelling, and digestibility of dry matter, organic matter, and crude protein of gilts and the recovery of gilts fed normal diets immediately after the exposure to contaminated diets by measuring growth performance and vulva swelling. In Exp. 1, four diets were prepared to contain 0%, 15%, 30%, or 45% contaminated barley containing 25.7 mg/kg deoxynivalenol and 26.0 μg/kg zearalenone. Sixteen gilts with an initial body weight (BW) of 33.3 kg (standard deviation = 3.0) were individually housed in a metabolism crate and assigned to 4 diets with 4 replicates in a randomized complete block design based on BW. During the 14-d feeding trial, individual BW and feed consumption were measured weekly and the vertical and horizontal lengths of vulva were measured every 3 d. From d 10, feces were collected by the maker-to-marker method for 4 d. Blood samples were collected on d 14. During the overall period, the average daily gain, average daily feed intake, and gain:feed of pigs linearly decreased (p<0.01) as the dietary concentration of contaminated barley increased. However, the digestibility of crude protein was linearly increased (p = 0.011) with the increasing amounts of contaminated barley. Increasing dietary Fusarium mycotoxin concentrations did not influence vulva size, blood characteristic as well as immunoglobulin level of pigs. In the Exp. 2, a corn-soybean meal-based diet was formulated as a recovery diet. Pigs were fed the recovery diet immediately after completion of the Exp. 1. During the 14-d of recovery period, the individual BW and feed consumption were measured weekly and the vertical and horizontal length of vulva were measured every 3 d from d 0. On d 7, the feed intake of pigs previously fed contaminated diets already reached that of pigs fed a diet with 0% contaminated barley and no significant difference in growth performance among treatments was observed during d 7 to 14 of the recovery period. In conclusion, increasing levels of mycotoxins in diets linearly decreased the growth performance of pigs, and these damages can be recovered in 7 d after the diet was replaced with a normal diet. The vulva size, blood characteristic, immune responses were not affected by increasing level of contaminated barley in the diets fed to pigs.

Alpha-fetoprotein, identified as a novel marker for the antioxidant effect of placental extract, exhibits synergistic antioxidant activity in the presence of estradiol.

Placenta, as a reservoir of nutrients, has been widely used in medical and cosmetic materials. Here, we focused on the antioxidant properties of placental extract and attempted to isolate and identify the main antioxidant factors. Porcine placental extracts were prepared through homogenization or acid hydrolysis, and their antioxidant activity was investigated in the human keratinocyte HaCaT cell line. Treatment with homogenized placental extract (H-PE) increased the cell viability of H2O2-treated HaCaT cells more than two-fold. H-PE treatment suppressed H2O2-induced apoptotic and necrotic cell death and decreased intracellular ROS levels in H2O2-treated HaCaT cells. The antioxidant factors in H-PE were found to be thermo-unstable and were thus expected to include proteins. The candidate antioxidant proteins were fractionated with cation-exchange, anion-exchange, and size-exclusion chromatography, and the antioxidant properties of the chromatographic fractions were investigated. We obtained specific antioxidant fractions that suppressed ROS generation and ROS-induced DNA strand breaks. From silver staining and MALDI-TOF analyses, alpha-fetoprotein (AFP) precursor was identified as a main marker for the antioxidant effect of H-PE. Purified AFP or ectopically expressed AFP exhibited synergistic antioxidant activity in the presence of estradiol. Taken together, our data suggest that AFP, a serum glycoprotein produced at high levels during fetal development, is a novel marker protein for the antioxidant effect of the placenta that exhibits synergistic antioxidant activity in the presence of estradiol.

Ileal Digestibility of Amino Acids in Meat Meal and Soybean Meal Fed to Growing Pigs

The objective of this experiment was to determine the concentration and digestibility of crude protein (CP) and amino acid (AA) in meat meal (MM), and to compare these values with the respective values in soybean meal (SBM). Six barrows (initial body weight = 66.9±3.8 kg) surgically fitted with a T-cannula at the distal ileum were allotted to a replicated 3×3 balanced Latin square design with 3 diets and 3 periods. Two experimental diets containing test ingredients as the sole source of AA were prepared to estimate the apparent ileal digestibility (AID) for CP and AA by the direct method. An N-free diet was also prepared to estimate basal endogenous losses of CP and AA. All experimental diets contained 5% chromic oxide as an indigestible index. Each period consisted of a 5-d adaptation period and a 2-d of ileal digesta collection period. Ileal digesta samples were collected from 0900 to 1700 on d 6 and 7 of each period. The concentrations of CP, Lys, Met, and Trp in MM and SBM were analyzed to be 64.1, 3.5, 1.1 and 0.6, and 45.6, 2.8, 0.8, and 0.3%, respectively. The AID of all AA except Gly in MM was less (p<0.05) than in SBM. The AID of Lys, Met, and Trp in MM was estimated to be 56.0, 71.7, and 47.1%, respectively. The SID of all AA in MM was less (p<0.05) than in SBM. The SID of Lys, Met, and Trp was 65.1, 79.2, and 78.5%, respectively. In conclusion, the CP and AA contents in MM were greater than those in SBM whereas the ileal digestibility of all AA in MM was less than in SBM.

Porcine nuclear transfer using somatic donor cells altered to express male germ cell function

Recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible. In the present study, we were able to modulate the cell fate of somatic cells to take on male germ cell function by introducing cell extracts derived from porcine testis tissue. Fibroblasts were treated with streptolysin O, which reversibly permeabilises the plasma membrane, and incubated with testis extracts. Our results showed that the testis extracts (TE) could activate expression of male germ cell-specific genes, implying that TE can provide regulatory components required for altering the cell fate of fibroblasts. Male germ cell function was sustained for more than 10 days after the introduction of TE. In addition, a single TE-treated cell was injected directly into the cytoplasm of in vitro-matured porcine oocytes. The rate of blastocyst formation was significantly higher in the TE-treated nuclear donor cell group than in the control cell group. The expression level of Nanog, Sox9 and Eomes was drastically increased when altered cells were used as donor nuclei. Our results suggest that TE can be used to alter the cell fate of fibroblasts to express male germ cell function and improve the developmental efficiency of the nuclear transfer porcine embryos.

In Vitro Solubility of Copper(II) Sulfate and Dicopper Chloride Trihydroxide for Pigs.

This study was conducted to determine the solubility of copper (Cu) in two sources of copper(II) sulfate (CuSO4) including monohydrate and pentahydrate and three sources of dicopper chloride trihydroxide (dCCTH) including α-form (dCCTH-α), β-form (dCCTH-β), and a mixture of α- and β-form (dCCTH-αβ) at different pH and a 3-step in vitro digestion assay for pigs. In Exp. 1, Cu sources were incubated in water-based buffers at pH 2.0, 3.0, 4.8, and 6.8 for 4 h using a shaking incubator at 39°C. The CuSO4 sources were completely dissolved within 15 min except at pH 6.8. The solubility of Cu in dCCTH-α was greater (p<0.05) than dCCTH-β but was not different from dCCTH-αβ during 3-h incubation at pH 2.0 and during 2-h incubation at pH 3.0. At pH 4.8, there were no significant differences in solubility of Cu in dCCTH sources. Copper in dCCTH sources were non-soluble at pH 6.8. In Exp. 2, the solubility of Cu was determined during the 3-step in vitro digestion assay for pigs. All sources of Cu were completely dissolved in step 1 which simulated digestion in the stomach. In Exp. 3, the solubility of Cu in experimental diets including a control diet and diets containing 250 mg/kg of additional Cu from five Cu sources was determined during the in vitro digestion assay. The solubility of Cu in diets containing additional Cu sources were greater (p<0.05) than the control diet in step 1. In conclusion, the solubility of Cu was influenced by pH of digesta but was not different among sources based on the in vitro digestion assay.

Different Coefficients and Exponents for Metabolic Body Weight in a Model to Estimate Individual Feed Intake for Growing-finishing Pigs.

Estimation of feed intake (FI) for individual animals within a pen is needed in situations where more than one animal share a feeder during feeding trials. A partitioning method (PM) was previously published as a model to estimate the individual FI (IFI). Briefly, the IFI of a pig within the pen was calculated by partitioning IFI into IFI for maintenance (IFIm) and IFI for growth. In the PM, IFIm is determined based on the metabolic body weight (BW), which is calculated using the coefficient of 106 and exponent of 0.75. Two simulation studies were conducted to test the hypothesis that the use of different coefficients and exponents for metabolic BW to calculate IFIm improves the accuracy of the estimates of IFI for pigs, and that PM is applied to pigs fed in group-housing systems. The accuracy of prediction represented by difference between actual and estimated IFI was compared using PM, ratio (RM), or averaging method (AM). In simulation studies 1 and 2, the PM estimated IFI better than the AM and RM during most of the periods (p<0.05). The use of 0.60 as the exponent and the coefficient of 197 to calculate metabolic BW did not improve the accuracy of the IFI estimates in both simulation studies 1 and 2. The results imply that the use of 197 kcal×kg BW0.60 as metabolizable energy for maintenance in PM does not improve the accuracy of IFI estimations compared with the use of 106 kcal×kg BW0.75 and that the PM estimates the IFI of pigs with greater accuracy compared with the averaging or ratio methods in group-housing systems.