The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with α1,3-galactosyltransferase gene (α1,3-GT gene), DT-A/pGT5’/neo/pGT3’, DT-A/NLS/pGT5’/neo/pGT3’ and pGT5’/neo/ pGT3’/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5’ recombination arm (pGT5’) and a 1.9-kb fragment as the 3’ recombination arm (pGT3’). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of α1,3-GT locus. DT-A/pGT5’/neo/pGT3’ vector contain only positive-nega-tive selection marker with conventional targeting vector. DT-A/NLS/pGT5’/neo/pGT3’ vector contain positive-negative selection marker and NLS sequences in upstream of 5’ recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5’/neo/pGT3’/NLS vector contain only positive selection marker and NLS sequence in downstream of 3’ recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with 300 μg/ml G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for α1,3-GT gene disruption in 3´ PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5’/neo/pGT3’ knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.
Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.
The Black pine bast scale, Matsucoccus thunbergianae is one of the most serious in black pine, Pinus thunbergii forests in Korea. Since this pest was first reported in Goheung, Korea in 1963, which is gradually spread into neighboring regions and now occurs in many regions of the southern and eastern part of the Korean peninsula. The monitoring for distribution of M. thunbergianae was able to observed by naked eye egg sacs and pupa of male on the host until now. Therefore, this monitoring was very difficult in the low density of M. thubergianae. This experiment was conducted to use simple and practical moving cross-shaped flat trap for monitoring of M. thunbergianae. The monitoring of M. thunbergianae using the device was carried out to southern regions of the Korean peninsula. The first emergence of male showed mid. March in Namhae and late march in Busan, Jinju and Pohang. The peak of emergence showed late March in Namhae and early April in the other regions. When the number of M. thunbergianae intermediate nymph showed 58~59, 11~44 and 8~25 on 39.25 ㎠ bark area of the black pine, Pinus thubergii for 1 week, the number of captured its male adult was 58~83, 67~488 and 1~55 on the moving cross-shaped flat trap (10× 13㎝), respectively. The low density of M. thunbergianae was some few the number of capture, but there were no significant difference in its high density. Also, the number of captured its male adult was no significant in the different color (yellow, red, white and blue) of the moving cross-shaped flat trap.
Several species of the genus Aphidius are used in biological control programs against aphid pests throughout the world and their behavior and physiology are well studied. But despite knowing the importance of sensory organs in their behavior, their antennal structure is largely unknown. In this study, the external morphology and distribution of the antennal sensilla on the antennal of both female and male adults of A. colemani were described using scanning electron microscopy (SEM). Generally, the filaform antennae of males (1,515.20±116.48 ㎛) are longer than females (1,275.06±116.42㎛). Antennae of this species is made up of scape, pedicel and flagellomeres. Male and female antennae differed in the total number of flagellomeres as 15 in males and 13 in females. Female and male antennae of A. colemani has samely seven types of sensilla. We classified sensilla placodea, Bohm bristles, 2 types of sensilla coeloconica, , 2 types of sensilla basiconica as with a tip pore and with wall pores, sensilla trichodea. In addition, the possible functions of the above sensilla types are discussed in light of previously published literature; mechanoreception(Bohm bristles, sensilla coeloconicaⅡ and sensilla trichodea) and chemoreception(sensilla coeloconicaⅠ, sensilla basiconicaⅠ,Ⅱ and sensilla placodea). Future studies on the functional morphology of the antennal sensilla of A. colemani using transmission electron microscopy (TEM) coupled with electrophysiological recordings will likely confirm the functions of the different sensilla identified in this study.
It has been reported that light-emitting diodes(LED) can be used in the treatment of oral diseases. Although bio-stimulatory effects of LED irradiation such as promotes stimulation of wound healing have been well known, there are few reports about molecular mechanism associated with cell cycle by LED irradiation. The purpose of present study was to examine the molecular event in cell cycle of LED irradiation on primary human gingival fibroblast(hGF) in vitro. The source of light for irradiation was a continuous-wave LED emitting at a wavelength of 635nm, and manufactured that energy density was 5mW/cm2 on sample surface. The hGF were irradiated for 1 hour at 37℃ in 5% CO2 humidified chamber. Experimental samples were acquired at 0 (right after irradiation), 8 and 24 hour after irradiation. To investigate the molecular mechanisms associated with cell cycle, growth phase was determined by flow cytometry and mRNA expression of cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, PCNA, p18, p27, p21, and p53 were determined by real time RT-PCR. Flow cytometric analysis demonstrated the percentage of cells in the G1 and S phase were decreased, but the G2 phase increased, which showed cells irradiated by LED were transitioned from S to G2 phase. For mRNA expression, cyclin B, cdc2, PCNA and p53 were increased at 0 hour after irradiation, and most of cell cycle molecules were increased at 8 hour after irradiation. At 24 hour after irradiation, cyclin A, cyclin E, PCNA and p18 were increased. Taken together, LED irradiation induced proliferation of hGF cells through transition from S to G2 phase.
Cloned calves derived from somatic cell nuclear transfer (SCNT) have been frequently lost by sudden death at 1 to 3 month following healthy birth. To address whether placental anomalies are responsible for the sudden death of cloned calves, we compared protein patterns of 2 placentae derived from SCNT of Korean Native calves died suddenly at two months after birth and those of 2 normal placentae obtained from AI fetuses. Placental proteins were separated using 2-Dimensional gel electrophoresis. Approximately 800 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis of Malanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, 8 spots were identified to be up-regulated proteins and 24 spots to be down-regulated proteins in SCNT placentae, among which proteins were high mobility group protein HMG1, apolipoprotein A-1 precursor, bactenecin 1, tropomyosin beta chain, H+-transporting ATPase, carbonic anhydrase II, peroxiredoxin 2, tyrosine-rich acidic matrix protein, serum albumin precursor and cathepsin D. These results suggested that the sudden death of cloned calves might be related to abnormal protein expression in placenta.
The aim of this study is to investigate the antioxidant and intracellular anti-inflammatory efficacy of blueberry leaf extracted with hot water (BLW), 70% ethanol (BLE), and 70% acetone (BLA) in RAW 264.7 macrophages. In order to evaluate the anti-inflammatory effect of blueberry leaf extracts, RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS) to induce the production of inflammation-related factors, which were measure by Western blotting and real-time PCR methods. i-NOS, COX-2 protein, and mRNA expression showed concentration-dependent decrease. The decreases in the mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) were concentration-dependent. Further, the antioxidant effects of blueberry leaf on total polyphenol contents, electron donating ability and ABTS+ radical scavenging activity were evaluated. The total polyphenol contents of BLW, BLE, and BLA were 217.04±2.98, 156.72±3.90, and 182.88±3.02 mg TAE/g, respectively, while the electron donating abilities at 1,000 μg/mL of BLW, BLE, and BLA were 81.7, 79.6, and 79.3%, respectively. The ABTS+ radical scavenging activity was fond to be concentration dependent. The nitric oxide (NO) production inhibition activities at 50 μg/mL of BLW, BLE, and BLA were 35.1, 42.4 and 42.7%, respectively. In conclusion, the antioxidant and anti-inflammatory test results indicate that blueberry leaf extracts (BLW, BLE, and BLA) can be used as potential anti-inflammatory agents.
Background : Undulatum Rhubarb, commonly produced in domestic, is rhizome of Rheum undulatum L. that belongs to the family Polygonaceae. It also can be used as a substitute of R. palmatum L., R. tanguticum Maximowicz ex Balf., and R. officinale Baillon which completely depend on import system. However, there should be clear clarification among Undulatum Rhubarb and Rhubarb, because Undulatum Rhubarb contains rhaponticin as marker compound that is not indicated at Rhubarb. Some of the recently imported Undulatum Rhubarbs have been found to be Rhubarb. Also, it is known that only Undulatum Rhubarb is cultivated at domestic environment. But some plant origins of Rhubarb are grown in Korea, too. Further study are needed to clarify clear origin between Undulatum Rhubarb and Rhubarb. Thus, we collected some domestically cultivated samples and identified them. Methods and Reseults : Rheum undulatum L., Rhubarb, Rheum tanguticum Maximowicz ex Balf. which were cultivated in Gangwondo Agricultural Research and Extension Services in Cheorwon were collected and anayzed the DNA sequences. We also compared DNA sequences in Rhubarb collected from England and R. rhabarbarum L., R. undulatum L., and R. franzenbachii on NCBI. As a result, two kinds of rhubarb cultivated in the test plantation were identified as R. rhabarbarum and R. officinale. In addition, R. undulatum (plant origin of Undulatum Rhubarb) was identified as Rhubarb (Rheum rhabarbarum) in England with 99 - 100% identical in nuclear ITS gene region. Conclusion : R. undulatum, plant origin of Undulatum Rhubarb, is reported as synonym of R. rhabarbarum, R. franzenbachii. Rheum speices which are cultivated as tester in Gangwondo Agricultural Research and Extension Services in Cheorwon are estimated as R. undulatum and R. officinale. Therefore, not only Undulatum Rhubarb but Rhubarb could be grown in Korea.
Background : Perilla frutescens L. is valuable as a medicinal plant as well as a natural medicine and functional food. Limonene perilla collected from various places showed 60% limonene compounds. However biological activity of these accession has not been reported before. Therefore, this study was conducted to investigate the biological activity of limonene perilla. Methods and Results : Fractional solvent extracts were obtained by using organic solvents such as n-hexane, chloroform, ethyl acetate, n-BuOH, and aqueous solvent from different parts of limonene perilla extracted initially in 70% EtOH. We investigated the effects of limonene perilla on total phenol and flavonoid contents, FRAP (Ferric Reducing Antioxidant Power), total saponin contents and tyrosinase inhibition activity. Leaves of limonene perilla produced the highest total phenolic contents (29.88 mg·CAE/g), flavonoid (8.39 mg·QE/g) and saponin contents (47.77 mg·GIE/g) than stems and roots of limonene perilla. FRAP of leaves was 823.00±3.58 μM·FeSO4·E/mg. Tyrosinase inhibition activity rate was 40.31% in 70% ethanol extracts from leaves of limonene perilla. Conclusion : This results suggest that leaf of limonene perilla fractions has significant antioxidant activity. Also, limonene perilla could be used as a functional biomaterial in developing cosmetics and functional foods.
Background : This study aimed to investigate the antioxidant and anti-inflammatory activities of water chestnut (Trapa japonica Flerow) extract. Methods and Results : The antioxidant and anti-inflammatory activities of 100% methanol extract of water chestnut were investigated. The methanol extract was evaluated for its total phenolic and flavonoid content, DPPH•(1,1-diphenyl-2-picrylhydrazyl) free-radical scavenging activity,reducing power, andeffect on nitric oxide (NO) production and cell viability using real-time polymerase chain reaction (qPCR). The total phenolic content was 438.31 ㎍ allic acid equivalent (GAE)/㎎ extract and the total flavonoid content was 61.40 ㎍ quercetin equivalent (QE)/㎎ extract. In addition, results revealed the extract possessed antioxidant activity (DPPH• free-radical scavenging activity) with IC50 value of 5.28 ㎍㎖ The reducing power of the extract was assayed spectro photometrically and showed Abs of 0.71 at 100 ㎍㎖ Furthermore, extracts of water chestnut exhibited no cytotoxicity in RAW 264.7 cells. In addition, the NO assay revealed that LPS-induced NO production was significantly inhibited following treatment with water chestnut extracts. The expression of pro-inflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 decreased in a concentration-dependent manner. The water chestnut extract also decreased tumor necrosis factor α (TNF-α) release. Conclusion : Therefore, the present findings provide scientific evidence for the nutritional potential, chemical composition, and biological activities of Trapa japonica Flerow anddemonstrate its potential use as a functional food forapplication in the pharmaceutical industry
Plants have evolved elaborate innate immune systems against invading pathogens, such as bacteria, fungi, oomycetes, viruses and insects. Among them, intracellular immune receptors known as nucleotide-binding site and leucine-rich repeat (NB-LRR) play critical roles in effector-triggered immunity (ETI) regarding to plant defense. Here, we identified potential NB-LRR coding sequences from pepper genome using bioinformatics analysis and performed comparative analysis with Solanaceae plants. As a result, we identified 267, 443, and 755 NBS-encoding genes in the genome of tomato, potato, and pepper, respectively. These may indicate that the Solanaceae NB-LRRs were evolved through species-specific unequal-duplication event. Further phylogenetic and clustering analyses revealed that Solanaceae NB-LRRs were classified into the 14 subgroups with 1 TNL and 13 CNL types. We found that the genes in CNL-G1 and CNL-G2 subgroup were highly expanded compared to other subgroup showing a large portion of NB-LRR in pepper genome. Among 755 NB-LRRs in pepper genome, 623 were physically mapped on all 12 pepper chromosome pseudomolecules. Furthermore, a number of NB-LRRs in the same group were physically clustered by tandem array in the specific chromosome. Genome-wide identification of pepper NB-LRR family and their evolutionary analysis could provide an important resource for identification and characterization of genes for breeding of disease resistance crops.
Diffuse involvement of the right pulmonary artery (PA) associated with fistula between the PA and coronary artery is report-ed in a woman with Takayasu’s arteritis. Both the subclavian arteries were totally occluded and drained by the meanderinged artery arising from both common carotid arteries. Lung perfusion scan revealed perfusion defect of right lung. Two fistulas were identified. A large fistula was between the right PA and left circumflex artery. A small fistula was between the right PA and left anterior descending artery. This is a rare case of Takayasu’s arteritis presenting with a coronary – pulmonary artery fistula that is secondary to a diffuse unilateral involvement of PA.
Health beneficial properties of soybean [Glycine max (L.) Merr.] isoflavones are well known. The objectives of this study were to determine and compare the isoflavone composition and concentrations of soybean varieties grown in different cultivated regions of Vietnam (i.e., upland and lowland). Total and individual isoflavone composition and concentrations were determined by high-performance liquid chromatography (HPLC). Total isoflavone concentrations varied from 1153 to 6604~mug~;g-1 and averaging 3354~mug~;g-1 across environments and varieties. In the lowland region, the highest total isoflavones concentration was observed in M103 cultivar (5653~mug~;g-1 ) and the lowest in VX9-3 (1153~mug~;g-1 ), whereas in the upland region the highest and lowest concentrations were in M103 (6604~mug~;g-1 ) and DT93 (1938~mug~;g-1 ), respectively. Across varieties, average total isoflavones concentration was higher in the upland than lowland region (3728 vs. 2980~mug~;g-1 ). The malonylglucosides and acetylglucosides concentrations in upland soybean varieties were higher than those from the lowland region. Despite the presence of Genotype (G) x Environment (E) interactions, varieties with consistently high (M103) and low (VX9-3, DT93) isoflavone concentrations across environments were identified. This is the first report of isoflavones in Vietnamese soybean varieties, revealing large variation in isoflavones concentration and profile among different varieties and cultivated regions. Results will be useful in selecting high-isoflavones soybean varieties for growth in tropical regions.