Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Effects of oral administration of different dosages of carvacrol essential oils on intestinal barrier function in broilers

Essential oils are widely used in the pharmaceutical, food and cosmetic industries, and many plant essential oils have shown that they have positive effects on broilers nutrition. This experiment was conducted to study the effects of orally administered different dosages of carvacrol essential oils on intestinal barrier function in broiler chickens. A total of eighty 28‐day‐old (1.28 ± 0.15 kg) ROSS 308 broilers were randomly allocated to four groups of 20 replicates each, with one chicken per replicate per cage, and all were fed with the same diet. Four experimental groups were orally administered 0, 200, 300 or 400 μl carvacrol essential oils at 18:00 hr every day during the 2‐week experimental period. As a result of which, the gene expression of the occludin, claudin‐1, claudin‐5, ZO‐1 and ZO‐2 in intestinal mucosa of small intestine (p < 0.05) and the goblet cell content in small intestine epithelium (p < 0.05) were significantly increased; test subjects with 300 or 400 μl carvacrol essential oils reduced the microbial counts of Salmonella spp. and Escherichia coli in the intestines (p < 0.05); Essential oils administration also significantly increased activity of the sucrase (p < 0.05) and lactase (p < 0.05) in intestinal mucosa. In conclusion, the carvacrol essential oils have positive effects on growth performance and intestinal barriers function of broilers; those effects may be related to the dosage, as administration of 300 or 400 μl was more effective than that of 200 μl.     

Therapeutic Potential of Phlorotannin-Rich Ecklonia cava Extract on Methylglyoxal-Induced Diabetic Nephropathy in In Vitro Model

Advanced glycation end-products (AGEs) play a vital role in the pathogenesis of diabetic complications. Methylglyoxal (MGO), one of the major precursors of AGEs, is a highly reactive dicarbonyl compound that plays an important role in the pathogenesis of diabetic nephropathy. This study was designed to evaluate the therapeutic potential of phlorotannin-rich Ecklonia cava extract (ECE) on MGO-induced diabetic nephropathy in in vitro models using mouse glomerular mesangial cells. ECE showed anti-glycation activity via breaking of AGEs-collagen cross-links and inhibition of AGEs formation and AGE-collagen cross-linking formation. The renoprotective effects were determined by assessing intracellular reactive oxygen species (ROS) and MGO accumulation, cell apoptosis, and the Nrf-2/ARE signaling pathway. MGO-induced renal damage, intracellular ROS production level, and MGO-protein adduct accumulation were significantly decreased by pretreating ECE. Moreover, ECE pretreatment exhibited preventive properties against MGO-induced dicarbonyl stress via activation of the Nrf2/ARE signaling pathway and reduction of RAGE protein expression in mouse glomerular mesangial cells. Collectively, these results indicated potential anti-glycation properties and prominent preventive effects of ECE against MGO-induced renal damage. Additionally, ECE may be utilized for the management of AGE-related diabetic nephropathy.     

Canine adenovirus type 1 infection of a Eurasian river otter (Lutra lutra)

A 10-year-old female Eurasian river otter (Lutra lutra) died after prolonged anorexia and weight loss in the Seoul Grand Park Zoo, Seoul, Republic of Korea. On necropsy, the liver was found to be swollen and friable with 1 lobe enlarged and necrotic. The other organs showed no significant alterations except for mild atrophy of the right kidney. Microscopically, there was multifocal hepatic necrosis. The hepatocytes around the necrotic areas were swollen and contained large basophilic intranuclear inclusions. Periportal infiltration by plasma cells and lymphocytes was also evident. Transmission electron microscopy revealed characteristic hexagonal virus particles sized approximately 70 nm in diameter in the nuclei of the hepatocytes, which were consistent with an adenovirus. Polymerase chain reaction of the formalin-fixed, paraffin-embedded liver sections was used to determine whether the virus was either the canine adenovirus type 1 (CAV-1), canine adenovirus type 2 (CAV-2), or some other viral agent. The results of these tests showed that the virus was CAV-1. To our knowledge, this is the first report on a CAV-1 infection in an otter.     

Chitosan nanoparticles enhance developmental competence of in vitro-matured porcine oocytes

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus–oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50 µg/ml) during in vitro maturation (IVM). Oocytes treated with 25 µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

Sacrocaudal agenesis in a Korean native calf (Bos Taurus Coreanae)

A male deformed Korean native calf was examined macroscopically. The deformed calf had no caudal vertebral columns from 5th lumbar vertebra, sacrum and coccygeal vertebrae. The spinal cord was terminated in the vertebral foramen of the 3rd lumbar vertebra. The cervical vertebrae had scoliosis and the 3rd and 4th cervical vertebrae were fused. The 2nd and 3rd lumbar vertebrae were fused and the left and right transverse processes of the 4th lumbar vertebra articulated with ala of the ilium. The rectum was greatly expanded by the imperforate anus and a rectourethral fistula was formed between the rectum and urethra. The deformed calf was recorded as a first documentation of sacrocaudal agenesis confirmed in a Korean native calf.