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.     

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.     

Leiomyosarcoma of urinary bladder in a Shih Tzu dog

A 10-year-old intact male Shih Tzu dog presented with hematuria. Double-contrast cystography revealed a polypoid filling defect at the apex of the urinary bladder. Ultrasonography revealed a heterogeneously hypoechoic intramural mass with minimal vascular flow beneath the submucosal layer. After partial cystectomy, a well-demarcated bladder leiomyosarcoma was diagnosed on histopathology. The patient was alive and well without any clinical signs, recurrence, or metastasis at the 29-month follow-up after the surgical removal of the bladder mass. Leiomyosarcoma should be considered as a differential diagnosis if mass-like lesions are observed in the urinary bladder, although this type of malignancy is rare in canines. Histopathological confirmation is important for predicting prognosis and determining further medical plans.     

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.     

Functional and Nutritional Characteristics of Soft Wheat Grown in No‐Till and Conventional Cropping Systems

The effects of no‐till versus conventional farming practices were evaluated on soft wheat functional and nutritional characteristics, including kernel physical properties, whole wheat composition, antioxidant activity, and end‐product quality. Soft white winter wheat cultivar ORCF 102 was evaluated over a two‐year period from three long‐term replicated no‐till versus conventional tillage studies in Oregon. Wheat from the no‐till cropping systems generally had greater test weight, kernel diameter, and kernel weight and had softer kernels compared with wheat from the conventional tillage systems. Compared with the conventional systems, no‐till whole wheat flour had lower protein and SDS sedimentation volume. Ash content as well as most minerals measured (calcium, copper, iron, magnesium, and zinc), except for manganese and phosphorus, were generally slightly lower in no‐till than in conventional wheat. Whole wheat flour from the no‐till cropping systems generally had slightly lower total phenolic content and total antioxidant capacity. Milling properties, including flour yield, break flour yield, and mill score, were not affected by tillage systems. Refined flour from no‐till systems had lower protein, SDS sedimentation volume, and lactic acid and sucrose solvent retention capacities compared with flour from conventional tillage. No‐till wheat generally had greater sugar‐snap cookie diameter than conventionally tilled wheat. In conclusion, no‐till soft white winter wheat generally had slightly reduced nutritional properties (protein, ash, most minerals, and total antioxidant content) compared with wheat from conventionally tilled systems, and it had equivalent or sometimes superior functional properties for baking cookie‐type products.     

Generation of Salmonella ghost cells expressing fimbrial antigens of enterotoxigenic Escherichia coli and evaluation of their antigenicity in a murine model

Salmonella Typhimurium ghost cells expressing K88ab, K88ac, K99, and FasA fimbriae of enterotoxigenic Escherichia coli (ETEC) in their envelopes were constructed. The genes encoding the fimbriae were individually cloned into an expression plasmid, pMMP81, carrying the asd gene, which was subsequently electroporated into the Δasd S. Typhimurium mutant. Plasmid pJHLP99, carrying the phiX174 lysis gene E, was also subsequently electroporated into the Salmonella mutant. The presence of the individual fimbriae on the ghost cells was examined by Western blot analysis. Forty BALB/c mice were equally divided into 2 groups of 20 mice each. Group A mice were intramuscularly vaccinated with a mixture of the 4 ghost cells expressing the individual fimbriae. The group B mice were inoculated with sterile phosphate-buffered saline as a control. The antigen-specific serum IgG concentrations were significantly higher in group A than in group B from week 2 until week 6 after inoculation. In addition, the antigen-specific IgA concentrations in fecal samples were significantly higher in group A than in group B at week 2 after inoculation. A large difference between the groups in the number of antigen-specific IgA-secreting cells in the small intestine was observed by immunohistochemical study. Also, the splenic lymphocyte proliferative responses were significantly greater in group A than in the control mice. These results suggest that vaccination with our Salmonella ghost cells can induce both humoral and cell-mediated immune responses and that the increased number of antigen-specific IgA-secreting cells in the small intestine may be correlated with the elevated fecal IgA immune response.     

Hydroxycinnamoylmalic acids and their methyl esters from pear (Pyrus pyrifolia Nakai) fruit peel

Two novel caffeoylmalic acid methyl esters, 2-O-(trans-caffeoyl)malic acid 1-methyl ester (6) and 2-O-(trans-caffeoyl)malic acid 4-methyl ester (7), were isolated from pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. In addition, 5 known hydroxycinnamoylmalic acids and their methyl esters were identified: 2-O-(trans-coumaroyl)malic acid (1), 2-O-(cis-coumaroyl)malic acid (2), 2-O-(cis-coumaroyl)malic acid 1-methyl ester (3), 2-O-(trans-coumaroyl)malic acid 1-methyl ester (4), and 2-O-(trans-caffeoyl)malic acid (phaselic acid, 5). The chemical structures of these compounds were determined by spectroscopic data from ESI MS and NMR. Of all the isolated compounds, five hydroxycinnamoylmalic acids and their methyl esters (2-4, 6, 7) were identified in the pear for the first time.