Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference

Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P < 0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning.     

Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP-32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP-32 and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Up-regulation of DARPP-32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5-fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. What's more, the results of subrenal capsule assay confirmed that DARPP-32 might play a certain role in MDR of gastric cancer. DARPP-32 could significantly down-regulate the expression of P-gp and zinc ribbon domain-containing 1 (ZNRD1), but not alter the expression of multidrug resistance-associated protein (MRP) or the glutathione S-transferase (GST). DARPP-32 could also significantly decrease the anti-apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP-32 might be helpful for understanding the mechanisms of MDR in gastric cancer.     

Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.     

Fine mapping and candidate gene analysis of dense and erect panicle 3, DEP3, which confers high grain yield in rice (Oryza sativa L.)

Architecture of the rice inflorescence, which is determined mainly by the morphology, number and length of primary and secondary inflorescence branches, is an important agronomical trait. In the current study, we characterized a novel dense and erect panicle (EP) mutant, dep3, derived from the Oryza sativa ssp. japonica cultivar Hwacheong treated with N-methyl-N-nitrosourea. The panicle of the dep3 mutant remained erect from flowering to full maturation, whereas the panicle of the wild type plant began to droop after flowering. The dep3 mutation also regulated other panicle characteristics, including panicle length, grain shape and grain number per panicle. Anatomical observations revealed that the dep3 mutant had more small vascular bundles and a thicker culm than wild type plants, explaining the EP phenotype. Genetic analysis indicated that the phenotype with the dense and EP was controlled by a single recessive gene, termed dep3. The DEP3 gene was identified as the candidate via a map-based cloning approach and was predicted to encode a patatin-like phospholipase A2 (PLA2) superfamily domain-containing protein. The mutant allele gene carried a 408?bp genomic deletion within LOC_Os06g46350, which included the last 47?bp coding region of the third exon and the first 361?bp of the 3??-untranslated region. Taken together, our results indicated that the patatin-like PLA2 might play a significant role in the formation of vascular bundles, and that the dep3 mutant may provide another EP resource for rice breeding programs.     

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r2 = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.     

Magnesium Deficiency Enhances Hydrogen Peroxide Production and Oxidative Damage in Chick Embryo Hepatocyte In Vitro

Magnesium deficiency and oxidative stress have been identified as correlative factors in many diseases. The origin of free radicals correlated with oxidative damage resulting from Mg-deficiency is unclear at the cellular level. To investigate whether hydrogen peroxide (H₂O₂) is associated in the oxidative stress induced by Mg-deficiency, the effect of Mg²⁺ deficiency (0, 0.4, 0.7 mM) on the metabolism of H₂O₂ was investigated in cultured chick embryo hepatocytes. After being cultured in the media with various concentrations of Mg²⁺ for 1, 2, 4, 6 and 10 days, parameters of H₂O₂ production, catalase activity, lipid peroxidation, intracellular total Mg and cell viability were analyzed. Results demonstrated that long-term incubation of chick embryo hepatocyte in extracellular Mg²⁺-deprivative and Mg²⁺-deficient (0.4 mM) states significantly enhanced the production of H₂O₂ (approximately twofold, respectively) and lipid peroxidation in the cell cultures, while decreasing the cell viability. Additionally, the reversing action of Mg²⁺ re-added to 1.0 mM and the partial reversing action of dimethylthiourea suggested that (i) [Mg²⁺]_e deficiency induced the increase of H₂O₂ production, (ii) [Mg²⁺]_e deficiency decreased catalase activity in chick embryo hepatocyte in vitro, subsequently causing oxidative stress and cell peroxidative damage.