Application of denaturing high-performance liquid chromatography for rice variety identification and seed purity assessment

Recent DNA sequencing projects and the establishment of high-throughput assays have provided an abundance of sequence information and data on nucleotide polymorphisms in rice. Based on previously identified single-nucleotide polymorphisms (SNPs) and insertions/deletions, we employed denaturing high-performance liquid chromatography (DHPLC) to genotype rice varieties using a chromatographic pattern-based strategy. In this study, 12 amplicons harboring multiple and informative SNPs were screened. PCR products of the 12 amplicons from 47 rice varieties were analyzed by DHPLC and DNA sequencing. Each homozygous sample with a single peak pattern in the initial DHPLC analysis was mixed with zhenshan97 for a second DHPLC analysis. The 12 amplicons were found to be polymorphic across the hybrids, and mixed homozygous samples with 43 distinct DHPLC elution profiles detected. Sequence analysis confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in six amplicons differentiated between all of the hybrids, inbred lines, and restorer lines and produced unique a fingerprint for these lines. In addition, we found that the DHPLC pattern of the hybrid was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. These results demonstrate that DHPLC can be efficiently applied for the rapid and automated identification of diverse rice varieties and could possibly be utilized for seed genetic purity testing on a high-throughput scale.     

GAS6 is an estrogen-inducible gene in mammary epithelial cells

To identify estrogen-responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERalpha interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERalpha was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells.     

S1P stimulates chemotactic migration and invasion in OVCAR3 ovarian cancer cells

OVCAR3 ovarian cancer cells express three sphingosine 1-phosphate (S1P) receptors, S1P(1), S1P(2), and S1P(3), but not S1P(4). Stimulation of OVCAR3 cells with S1P induced intracellular calcium increases, which were partly inhibited by VPC 23019 (an S1P(1/3) antagonist). S1P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S1P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S1P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S1P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 23019 (an S1P(1/3) antagonist) completely inhibited S1P-induced chemotaxis. S1P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 23019. Taken together, this study suggests that S1P stimulate chemotactic migration and cellular invasion, and VPC 23019-sensitive S1P receptor(s) might be involved in the processes.     

Arginine 177 is involved in Mn(II) binding by manganese peroxidase.

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

杀虫剂轮用和混用对害虫种群抗性演化的影响

杀虫剂轮用和混用是当前害虫抗药性治理中最常采用的两种用药策略。该文用抗性模型和室内试验对轮用和混用延缓害虫抗性演化的效果及轮用最佳间隔期问题作了研究。模型模拟结果表明,在延缓害虫抗性演化方面轮用和混用对抗性演化的影响主要取决于杀虫剂作用强度、抗性基因型个体的适合度大小和杀虫剂混用后的毒理效应类型。两种杀虫剂轮用时,轮用间隔期以1(即两种杀虫剂隔次施用)为佳。以模型昆虫小菜蛾Plutella xylostella在室内试验结果表明,氰戊菊酯单用(I)及与杀虫单轮用(II)和混用(1∶1)(III)连续处理8代后,抗性个体频率为0.01的小菜蛾种群其3龄幼虫对氰戊菊酯的抗性分别上升了75.87(I)、28.67(II)和58.72(III)倍,与模型模拟结果表现出较好的一致性。据此,作者认为抗性模型可用于预测害虫抗性演化和评价抗性治理策略。     

The first baleen whale marine protected area proposed for Bryde's whales in the Beibu Gulf,China

The establishment of marine protected areas (MPAs) for cetaceans is an important strategy to mitigate human disturbance and protect biodiversity. Despite abundant cetacean species, there are only a few MPAs dedicated to cetacean conservation in China, all of which are for inshore dolphins. Bryde's whales, the only nearshore baleen whale population in mainland China, are conflicting with intensive human activities, yet an effective conservation strategy is lacking. This study used species distribution models to analyze distribution patterns and suitable habitats of Bryde's whales in the Beibu Gulf and proposes the first baleen whale MPA in China. Our results showed Bryde's whales have a seasonal distribution pattern in the Beibu Gulf, and that the waters around Weizhou Island and the southeastern coast of Vietnam were their core habitats. The seasonal nighttime light data indicated a negative relationship between the number of ship lights and Bryde's whale sightings and suggest that Bryde's whales might be threatened by fisheries. We proposed an MPA based on the results, suggesting that the waters within 20 km around Weizhou Island should be declared a protected area. Furthermore, we recommend that anthropogenic activities in the waters around Weizhou Island are better managed to reduce negative impacts on marine life.