The Ubiquitin Receptor DA1 Regulates Seed and Organ Size by Modulating the Stability of the Ubiquitin-Specific Protease UBP15/SOD2 in Arabidopsis

Although the control of organ size is a fundamental question in developmental biology, little is known about the genetic and molecular mechanisms that determine the final size of seeds in plants. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligases DA2 and ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to restrict seed growth in Arabidopsis thaliana. Here, we describe UBIQUITIN-SPECIFIC PROTEASE15 (UBP15), encoded by SUPPRESSOR2 OF DA1 (SOD2), which acts maternally to regulate seed size by promoting cell proliferation in the integuments of ovules and developing seeds. The sod2/ubp15 mutants form small seeds, while overexpression of UBP15 increases seed size of wild-type plants. Genetic analyses indicate that UBP15 functions antagonistically in a common pathway with DA1 to influence seed size, but does so independently of DA2 and EOD1. Further results reveal that DA1 physically associates with UBP15 in vitro and in vivo and modulates the stability of UBP15. Therefore, our findings establish a genetic and molecular framework for the regulation of seed size by four ubiquitin-related proteins DA1, DA2, EOD1, and UBP15 and suggest that they are promising targets for increasing seed size in crops.     

A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.     

Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

The purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 +/- 2.70 versus 87.40 +/- 5.13; 44.10 +/- 8.62 versus 58.38 +/- 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.     

DEM Numerical Simulation of Abrasive Wear Characteristics of a Bioinspired Ridged Surface

<正> This paper presents numerical investigations into a ridged surface whose design is inspired by the geometry of a Farrer'sscallop.The objective of the performed research is to assess if the proposed Bioinspired Ridged Surface (BRS) can potentiallyimprove wear resistance of soil-engaging components used in agricultural machinery and to validate numerical simulationsperformed using software based on the Discrete Element Method (DEM).The wear performance of the BRS is experimentallydetermined and also compared with a conventional flat surface.Different size of soil particles and relative velocities between theabrasive sand and the testing surfaces are used.Comparative results show that the numerical simulations are in agreement withthe experimental results and support the hypothesis that abrasive wear is greatly reduced by substituting a conventional flatsurface with the BRS.     

冷冻电镜密度图可视化软件VAT4M

基于冷冻电镜三维密度图的特征分析,研究开发了冷冻电镜密度图可视化软件VAT4M(Visual Analysis Tools for Macro-Molecule Map and Model),实现了对三维冷冻电镜密度图的可视化分析,其主要功能包括密度图和分子模型的可视化、密度图的分割和晶体结构在密度图中的匹配。在可视化方面,在实现等值面可视化方法的同时还实现了体绘制可视化模式。对于结构分析,VAT4M提供了自动分割工具,和所见即所得的分割浏览器。同时,VAT4M还提供了极值吸收法与梯度法相结合的自动匹配方法,而且,对于具有对称性的密度图,可根据一个亚基的匹配结果,迅速得到其准原子模型。最后给出了若干应用实例。实验表明,本平台对冷冻电镜密度数据具有良好的可视与分析功能。     

Preparation and PCR-amplification properties of a novel amphiphilic poly(N-vinylpyrrolidone) (PVP) copolymer

A novel amphiphilic copolymer, P(NVP-co-TrpAMT) (9) was prepared, comprising hydrophilic N-vinylpyrrolidone (NVP; 8) and hydrophobic 'N-[(tert-butoxy)carbonyl]tryptophanamido-N'-methacryl thiourea' (TrpAMT; 7) segments. The amphiphilic copolymer 9 was characterized by (1)H-NMR, GPC-MALLS, TEM, and MTT assay. It has a critical micelle concentration (cmc) of 45.7 mg/l in aqueous solution, and good biocompatibility in vivo. According to TEM, the polymer is mostly present as spherical micelles in water, with a diameter of ca. 60-90 nm. In the presence of 0.1 mug/ml of 9, the PCR amplification of the GC-rich beta-actin was efficiently enhanced. Also, the fluorescence intensity of the reporter dye SYBR Green I was increased by 26% at the 14th cycle during real-time PCR of plasmid pUC18 DNA.     

Autophagy is involved in ethanol-induced cardia bifida during chick cardiogenesis

Excess alcohol consumption during pregnancy has been acknowledged to increase the incidence of congenital disorders, especially the cardiovascular system. However, the mechanism involved in ethanol-induced cardiac malformation in prenatal fetus is still unknown. We demonstrated that ethanol exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Previously, we reported that autophagy was involved in heart tube formation. In this context, we demonstrated that ethanol exposure increased ATG7 and LC3 expression. mTOR was found to be inhibited by ethanol exposure. We activated autophagy using exogenous rapamycin (RAPA) and observed that it induced cardiac bifida and increased GATA5 expression. RAPA beads implantation experiments revealed that RAPA restricted ventricular myosin heavy chain (VMHC) expression. In vitro explant cultures of anterior primitive streak demonstrated that both ethanol and RAPA treatments could reduce cell differentiation and the spontaneous beating of cardiac precursor cells. In addition, the bead experiments showed that RAPA inhibited GATA5 expression during heart tube formation. Semiquantitative RT-PCR analysis indicated that BMP2 expression was increased while GATA4 expression was suppressed. In the embryos exposed to excess ethanol, BMP2, GATA4 and FGF8 expression was repressed. These genes are associated with cardiomyocyte differentiation, while heart tube fusion is associated with increased Wnt3a but reduced VEGF and Slit2 expression. Furthermore, the ethanol exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. In sum, our results revealed that disrupting autophagy and excess ROS generation are responsible for inducing abnormal cardiogenesis in ethanol-treated chick embryos.