Transgenic silkworms (<Emphasis Type="Italic">Bombyx mori</Emphasis>) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

蔗糖对桃幼苗生长发育及其SnRK1酶活性的影响

以实生桃(Prunus persica)苗为试材, 探讨SnRK1对不同浓度蔗糖及处理时间的响应特性, 揭示蔗糖对植株生长发育的影响, 以期为果树生产提供理论依据及技术支持。结果表明, 施加5%蔗糖时, 植株体内SnRK1酶活性最高, 且在一定时间内, 酶活性持续升高; 与对照(清水和甘露醇)相比, 5%蔗糖处理显著提高植株可溶性糖、淀粉和叶片叶绿素含量, 增加植株地上部和地下部生物量, 显著加快植株净光合速率; 通过观察根系构型, 发现5%蔗糖可以显著增加根系总表面积、总体积和侧根数量, 并可促进根系加粗加长生长。qRT-PCR分析表明, 外源蔗糖能促进根系中生长素的合成和转运。综上, 一定浓度蔗糖可以提高植株体内SnRK1酶活性, 影响植株碳代谢, 促进植株生长发育, 且增加根系生长素的合成与转运, 进而影响根系构型。     

Determination and comparison of sludge reduction rates caused by microfaunas' predation

Using micorfauna to reduce excess sludge is a potentially effective ecological technology and scaling the rate of sludge reduction rate is the first step. A method to scale the rate of sludge reduction caused by microfauna was proposed, and comparison of sludge reduction rates induced by four microfaunas was carried out. The principle of this method is based on the change of carbon forms. The rate of sludge reduction was correlated with the rate at which solids were changed into liquid and gas. Four microfaunas, including Aeolosoma hemprichi, Daphnia magna, Tubifex tubifex and Physa acuta, were cultured with sterilized sludge in a covered sterilized bottle and were then isolated from the atmosphere above the liquid phase. The rates of sludge reduction using the four microfaunas were 0.8, 0.18, 0.54 and 0.1 mg-sludge/(mg-Microfauna d), respectively, changing with the microfaunas' phylum or class and body size. Based on the change of carbon (C) forms, the proposed method produced accurate results similar to those produced using the direct measuring method.     

PLCζ causes Ca(2+) oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2)

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.     

Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT+Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

蔗糖对桃幼苗生长发育及其SnRK1酶活性的影响

以实生桃(Prunus persica)苗为试材, 探讨SnRK1对不同浓度蔗糖及处理时间的响应特性, 揭示蔗糖对植株生长发育的影响, 以期为果树生产提供理论依据及技术支持。结果表明, 施加5%蔗糖时, 植株体内SnRK1酶活性最高, 且在一定时间内, 酶活性持续升高; 与对照(清水和甘露醇)相比, 5%蔗糖处理显著提高植株可溶性糖、淀粉和叶片叶绿素含量, 增加植株地上部和地下部生物量, 显著加快植株净光合速率; 通过观察根系构型, 发现5%蔗糖可以显著增加根系总表面积、总体积和侧根数量, 并可促进根系加粗加长生长。qRT-PCR分析表明, 外源蔗糖能促进根系中生长素的合成和转运。综上, 一定浓度蔗糖可以提高植株体内SnRK1酶活性, 影响植株碳代谢, 促进植株生长发育, 且增加根系生长素的合成与转运, 进而影响根系构型。