Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.     

Structural chemoproteomics and drug discovery

Our laboratories have developed several technologies to accelerate drug discovery process on the basis of structural chemoproteomics. They include SPS technology for the efficient determination of protein structures, SCP technology for the rapid lead generation and SDF technology for the productive lead optimization. Using these technologies, we could determine many 3D structures of target proteins bound with biologically active chemicals including the structure of phosphodiesterase 5/Viagra complex and obtain highly potent compounds in animal models of obesity, diabetes, cancer and inflammation. In this paper, we will discuss concepts and applications of structural chemoproteomics for drug discovery.     

Membrane fusion induced by neuronal SNAREs transits through hemifusion

Synaptic transmission requires the controlled release of neurotransmitter from synaptic vesicles by membrane fusion with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for synaptic membrane fusion. The mechanism by which SNAREs drive membrane fusion is thought to involve a hemifusion intermediate, a condition in which the outer leaflets of two bilayers are combined and the inner leaflets remain intact; however, hemifusion has been observed only as an end point rather than as an intermediate. Here, we examined the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs using fluorescence assays that monitor both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is dominant at the early stage of the fusion reaction. Over time, hemifusion transitioned to complete fusion, showing that hemifusion is a true intermediate. We also show that hemifusion intermediates can be trapped, likely as unproductive outcomes, by modulating the surface concentration of the SNARE proteins.     

A comparison of in vitro anticancerous activity and mechanism of ethanolic extracts from different Ganoderma genus

Five ethanolic extracts from the mycelia of Ganoderma lucidum, G. tsugae, G. oerstedii, G. subamboinense, and G. resinaceum were respectively studied on their anticancerous activities against leukemic HL-60 cell line in vitro. Results showed that all five extracts potently inhibited HL-60 proliferation. The extract from G. lucidum mycelia exerted the highest activity. Annexin V/PI bivariate flow cytometric analysis further revealed that the five extracts significantly induced early apoptosis in HL-60 cells. The results illustrate that not only G. lucidum but also other Ganoderma species can inhibit cancer cells, and their mechanisms are related to induction of apoptosis. __________ Translated from Journal of Shanghai Normal University (Natural Sciences), 2005, 34(2): 77–81 [译自: 上海师范大学学报 (自然科学版), 2005, 34(2): 77–81]     

Antioxidant and antidiabetic activities of mycelial and fruit-body extracts from <Emphasis Type="Italic">Mycoleptodonoides aitchisonii</Emphasis>

Mycoleptodonoides aitchisonii (Berk.) Maas Geest. is a culinary mushroom that is recognized as both a nutritious food and an excellent source of bioactive compounds. The purpose of this study was to investigate the antioxidant and antidiabetic properties of M. aitchisonii (MA) both in vitro and in vivo. Total oxyradical scavenging capacity (TOSC) assays revealed that fruit-body extracts had higher antioxidant capacity than mycelial extracts, 0.9-fold higher as measured by peroxynitrite (PN) scavenging assay, 3.7-fold higher as measured by peroxyl radical (PR) scavenging assay, and 1.6-fold as measured by hydroxyl radical (HR) scavenging assay, respectively. The assay of Akt phosphorylation, which is inhibited by Interleukin 6 (IL-6) in the signal transduction pathway for diabetes, was employed to evaluate the antidiabetic activity. Fruit-body extracts significantly increased Akt phosphorylation according to the fruit-body extract concentration, with a maximum increment of 77% at a concentration of 100 μg/mL compared to 51.4% decrement caused by IL-6, but there was no effect of mycelial extracts. Treatment with 5% MA fruit-body powder and streptozotocin (STZ) decreased the blood sugar level to 233.8 mg/dL in diabetic mice compared to 333.8 mg/dL after treatment with STZ alone. Additionally, MA treatment lowered total cholesterol (TC), triglyceride (TG), and LDL-cholesterol levels, while it increased the HDL-cholesterol level. All these findings indicate that fruit-body of M. aitchisonii has potential utility in preventing various diseases such as disorders of sugar and lipid metabolism.     

Genetic engineering of coryneform bacteria

The coryneforms are a diverse group of bacteria which includes animal and plant pathogens as well as non-pathogenic bacteria. Although they are of significant economic and health importance, their genetics is poorly understood. The development of genetic engineering techniques for coryneforms and initial gene cloning studies are discussed.     

紫羊茅重金属抗性和敏感品种中类金属硫蛋白基因的鉴定及表达初探

重金属对生命机体的作用具有双重性。一方面,作为多数辅酶的辅助因子对细胞的正常代谢必不可少;另一方面,当重金属超过一定的浓度时对细胞有较大的毒性。在长期的进化过程中,生物可能形成一种调节细胞内重金属浓度的机制。这种机制在动物和真菌中被认为同金属硫蛋白(metallothionein,MT)的作用密切相关。植物中也存在类似的与重金属结合的低分子量蛋白(heavy metal binding pep-tide)。最近对拟南芥菜和水稻中类金属硫蛋白(MT-like)基因的研究证实其作用与动物MT相似。紫羊茅品种“Merlin”是一种从锌铅矿区的重金属污染地采集的单子叶草种,对镉和铜的抗性都较高,分别达到50mg/L和30mg/L,而5mg/L Cd~(2 )或2mg/L Cu~(2 )便可抑制敏感品种“S59”的正常生长。目前