p38-Mediated phosphorylation of Eps15 endocytic adaptor protein

Epidermal growth factor receptor pathway substrate 15 (Eps15) has been suggested to be involved in the endocytosis of cell surface receptors, including epidermal growth factor receptor (EGFR). Eps15 is phosphorylated at Tyr-849 upon stimulation with EGF during endocytic processes. In the present study, we found that stimulation of HeLa cells with EGF or TNF-α induced transient phosphorylation of Eps15 at Ser-796. Inhibition of p38 completely blocked phosphorylation and recombinant p38α directly phosphorylated the residue. These results demonstrate a novel stress kinase-mediated signaling pathway to Eps15 endocytic adapter protein.     

Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells

It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway.     

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC–MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.     

Monoclonal antibodies against recombinant Der f 3 reveal localization of Der f 3 in the gut and faecal pellets of <Emphasis Type="Italic">Dermatophagoides farinae</Emphasis>

Home dust mite derived materials are known to be a major source of problematic inhalant allergens. The aim of this study was to determine the localization of the group 3 allergen, Der f 3, within Dermatophagoides farinae, in order to assess the relative importance of excreted materials and nonexcreted body components as allergen sources. Recombinant Der f 3 (rDer f 3) was expressed in bacteria and purified as an immunogen for production of monoclonal antibodies (mAb) against it. Dermatophagoides farinae mites and their faecal pellets were embedded in paraffin, and serial sections were immunoprobed with mAb clone 3D3 against Der f 3. D. farinae midgut mucosa, gut contents and faecal pellets were strongly immunopositive for Der f 3. Der f 3 immunoreactive products were not detected in any other internal organs of the mite. These results suggest that Der f 3 allergen may be synthesized in and secreted from the digestive tract and excreted from the mite’s body in the faecal pellets.     

Production of biologically active recombinant annexin B1 with enhanced stability via a tagging system

Annexin B1 is a novel Ca²⁺-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure–function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.     

柞蚕蛹期灵菌败血病Serratia marcescens C3菌株分离鉴定

以柞蚕(Antheraea pernyi)蛹为替代寄主繁殖白蛾周氏啮小蜂(Chouioia cunea)技术在辽宁、北京、天津、上海、河北、山东等地美国白蛾(Hyphantria cunea)的生物防治中发挥了重要作用。利用柞蚕蛹繁殖白蛾周氏啮小蜂时,柞蚕蛹期软化病是繁蜂的主要障碍。通过对利用柞蚕蛹繁蜂时蛹内组织液化后呈粉红色这一未知软化病的典型症状进行病原细菌的分离和纯化,得到C3菌株。经Biolog系统和16S rRNA序列分析,鉴定C3菌株为灵菌(Serratia marcescens),经过柯赫法则检验,确定灵菌C3菌株是导致柞蚕蛹期灵菌败血病的病原菌。描述了繁蜂时柞蚕蛹期灵菌败血病发病期的认别特征。