A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.     

Antiinflammatory flavonoids from Artocarpus heterophyllus and Artocarpus communis

The antiinflammatory activities of the isolated flavonoids, including cycloartomunin (1), cyclomorusin (2), dihydrocycloartomunin (3), dihydroisocycloartomunin (4), cudraflavone A (5), cyclocommunin (6), and artomunoxanthone (7), and cycloheterohyllin (8), artonins A (9) and B (10), artocarpanone (11), artocarpanone A (12), and heteroflavanones A (13), B (14), and C (15) from Artocarpus communis and A. heterophyllus, were assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, and macrophages. Compound 4 significantly inhibited the release of beta-glucuronidase and histamine from rat peritoneal mast cells stimulated with P-methoxy-N-methylphenethylamine (compound 48/80). Compound 11 significantly inhibited the release of lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Compounds 8, 10, and 11 significantly inhibited superoxide anion formation in fMLP-stimulated rat neutrophils while compounds 2, 3, 5, and 6 evoked the stimulation of superoxide anion generation. Compound 11 exhibited significant inhibitory effect on NO production and iNOS protein expression in RAW 264.7 cells. The potent inhibitory effect of compound 11 on NO production in lipopolysaccharide (LPS)-activated macrophages, probably through the suppression of iNOS protein expression.     

Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts

The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.     

Spatial changes of soil fungal and bacterial biomass from a sub-alpine coniferous forest to grassland in a humid, sub-tropical region

 Fungal and bacterial biomass were determined across a gradient from a forest to grassland in a sub-alpine region in central Taiwan. The respiration-inhibition and ergosterol methods for the evaluation of the microbial biomass were compared. Soil fungal and bacterial biomass both significantly decreased (P<0.05) with the shift of vegetation from forest to grassland. Fungal and bacterial respiration rates (evolved CO₂) were, respectively, 89.1 μl CO₂ g^–1 soil h^–1 and 55.1 μl CO₂ g^–1 soil h^–1 in the forest and 36.7 μl CO₂ g^–1 soil h^–1 and 35.7 μl CO₂ g^–1 soil h^–1 in the grassland surface soils (0–10 cm). The fungal ergosterol content in the surface soil decreased from the forest zone (108 μg g^–1) to the grassland zone (15.9 μg g^–1). A good correlation (R ²=0.90) was exhibited between the soil fungal ergosterol content and soil fungal CO₂ production (respiration) for all sampling sites. For the forest and grassland soil profiles, microbial biomass (respiration and ergosterol) declined dramatically with depth, ten- to 100-fold from the surface organic horizon to the deepest mineral horizon. With respect to fungal to bacterial ratios for the surface soil (0–10 cm), the forest zone had a significantly (P<0.05) higher ratio (1.65) than the grassland zone (1.05). However, there was no fungal to bacterial ratio trend from the surface horizon to the deeper mineral horizons of the soil profiles. Received: 30 March 2000     

Plasticity of the differentiated state

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.     

苦楝果实中化学成分进一步研究及生物活性测定

采用生物活性追踪法,从苦楝果实中分离得苦楝新醇(Ⅰ)、苦楝酮(Ⅱ)、苦楝醇(Ⅲ)、苦楝二醇(Ⅳ)、苦楝三醇(Ⅴ)、香草酸(Ⅵ)、香草醛(Ⅶ)和川楝素(Ⅷ),并根据波谱(1R,MS,′H—NMR,~(13)C—NMR)分析和理化常数测定确定其结构.其中Ⅱ、Ⅲ、Ⅳ和Ⅴ的21—H存在差向异构现象.生测结果表明,Ⅱ～Ⅴ和Ⅷ对菜青虫(Pieris rapae)有明显的拒食活性,Ⅷ还表现出明显的胃毒活性;Ⅰ、Ⅳ和Ⅶ仅表现出一定的拒食活性.     

Chemical transformations in individual ultrasmall biomimetic containers

Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.     

楝属川楝素含量与生物活性的关系

本文报道用高速液相色谱测定了川楝素含量的各地楝树样品水提物及几种川楝素产品对菜青虫的生物活性,讨论了川楝素含量与生物活性的关系。叶、果样品中虽不含或仅含少量川楝素,但对试虫仍表现出一定的活性。皮中含有较丰富的川楝素,其含量与拒食活性呈正相关,但与毒效作用不相关。四川样品川楝素含量及生物活性均很高而值得重视。川楝素粗产品相对于纯川楝素而言对试虫具更高的生物活性。这种川楝素含量与活性的非线性关系说明样品中其它成份也具活性或与川楝素有着协同作用。通过测定、分析和讨论,指出用川楝素粗产品防治菜青虫是一经济、有效,可行的措施。文中应用了评判援效型杀虫剂的新方法,供讨论和参考。