Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

Distribution of dissolved organic carbon and dissolved fulvic acid in mesotrophic Lake Biwa, Japan

The dissolved organic carbon (DOC) concentrations in mesotrophic Lake Biwa were determined by a total organic carbon (TOC) analyzer, and DOC molecular size distributions were determined by size exclusion chromatography (SEC) using a fluorescence detector at excitation/emission (Ex/Em) levels of 300/425 nm with the eluent at pH 9.7. The fluorescence wavelengths for detection were chosen from the result of excitation–emission matrix spectrometry (EEM) analysis for dissolved fulvic acid (DFA) extracted from Ado River (peak A, Ex/Em = 260–270/430–440 nm; peak B, Ex/Em = 300–310/420–430 nm). Ado River DFA was eluted with a retention time (RT) of 7.4–8.9 min and the apparent molecular weight was estimated at 22–87 kDa based on the elution curve for the spherical protein molecular weight standard. A DFA peak eluted at the same retention time as Ado River DFA also appeared in all the samples of Lake Biwa water. From the linear relationship between the peak areas with an RT of 7.4–8.9 min by SEC analysis and DOC values of DFA by TOC analysis of a series of DFA samples (r² = 0.9995), the concentrations of DFA in the lake water were roughly calculated. DFA was distributed within the range 0.25–0.43 mg C l⁻¹ and accounted for 15%–41% of DOC, with the highest ratios observed at a depth of 70 m in August and the lowest at 2.5 m in May.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

Identification of the sex pheromone components secreted by female moths of Peridroma saucia (Noctuidae: Noctuinae)

The variegated cutworm, Peridroma saucia Hübner, is a lepidopteran pest to a large number of crops in Canada, the United States, and Europe. It was probably naturalized in Japan in the 1970s. The pheromone glands of the female moth include two components with electroantennographic activity in a ratio of 3:1. GC-MS analyses of pheromone extracts untreated and treated with dimethyl disulfide revealed the major component to be (Z)-11-hexadecenyl acetate and the minor component to be (Z)-9-tetradecenyl acetate. The synthetic pheromone was used to attract a large number of males in a vegetable field in Tokyo, which suggests that this species has already become a harmful pest in Japan.     

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.     

Adhesive Property of Toxin-Coregulated Pilus of Vibrio cholerae O1

The adhesive property of toxin-coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.     

Prostaglandin E(2) protects human lung fibroblasts from cigarette smoke extract-induced apoptosis via EP(2) receptor activation

Prostaglandin E(2) (PGE(2)) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE(2) has a protective effect on cigarette smoke extract (CSE)-induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%-20%) led to apoptosis and induced imbalance in favor of pro- over anti-apoptotic protein expression and activated caspases. PGE(2) blocked CSE-induced apoptosis and modulated the balance of pro- and anti-apoptotic proteins and decreased the activation of caspases. This anti-apoptotic effect was mediated via EP(2) receptor activation as the EP(2) agonist butaprost mimicked PGE(2) activity and siRNA for the EP(2) receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE(2)-mediated cytoprotective effect. Correspondingly, c-AMP analogs blocked CSE-induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT-5720 abolished PGE(2)-mediated protection. PGE(2) and butaprost phosphorylated Bad and KT-5720 blocked phosphorylation. These results suggest that PGE(2) inhibits CSE-induced apoptosis via EP(2) receptor activation and activation of PKA, which leads to an alteration in the balance between pro- and anti-apoptotic factors. Through such a mechanism, PGE(2) may alter survival of cells in the smoke-exposed lungs, thus affecting the pathogenesis of cigarette smoke-induced disease.     

The role of carbon flux and biometric observations in constraining a terrestrial ecosystem model: a case study in disturbed forests in East Asia

The process of confining unnecessary freedom is a step toward advanced ecosystem modeling. This study demonstrates the importance of carbon flux and biometric observation in constraining a terrestrial ecosystem model with a simple optimization scheme. At the selected sites from AsiaFlux network, a simultaneous optimization scheme for both carbon flux and biomass was compared with carbon flux-oriented and biomass-oriented optimization schemes using the Biome-BGC model. The optimization scheme oriented to either carbon flux or biomass provided simulation results that were consistent with observations, but with reduced performance in unconstrained variables. The simultaneous optimization scheme yielded results that were consistent with observations for both carbon flux and biomass. By comparing long-term projections simulated by three schemes, it was found that the optimization oriented only to carbon flux has limited performance because misrepresented biomass significantly affected a projection of carbon exchange through heterotrophic respiration. From these experiments, we found that (1) a process model like Biome-BGC is capable of reproducing both carbon flux and biomass within acceptable proximity, (2) constraining biomass is importance not just because it is one of carbon cycle components, but also it significantly affects simulations of carbon flux. Thus, it is important to invest more effort to improve simulation of biomass as well as carbon flux.