Autocrine TGF-alpha is associated with Benzo(a)pyrene-induced mucus production and MUC5AC expression during allergic asthma.

OBJECTS: Benzo(a)pyrene (BaP), an environmental pollutant, is present in high concentrations in urban smog and cigarette smoke and has been reported to promote high mucin 5AC (MUC5AC) expression. Epithelium-derived inflammatory cytokines are considered an important modulator of mucus oversecretion and MUC5AC overexpression. Here, we investigated whether the effect of BaP on MUC5AC overexpression was associated with cytokine autocrine activity in vivo and in vitro. METHODS: In vivo, BALB/c mice were treated with ovalbumin (OVA) in the presence or absence of BaP. Allergy-induced mucus production was assessed by Alcian Blue Periodic acid Schiff (AB-PAS) staining. The human airway epithelial cell line NCI-H292 was used in vitro. MUC5AC and transforming growth factor (TGF)-α mRNA levels were assessed with real-time quantitative PCR. The concentration of cytokines was measured by ELISA. The MUC5AC, p-ERK, ERK, p-EGFR and EGFR proteins were detected by Western blotting in cells or by immunohistochemistry in mouse lungs. Small-interfering RNAs were used for gene silencing. RESULTS: TGF-α was overproduced in the supernatant of NCI-H292 cells treated with BaP. Knockdown of TGF-α expression inhibited the BaP-induced increase in MUC5AC expression and subsequent activation of the EGFR-ERK signalling pathway. Knocking down aryl hydrocarbon receptor (AhR) expression or treatment with an ROS inhibitor (N-acetyl-L-cysteine) could relieve the TGF-α secretion induced by BaP in epithelial cells. In an animal study, coexposure to BaP with OVA increased mucus production, MUC5AC expression and ROS-EGFR-ERK activation in the lung as well as TGF-α levels in bronchoalveolar lavage fluid (BALF). Furthermore, the concentration of TGF-α in BALF was correlated with MUC5AC mRNA levels. Additionally, TGF-α expression was found to be positively correlated with MUC5AC expression in the airway epithelial cells of smokers. Compared with non-smoker asthma patients, TGF-α serum levels were also elevated in smoker asthma patients. CONCLUSION: Autocrine TGF-α was associated with BaP-induced MUC5AC expression in vitro and in vivo. BaP induced TGF-α secretion by activating AhR and producing ROS, which led to activation of the EGFR-ERK pathway.

Solving probability reasoning based on DNA strand displacement and probability modules.

In computation biology, DNA strand displacement technology is used to simulate the computation process and has shown strong computing ability. Most researchers use it to solve logic problems, but it is only rarely used in probabilistic reasoning. To process probabilistic reasoning, a conditional probability derivation model and total probability model based on DNA strand displacement were established in this paper. The models were assessed through the game "read your mind." It has been shown to enable the application of probabilistic reasoning in genetic diagnosis.

Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture.

We generated transgenic silkworms that synthesized human type I collagen alpha1 chain [alpha1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant alpha1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the alpha1(I) chain content to 8.0% (4.24 mg per cocoon). The alpha1(I) chain was extracted and purified from the cocoons using a very simple method. The alpha1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the alpha1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the alpha1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the alpha1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human alpha1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture.

Integrin signal masks growth-promotion activity of HB-EGF in monolayer cell cultures.

The extracellular environment and tissue architecture contribute to proper cell function and growth control. Cells growing in monolayers on standard polystyrene tissue culture plates differ in their shape, growth rate and response to external stimuli, compared with cells growing in vivo. Here, we showed that the EGFR (epidermal growth factor receptor) ligand heparin-binding EGF-like growth factor (HB-EGF) strongly stimulated cell growth in nude mice, but not in cells cultured in vitro. We explored the effects of HB-EGF on cell growth under various cell culture conditions and found that growth promotion by HB-EGF was needed in three-dimensional (3D) or two-dimensional (2D) culture systems in which cell-matrix adhesion was reduced. Under such conditions, cell growth was extremely suppressed in the absence of HB-EGF, but markedly potentiated in the presence of HB-EGF. When the integrin signal was reduced using antibodies or knockout of either integrin beta1 or focal adhesion kinase (FAK), cells showed HB-EGF-dependent growth. We also showed that EGF, transforming growth factor-alpha (TGFalpha) or ligands of other receptor tyrosine kinases (RTKs) stimulated cell growth in 3D culture, but not in tissue culture plates. These results indicate that the integrin signal was sufficient to support cell growth in 2D tissue culture plates without addition of the growth factor, whereas stimulation by growth factors was clearly demonstrated in culture systems in which integrin signals were attenuated.

The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice.

Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

IndexToolkit: an open source toolbox to index protein databases for high-throughput proteomics.

UNLABELLED: A software package, IndexToolkit, aimed at overcoming the disadvantage of FASTA-format databases for frequent searching, is developed to utilize an indexing strategy to substantially accelerate sequence queries. IndexToolkit includes user-friendly tools and an Application Programming Interface (API) to facilitate indexing, storage and retrieval of protein sequence databases. As open source, it provides a sequence-retrieval developing framework, which is easily extensible for high-speed-request proteomic applications, such as database searching or modification discovering. We applied IndexToolkit to database searching engine pFind to demonstrate its effect. Experimental studies show that IndexToolkit is able to support significantly faster searches of protein database. AVAILABILITY: The IndexToolkit is free to use under the open source GNU GPL license. The source code and the compiled binary can be freely accessed through the website http://pfind.jdl.ac.cn/IndexToolkit. In this website, the more detailed information including screenshots and documentations for users and developers is also available.

Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor.

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.