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Acetylation of extracellular proteins has been observed in many independent studies where particular attention has been given to the dynamic change of the microenvironmental protein post-translational modifications. While extracellular proteins can be acetylated within the cells prior to their micro-environmental distribution, their deacetylation in a tumor microenvironment remains elusive. Here it is described that multiple acetyl-vWA domain-carrying proteins including integrin β3 (ITGB3) and collagen 6A (COL6A) are deacetylated by Sirtuin family member SIRT2 in extracellular space. SIRT2 is secreted by macrophages following toll-like receptor (TLR) family member TLR4 or TLR2 activation. TLR-activated SIRT2 undergoes autophagosome translocation. TNF receptor associated factor 6 (TRAF6)-mediated autophagy flux in response to TLR2/4 activation can then pump SIRT2 into the microenvironment to function as extracellular SIRT2 (eSIRT2). In the extracellular space, eSIRT2 deacetylates ITGB3 on aK416 involved in cell attachment and migration, leading to a promotion of cancer cell metastasis. In lung cancer patients, significantly increased serum eSIRT2 level correlates with dramatically decreased ITGB3-K416 acetylation in cancer cells. Thus, the extracellular space is a subcellular organelle-like arena where eSIRT2 promotes cancer cell metastasis via catalyzing extracellular protein deacetylation.

Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (TC, L267P) and rs3746803 (C T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.

The temperature of a minute object is typically measured by an infrared thermograph, a precise and expensive device. This study presents an image–temperature model constructed by particle swarm optimization algorithm to estimate the temperature according to the color image data on object’s surface. The demonstrated case was a palladium alloy microprobe used in wafer testing. Two series of conduction experiments were implemented to imitate the probing condition of the microprobe in wafer testing. Collecting the images captured from a digital video camera and the temperatures measured by the infrared thermograph, the correlations between the color image data and their corresponding temperatures were investigated. An image–temperature model is further constructed by the particle swarm optimization algorithm based on the image components of the RGB and HSV models of the examined pixel. The model developed provides valuable references for making out the electrothermal effect of the palladium alloy microprobe used in wafer testing.

To establish a mice model of cisplatin-induced ototoxicity, and to investigate the effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea.Terminal deoxynucleotidyl transferase-mediated nick end labeling method (TUNEL) was used to monitor the apoptosis of spiral ganglion cell. Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea. Auditory brainstem response (ABR) was measured to observe the change of hearing.The weight and hearing of mice in different dose of cisplatin groups were declined significantly as compared with those of control group (P0.05, P0.01), and the TUNEL positive cell number and expression of caspase-3 were greater remarkably with the more cisplatin injected.A mouse model of cisplatin-induced ototoxicity can be established. Cisplatin can lead to the apoptosis of spiral ganglion cells, and caspase-3 has participated in this apoptosis process, which approves further that apoptosis might be one of the mechanisms of cisplatin ototoxicity.

To construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.Three RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated.All the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way.Plasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.

Physiological level of reactive oxygen species (ROS) has been shown to play an important role in mitogen-stimulated cell signaling in many cell types. Both EGF and bFGF can induce ROS generation in human lens epithelial cells. But the role of ROS and Redox signaling on EGF and bFGF-stimulated cell proliferation is not clear. This study was to investigate the control of EGF and bFGF-induced cell proliferation by Redox signaling in human lens epithelial cells (SRA 01/04), using specific inhibitors to Redox signaling.EGF and bFGF-induced cell proliferation was measured by [methyl-3H] thymidine incorporation assay. In some experiments, cell proliferation was also measured by trypan blue negative cell counting parallel with 3H-thymidine incorporation assay. The inhibitors used in this study include: catalase (specific enzyme to detoxify hydrogen peroxide), N-acetyl-L-cysteine (free radical scavenger), DPI (inhibitor for NADPH oxidase) and AACOCF3 (specific inhibitor for cytosolic phospholipase A2, which had been shown to play important role in ROS generation in our previous study). Serum starved SRA 01/04 cells were pretreated with these inhibitors for 30 minutes before exposure to EGF or bFGF (20 microg/L). In short term study, all these inhibitors were removed before adding growth factor, while in long term study, inhibitors were maintained in the medium along with growth factor. Cells were kept growing in the medium with 20 microg/L EGF or 20 microg/L bFGF for 48 hours. Then cell proliferation was quantified by [methyl-3H] thymidine incorporation assay or by cell counting.We found that catalase, NAC, DPI and AACOCF3 were able to suppress EGF and bFGF-induced cell proliferation in both short term and long term study. In EGF study, 20 microg/L EGF produced about 26% (t = 7.093, P0.01) increase in DNA synthesis after 48 hours. Pretreatment of the cells for 30 minutes with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 inhibited EGF-stimulated DNA synthesis by 18.0% (t=6.132, P0.01), 24.6% (t=6.188, P0.01), 28.5% (t=6.386, P0.01) and 16.4% (t =3.705, P =0.001) respectively. The inhibition was dose-dependent and was proved by trypan-blue negative cell counting. If the cells were treated with inhibitors for 48.5 hours (long term study), the lowest concentrations to inhibit cell proliferation were much lower than those used in short term study. Treatment of the cells with 0.5 x 10(5) U/L catalase, 0.2 mmol/L NAC, 0.01 micromol/L DPI and 0.1 micromol/L AACOCF3 led to suppression on DNA synthesis significantly. Similar results were detected in bFGF study. 48 hours treatment with 20 microg/L bFGF induced about 28.8% (t =9.523, P0.01) increase in cell proliferation. If the cells were pretreated with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 for 30 minutes, bFGF-stimulated cell proliferation was suppressed by 24.5% (t = 6.697, P0.01), 22.2% (t = 6.693, P0.01), 23.9% (t =6.661, P0.01) and 30.5% (t =8.959, P0.01) respectively. If cells were treated with inhibitors for 48.5 hours, the lowest concentration of catalase, NAC, DPI and AACOCF3 to inhibit cell proliferation significantly was 0.5 x 10(5) U/L( t =21.641, P0.01), 0.2 mmol/L (t =11.218, P0.01), 0.01 micromol/L (t = 4.570, P0.01) and 0.1 micromol/L (t = 5.426, P0.01) respectively, lower than those used in short term study.We conclude that mitogenic stimulus function of EGF and bFGF in human lens epithelial cells appears to be mediated via ROS to activate cell proliferation. Inhibition of Redox signaling, either by removal of ROS (the role of catalase and NAC) or blocking ROS generation (the role of DPI and AACOCF3), eradicate EGF and bFGF-stimulated cell proliferation. It is proposed that Redox signaling may play an important role in cell proliferation in human lens epithelial cells.

Dishevelled proteins are multifunctional and highly conserved. These proteins are also required for the specification of cell fate and polarity by secreted Wnt proteins. To investigate the molecular mechanism of Dishevelled in mediating Wnt signal transduction, a mouse 11.5dpc embryo library was screened by yeast-two-hybrid system to find mouse Dishevelled2 DEP domain and C-terminal interacting proteins. 15 possitive clones were identified from 4.1 x 10(6) transformants. The DNA sequences of the positive AD/library plasmids were determined. The BLAST results revealed that one of the positive clones contained N-terminus cDNA fragments (amino acids 6-122) of Gli3 protein. The interaction between Dv12 and Gli3 detected by yeast two-hybrid system suggests that Gli3 might play a role in some biological processes with Dishevelled.

To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established.Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL.The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility.The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.

Leukemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, which was discovered to inhibit the proliferation of murine myeloid leukemic cell line M1, has multiple functions in various biological processes. This factor is highly glycosylated when it binds to receptor to activate the signal transduction. Therefore, expression of LIF through eukaryotic system is the best way to obtain the correct glycosylation. In this study, human LIF cDNA with the sequence of signal peptide was cloned from adult blood cells by RT-PCR, and then subcloned into pcDNA3 for expression in HEK-293T cells. After transfection of the recombinant plasmid pcDNA3/LIF into HEK-293T cells, the conditioned medium containing the secreted LIF was obtained. The activity of the secreted LIF in the conditioned medium was detected through the phosphorylation of STAT3, EMSA and luciferase reporter (pGL2-APRE-luc) assays in Hep3B and 293T cells. Furthermore, to investigate the biological functions of the overexpressed recombinant LIF, a [(3)H]-thymedine incorporation assay was performed, and the results showed that the recombinant LIF inhibited the growth of M1 cells strongly. Taken together, all the function assays suggest that the recombinant LIF has the normal functions, suggesting that the recombinant LIF could be used for further studies.