谷氨酸及NMDA受体拮抗剂MK-801对大鼠伏核痛兴奋神经元电活动的影响

本文研究了谷氨酸(glutamic acid,Glu)及其NMDA受体拮抗剂5-甲基二氢丙环庚烯亚胺马来酸(MK-801)对人鼠伏核(nucleus accumbens,NAc)痛兴奋神经元(pain-excitation neurons,PEN)痛诱发反应的影响。电刺激坐骨神经作为伤害性刺激,用玻璃微电极记录NAc的PEN放电,观察脑室内注射Glu和NAc内注射MK-801对大鼠NAc中PEN伤害性诱发活动的影响。结果显示,伤害性刺激可使NAc的PEN电活动增强;脑室内注射Glu(10nmol／10μl)可使NAc的PEN伤害性诱发放电频率增加;NAc内注射MK-801(1．0nmol／0．5μl)可阻断这种作用;MK-801本身也可部分抑制PEN伤害性诱发反应。上述结果表明,Glu对PEN伤害性反应的易化作用是通过NMDA受体介导的：Glu和NMDA受体参与NAc伤害性信息传递的调制。     

Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer

Background

Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients.

Methods and Findings

To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples.

Conclusions

Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.     

植物激素相关核酸和蛋白质二级数据库的构建与应用

以NCBI维护的一级数据库为数据源建立植物激素相关核酸和蛋白质二级数据库。将该二级数据库设计为基因、蛋白质和文献三部分, 编写软件从上述数据源中采集数据, 并以XML作为中间格式保存, 通过解析提交到二级数据库中并集成部分生物信息学工具软件, 初步实现了数据检索、统计分析、基于Web的本地化BLAST同源序列检索、序列的自动拼接以及蛋白质结构和功能位点的分析等功能。该二级数据库的构建为植物激素作用分子机理研究提供了高针对性的植物激素数据源和生物信息学辅助工具。     

Activation of the Imprinted Dlk1-Dio3 Region Correlates with Pluripotency Levels of Mouse Stem Cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.     

IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

The Imaging of Insulinomas Using a Radionuclide-Labelled Molecule of the GLP-1 Analogue Liraglutide: A New Application of Liraglutide

Objective

This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of ¹²⁵I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas.

Methods

Liraglutide was labelled with ¹²⁵I by the Iodogen method. The labelled ¹²⁵I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of ¹²⁵I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined.

Results

The labelling of liraglutide with ¹²⁵I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between ¹²⁵I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (K_d) was 128.8±30.4 nmol/L(N = 3), and the half-inhibition concentration (IC₅₀) was 542.4±187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with ¹²⁵I-liraglutide indicated that the tumour imaging was clearest at 90 min after the ¹²⁵I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the ¹²⁵I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83±1.30(N = 3). Our study suggested that ¹²⁵I-liraglutide was predominantly metabolised and cleared by the liver and kidneys.

Conclusion

The radionuclide ¹²⁵I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.     

Anthraquinone G503 Induces Apoptosis in Gastric Cancer Cells through the Mitochondrial Pathway

G503 is an anthraquinone compound isolated from the secondary metabolites of a mangrove endophytic fungus from the South China Sea. The present study elucidates the anti-tumor activity and the underlying mechanism of G503. Cell viability assay performed in nine cancer cell lines and two normal cell lines demonstrated that the gastric cancer cell line SGC7901 is the most G503-sensitive cancer cells. G503 induced SGC7901 cell death via apoptosis. G503 exposure activated caspases-3, -8 and -9. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not caspase-8 inbibitor Z-IETD-FMK, attenuated the effect of G503. These results suggested that the intrinsic mitochondrial apoptosis pathway, rather than the extrinsic pathway, was involved in G503-induced apoptosis. Furthermore, G503 increased the ratio of Bax to Bcl-2 in the mitochondria and decreased the ratio in the cytosol. G503 treatment resulted in mitochondrial depolarization, cytochrome c release and the subsequent cleavage of caspase -9 and -3. Moreover, it is reported that the endoplasmic reticulum apoptosis pathway may also be activated by G503 by inducing capase-4 cleavage. In consideration of the lower 50% inhibitory concentration for gastric cancer cells, G503 may serve as a promising candidate for gastric cancer chemotherapy.     

Influence of Al3+ on the titer of spiramycin and effective components in fermentor

Spiramycin is a multicomponent antibiotic, and different components have different antibacterial activities. In Streptomyces spiramyceticus 16-10-2, spiramycin II and spiramycin III (SPMII and SPMIII) are the main components, while spiramycin I (SPMI) needs to be controlled below 12%. Based on this, the influences of Al³⁺ on total spiramycin titer and components were investigated in this work. Those experiments were mainly performed in 15?L fermentor and Al³⁺ made a great improvement in spiramycin titer. The optimal adding concentration and adding time of Al³⁺ were 0.32?g/L at 12?hr. Under this condition, spiramycin titer was increased by 19.51% compared with the control. Moreover, the percentage of SPMII and SPMIII was increased by 7.14%. At the same time, the time of mycelia autolysis was lengthened. In addition, the specific activities of acetyl-CoA synthetase, acetate kinase, acetylphosphotransferase, and acylating enzyme were much higher than those of control. The content of acetic acid and succinic acid was beyond 3 and 4.5 times than that of control, respectively.