Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.     

Intervention of cardiomyocyte death based on real-time monitoring of cell adhesion through impedance sensing

Cardiomyocyte death caused by proinflammatory cytokines, such as Tumor necrosis factor α (TNF-α), is one of the hot topics in cardiovascular research. TNF-α can induce multiple cell processes that are dependent on the treatment time although the long-term treatment definitely leads to cell death. The ability to intervene in cell death will be invaluable to reveal the effects of short-term TNF-α treatment to cardiomyocytes. However, a real-time monitoring technique is needed to guide the intervention of cell responses. In this work, we employed the impedance-sensing technique to real-time monitor the equivalent cell–substrate distance of cardiomyocytes via electrochemical impedance spectroscopy (EIS) and electrical cell–substrate impedance sensing (ECIS). In the stabilized cardiomyocyte culture, the sustained TNF-α treatment caused strengthened cell adhesion in the first 2 h which was followed by the transition to cell detachment afterwards. Considering cell detachment was an early morphological evidence of cell death, we removed TNF-α from the cardiomyocyte culture before the transition to achieve the intervention of cell responses. The result of this intervention showed that cell adhesion was continuously strengthened before and after the removal of TNF-α, indicating the short-term treated cardiomyocytes did not undergo death processes. It was also demonstrated in TUNEL and TBE tests that the percentages of apoptosis and cell death were both lowered.     

Parallel gene cloning and protein production in multiple expression systems

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5′‐EcoRI/3′‐XhoI identical in these vectors for ligating with the sticky‐end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

椒江口滩涂大型底栖动物群落格局与多样性

为了解椒江口滩涂大型底栖动物群落格局与多样性, 揭示其对环境变化的响应规律, 作者于2007年10月、2008年1月、4月和7月在椒江口南岸和北岸潮间带, 沿河流到海洋方向共布设6条采样断面进行大型底栖动物调查。分析了大型底栖动物种类组成、栖息密度和生物量的时空变化特征, 在此基础上运用α, β和γ多样性测度方法对大型底栖动物多样性进行分析, 同时探讨了大型底栖动物群落结构对环境变化的响应方向及程度, 结果显示: (1) 6条断面共记录到大型底栖动物78种, 总种数随季节变化显著, 在空间上沿河流到海洋方向呈升高趋势; (2) 栖息密度的季节变化不显著(P=0.145>0.05), 但空间变化显著(P=0.017<0.05), 生物量的季节变化显著(P=0.012<0.05), 空间变化极显著(P=0.004<0.01); (3) β和γ多样性指数定量显示了椒江河口区域滩涂环境的多变性和大型底栖动物群落的多样性和更替性。     

再论DG指数的性质与应用

密度-类群指数(DG指数)是为研究复杂的土壤动物群落多样性而创建的, 其理论基础是土壤动物群落中各类群互不干扰和互利的关系大于互相竞争的关系, 因而公式中各类群是互相独立、互不影响的.DG指数自1990年建立以来, 经作者及同行学者在不同地带、不同生态系统和不同级别的土壤动物群落研究中运用, 都获得良好的效果, 能真实地反映群落的多样性状况,显示出它具有广泛的适用性.用情景分析法(scenario analysis)探讨DG指数与Shannon-Wiener多样性指数(H')的预期效果, 结果显示, 当群落间种数差和均匀度(J)差为正负相反的数值时, Shannon-Wiener指数与DG指数不一致.文中还对土壤动物群落多样性的概念、性质及指数适用性进行了讨论, 认为土壤动物群落在客观上不存在均匀的数量分布, 把"均匀度"看作是多样性的重要指标是不适宜的.DG指数虽然没有直接包含许多信息, 但能在广泛的情况下经得起检验, 因而具有重要的应用价值.     

Molecular events associated with epithelial to mesenchymal transition of nasopharyngeal carcinoma cells in the absence of Epstein-Barr virus genome

Epithelial-mesenchymal transition (EMT) is an important process in tumor metastasis. The EMT-related events associated with metastasis of NPC in the absence of EBV have not been elucidated. We established an EBV-negative NPC cell line from a bone marrow biopsy of an NPC patient. Using a Matrigel system we isolated an invasive and non-invasive sublines, designated NPC-BM29 and NPC-BM00. NPC-BM29 acquired an invasive-like phenotype characterized by EMT, marked by down-regulation of E-cadherin and β-catenin with concomitant increased expression of Ets1. NPC-BM29 cells expressed ≥ 10-fold higher of MMP-9 than NPC-BM00 cells. NPC-BM29 cells grew better in 2% serum than NPC-BM00 cells, with a population doubling-time of 26.8 h and 30.7 h, respectively. A marked reduction in colony-formation ability of NPC-BM00 cells compared to NPC-BM29 was observed. Wound-healing assay revealed that NPC-BM29 cells displayed higher motility than NPC-BM00 and the motility was further enhanced by cell treatment with TPA, a PKC activator. Cell surface markers and tumor-associated molecules, AE3, MAK6 and sialyl-Tn, were up-regulated in NPC-BM29 cells, whereas the expression of HLA-DR and CD54 was significantly increased in NPC-BM00 cells. NPC-BM29 consistently released higher levels of IL-8 and IL-10 than NPC-BM00, with low levels of IL-1α expression in both cell lines. Higher level of VEGF production was detected in NPC-BM00 than NPC-BM29 cells. These data show that EBV is not required for exhibiting multiple metastatic phenotypes associated with EMT. More studies that target right molecules/signalings associated with the EMT may offer new therapeutic intervention options for NPC invasion and metastasis.     

Critical Factors Determining Dimerization of Human Antizyme Inhibitor

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K_d, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K_d value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K_d value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1–3). Ornithine decarboxylase (ODC,³ EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8–15). They also have been associated with human diseases and a variety of cancers (16–26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6, 11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity requires the formation of a dimer (29–31). X-ray structures of the ODC enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in PLP interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate (27, 32–41).ODC undergoes a unique ubiquitin-independent proteasomal degradation via a direct interaction with the regulatory protein antizyme (AZ). Binding of AZ promotes the dissociation of the ODC homodimers and targets ODC for degradation by the 26 S proteasome (42–46). Current models of antizyme function indicate that increased polyamine levels promote the fidelity of the AZ mRNA translational frameshift, leading to increased concentrations of AZ (47). The AZ monomer selectively binds to dimeric ODC, thereby inactivating ODC by forming inactive AZ-ODC heterodimers (44, 48–50). AZ acts as a regulator of polyamine metabolism that inhibits ODC activity and polyamine transport, thus restricting polyamine levels (4, 5, 51, 52). When antizymes are overexpressed, they inhibit ODC and promote ubiquitin-independent proteolytic degradation of ODC. Because elevated ODC activity is associated with most forms of human malignancies (1), it has been suggested that antizymes may function as tumor suppressors.In contrast to the extensive studies on the oncogene ODC, the endogenous antizyme inhibitor (AZI) is less well understood. AZI is homologous to the enzyme ODC. It is a 448-amino acid protein with a molecular mass of 50 kDa. However, despite the homology between these proteins, AZI does not possess any decarboxylase activity. It binds to antizyme more tightly than does ODC and releases ODC from the ODC-antizyme complex (53, 54). Both the AZI and AZ proteins display rapid ubiquitin-dependent turnover within a few minutes to 1 h in vivo (5). However, AZ binding actually stabilizes AZI by inhibiting its ubiquitination (55).AZI, which inactivates all members of the AZ family (53, 56), restores ODC activity (54), and prevents the proteolytic degradation of ODC, may play a role in tumor progression. It has been reported that down-regulation of AZI is associated with the inhibition of cell proliferation and reduced ODC activity, presumably through the modulation of AZ function (57). Moreover, overexpression of AZI has been shown to increase cell proliferation and promote cell transformation (58–60). Furthermore, AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function (60, 61). These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis.It is now known that ODC exists as a dimer and that AZI may exist as a monomer physiologically (62). Fig. 1 shows the dimeric structures of ODC (Fig. 1A) and AZI (Fig. 1B). Although structural studies indicate that both ODC and AZI crystallize as dimers, the dimeric AZI structure has fewer interactions at the dimer interface, a smaller buried surface area, and a lack of symmetry of the interactions between residues from the two monomers, suggesting that the AZI dimer may be nonphysiological (62). In this study, we identify the critical amino acid residues governing the difference in dimer formation between ODC and AZI. Our preliminary studies using analytical ultracentrifugation indicated that ODC exists as a dimer, whereas AZI exists in a concentration-dependent monomer-dimer equilibrium. Multiple sequence alignments of ODC and AZI from various species have shown that residues 277, 331, 332, and 389 are not conserved between ODC and AZI (Open in a separate windowFIGURE 1.Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).

TABLE 1

Amino acid residues at the dimer interface of human ODC and AZI