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991.
Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis 总被引:1,自引:0,他引:1
Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression. 相似文献
992.
Effect of IFNgamma on caspase-3, Bcl-2 and Bax expression, and apoptosis in rabbit placenta 总被引:1,自引:0,他引:1
The purpose of this study was to determine whether apoptosis in placenta was affected by IFNgamma, which can induce abortion, and whether the effect of IFNgamma on apoptosis resulted from an intrinsic program of apoptosis, which was regulated by Bcl-2 and Bax. DNA fragmentation analysis indicated that cleavage of DNA into 180 bp and its polymers were recognized in placenta in control and IFNgamma treated groups. Quantitative analysis of low molecular weight fragments of DNA revealed a significant increase in cases of 100,000 IU IFNgamma treatment compared with those in normal pregnancy (P<0.05). An analysis in situ revealed that apoptosis occurred predominantly in syncytiotrophoblast. Expression of Bcl-2 and Bax in placenta was evaluated by immunoblot analysis and immunohistochemistry study. Bcl-2 was expressed predominantly in syncytiotrophoblast, and was not expressed in cytotrophoblast of all cases. Whereas Bax was expressed in cytotrophoblast, syncytiotrophoblasts were found to be negative for Bax protein expression in all cases. Both Bcl-2 and Bax expression was decreased 0.44 fold and 0.46 fold by 50,000 IU IFNgamma and 0.41 fold and 0.03 fold by 100,000 IU IFNgamma. This resulted in change of a 0.07 fold increase in the Bax:Bcl-2 ratio in 50,000 IU IFNgamma treated groups and 0.41 fold increase in 100,000 IU IFNgamma treated groups as compared with those in control groups. The difference in Bax to Bcl-2 ratio between control and 100,000 IU IFNgamma treated groups was significant (P<0.05). The localization of caspase-3, the executioner of apoptosis, was detected in some cytotrophoblast and syncytiotrophoblast and increased 0.03 fold and 0.68 fold in 50,000 IU IFNgamma and 100,000 IU IFNgamma treated groups, respectively. There was significant difference between control and 100,000 IU IFNgamma treated groups (P<0.05). The results showed that high dose of IFNgamma administration increased the extent of apoptosis in placenta, the Bax to Bcl-2 ratio, and the activated caspase-3. 相似文献
993.
Shen YH Godlewski J Bronisz A Zhu J Comb MJ Avruch J Tzivion G 《Molecular biology of the cell》2003,14(11):4721-4733
14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other. 相似文献
994.
Murata Y Nikaido M Sasaki T Cao Y Fukumoto Y Hasegawa M Okada N 《Molecular phylogenetics and evolution》2003,28(2):253-260
Afrotheria is a huge assemblage of various mammals encompassing six orders that were once classified as distantly related groups. This superordinal relationship may have resulted from the break-up of Gondowanaland followed by the isolation of the African continent between 105 and 40 million years ago. Although the monophyly of Afrotheria is well supported by recent molecular studies, the interrelationships within afrotherian mammals remain unclarified. In this study, we determined the sequence of the complete mitochondrial genomes of hyrax, golden mole, and elephant shrew. These sequences were compared with those of other eutherians to analyze the phylogenetic relationships among afrotherians and, in particular, those among paenungulates. Our mitochondrial genome analysis supports the monophyly of Tethytheria. 相似文献
995.
Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture 总被引:32,自引:0,他引:32
Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells. 相似文献
996.
Fiscella M Perry JW Teng B Bloom M Zhang C Leung K Pukac L Florence K Concepcion A Liu B Meng Y Chen C Elgin EC Kanakaraj P Kaufmann TE Porter J Cibotti R Mei Y Zhou J Chen G Roschke V Komatsoulis G Mansfield B Ruben S Sanyal I Migone TS 《Nature biotechnology》2003,21(3):302-307
A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins. 相似文献
997.
Background
Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources. 相似文献998.
Synchrotron infrared microspectroscopy (SIRMS) was used for the first time to image the distribution and secondary structure of an enzyme (lipase B from Candida antarctica, CALB) immobilized within a macroporous polymer matrix (poly(methyl methacrylate)) at 10 microm resolution. The beads of this catalyst (Novozyme435) were cut into thin sections (12 microm). SIRMS imaging of these thin sections revealed that the enzyme is localized in an external shell of the bead with a thickness of 80-100 microm. Also, the enzyme was unevenly distributed throughout this shell. Furthermore, by SIRMS-generated spectra, it was found that CALB secondary structure was not altered by immobilization. Unlike CALB, polystyrene molecules of similar molecular weight diffuse easily throughout Novozyme435 beads. Scanning electron micrograph (SEM) images of the Novozyme435 beads showed that the average pore size is 10 times larger than CALB or polystyrene molecules, implying that there is no physical barrier to enzyme or substrate diffusion throughout the bead. Thus, the difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin. 相似文献
999.
Conformation-dependent effects of VIP on nociception in rats 总被引:1,自引:0,他引:1
The purpose of this study was to determine whether intrathecal injection of aqueous (random coil) vasoactive intestinal peptide (VIP) and VIP self-associated with sterically stabilized phospholipid micelles (alpha-helix VIP) at the lower lumbar vertebral level modulates foot withdrawal latency to low and high rate noxious radiant skin heating in anesthetized rats. We found that intrathecal random coil VIP evoked a significant bimodal, concentration-dependent response, early potent antinociception followed by hyperalgesia, during exposure to low and high rates of skin heating (P<0.05). Intrathecal alpha-helix VIP elicited a qualitatively similar response to that of random coil VIP except that the rate of decay of antinociception was faster and slower at low and high rates of skin heating, respectively. In addition, a low concentration of alpha-helix VIP evoked a potent late antinociception not observed with random coil VIP. Taken together, these data indicate that VIP modulates somatosensory processing in the lumbosacral spinal cord of rats in a complex fashion, and that this response is dependent, in part, on the conformation of VIP in the vicinity of target cells in the peripheral nervous system. 相似文献
1000.
A new method for fold recognition is developed and added to the general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/). The new method (PROSPECT II) has four key features. (i) We have developed an efficient way to utilize the evolutionary information for evaluating the threading potentials including singleton and pairwise energies. (ii) We have developed a two-stage threading strategy: (a) threading using dynamic programming without considering the pairwise energy and (b) fold recognition considering all the energy terms, including the pairwise energy calculated from the dynamic programming threading alignments. (iii) We have developed a combined z-score scheme for fold recognition, which takes into consideration the z-scores of each energy term. (iv) Based on the z-scores, we have developed a confidence index, which measures the reliability of a prediction and a possible structure-function relationship based on a statistical analysis of a large data set consisting of threadings of 600 query proteins against the entire FSSP templates. Tests on several benchmark sets indicate that the evolutionary information and other new features of PROSPECT II greatly improve the alignment accuracy. We also demonstrate that the performance of PROSPECT II on fold recognition is significantly better than any other method available at all levels of similarity. Improvement in the sensitivity of the fold recognition, especially at the superfamily and fold levels, makes PROSPECT II a reliable and fully automated protein structure and function prediction program for genome-scale applications. 相似文献