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Michael S. Christodoulou Sandra Liekens Konstantinos M. Kasiotis Serkos A. Haroutounian 《Bioorganic & medicinal chemistry》2010,18(12):4338-4350
The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro. 相似文献
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Codorean E Nichita C Albulescu L Răducan E Popescu ID Lonită AC Albulescu R 《Roumanian archives of microbiology and immunology》2010,69(1):13-19
There is an emerging trend in immunotoxicological studies to use the multiplex technologies for testing the safety and the efficacy of new pharmaceuticals by using cytokines profiling as biomarker. The Luminex 200 xMAP (multi-analyte profiling) technology provides simultaneous measurement of multiple cytokines in small sample volumes, expressing rapidly the differences between various test compounds. The aim is to develop and validate the Luminex 200 multiplex immunoassays by correlation with ELISA (enzyme-linked immunosorbent assays) for implementation in evaluating cytokine profiling in immunotoxicological studies in vitro. METHODS: Human peripheral whole blood from healthy subject diluted 1+4 with RPMI 1640 was cultured 48 hours in 28 experimental variants: control, in presence of mitogens, bioflavonoid extracts (from Crataegus monogyna and Echinacea purpurea) as cytoprotectors and with a toxic compound [Pb(NO3)2]), separately or variously combined. IL-1beta and IL-2 were comparatively performed by xMAP and ELISA immunoassays from the same sample to initialize validation of multiplex cytokine panel: IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, usually performed by Luminex 200 system in our immunotoxicological studies. The results indicate similarly typed trends of cytokine values obtained by both methods, with comparable relative changes in presence of mitogens, bioflavonoids and toxic, respectively. Although xMAP absolute cytokine values were higher than ELISA values, the correlation between multiplexed assay and ELISA was good for IL-1beta and IL-2 with positive correlation coefficients near to 1. Conclusions. Quantitative differences between absolute values for IL-1beta and IL-2 obtained by xMAP and ELISA assays are found, but the relative values are comparable and the two methods keep similar trends in similar exposure conditions. The performance parameters of the xMAP assay and the good correlation coefficients with the "gold standard" ELISA recommend to validate the multiplex assay for analyzing cytokine profiles in immunotoxicological studies in vitro. 相似文献
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An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states 总被引:1,自引:0,他引:1
Blombach F Launay H Zorraquino V Swarts DC Cabrita LD Benelli D Christodoulou J Londei P van der Oost J 《Journal of bacteriology》2011,193(11):2861-2867
HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins. 相似文献
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Constantina Constantinou Androulla Elia Michael J Clemens 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(5):279-289
BACKGROUND INFORMATION: The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. RESULTS: We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. CONCLUSIONS: The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53. 相似文献
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Arturo Casini James T. MacDonald Joachim De Jonghe Georgia Christodoulou Paul S. Freemont Geoff S. Baldwin Tom Ellis 《Nucleic acids research》2014,42(1):e7
Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. 相似文献
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Christodoulou J Malmendal A Harper JF Chazin WJ 《The Journal of biological chemistry》2004,279(28):29092-29100
Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca(2+) binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca(2+). The addition of substoichiometric amounts of Ca(2+) is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca(2+)-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca(2+) affinity for JC reveal two cooperatively interacting high affinity binding sites (K(d)(,mean) = 5.6 nm at 20 mm KCl) in the C-lobe and two weaker sites in the N-lobe (K(d,mean) = 110 nm at 20 mm KCl). The corresponding Ca(2+) binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca(2+), the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca(2+) levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region. 相似文献