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961.
Sarno S Ruzzene M Frascella P Pagano MA Meggio F Zambon A Mazzorana M Di Maira G Lucchini V Pinna LA 《Molecular and cellular biochemistry》2005,274(1-2):69-76
A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (Ki = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (Ki = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (Ki = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (Ki = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs. 相似文献
962.
Pagano MA Sarno S Poletto G Cozza G Pinna LA Meggio F 《Molecular and cellular biochemistry》2005,274(1-2):23-29
Among the features of protein kinase CK2, autophosphorylation at its β-subunit(s) upon incubation with ATP/Mg++ was early detected as a rapid and stoichiometric event occurring through an intramolecular mechanism as judged from kinetic analyses. The autophosphorylation site was mapped to Ser2 and, to a lesser extent, Ser3 both fulfilling the CK2 consensus sequence (MSSSEEV). The crystal structure of the heterotetrameric holoenzyme, however, is not compatible with an intramolecular autophosphorylation of the N-terminal stretch of either of the two β subunits. Here we show that efficient “intramolecular” autophosphorylation of the β subunit is crucially dependent on the formation of oligomers composed by several holoenzyme heterotetrameric protomers. Increasing ionic strength of the incubation medium promoting dissociation of the supramolecular oligomers abrogates β subunit autophosphorylation, although CK2 catalytic activity, as judged from the phosphorylation of exogenous substrates, is still quite evident. These findings, in conjunction with graphic modelization, support the view that CK2 autophosphorylation at its β subunits takes place through an “intraoligomeric” mechanism where the β subunits of a protomer are phosphorylated by the catalytic subunits of another adjacent protomer. It appears therefore that in vivo β autophosphorylation is symptomatic of supramolecular CK2 oligomers. 相似文献
963.
Llorens F Sarno S Sarró E Duarri A Roher N Meggio F Plana M Pinna LA Itarte E 《Molecular and cellular biochemistry》2005,274(1-2):53-61
The β-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138–333 of eIF2β (eIF2β-CT) interacts with CK2α as efficiently as full length eIF2β, whereas the form corresponding to residues 1–137, which contains the CK2 phosphorylation sites, (eIF2β-NT) does not bind. The use of different mutants and truncated forms of CK2α allowed us to map the basic segment K74–K83 at the beginning of helix αC and residues R191R195K198 in the p+1 loop as the main determinants for the binding to eIF2β-CT of either the isolated CK2α subunit or the CK2 holoenzyme. The presence of eIF2β-CT stimulated the activity of CK2α towards the RRRAADSDDDDD peptide substrate; effect that was not observed with the CK2α K74-77A whose ability to bind to eIF2β-CT is severely impaired. Gel filtration analysis confirmed the ability of CK2α to form complexes with eIF2β-CT, and the contribution of the basic cluster in CK2α (K74–K77) in this association. 相似文献
964.
Girosi L Ramoino P Diaspro A Gallus L Ciarcia G Tagliafierro G 《Cell and tissue research》2005,320(2):331-336
The presence of FMRFamide-related peptides (FaRPs) was investigated, by immunohistochemical methods with a polyclonal FMRFamide antiserum, in the sea-fan Eunicella cavolini (Van Koch 1887), a representative of the cnidarians (octocorallians). The identification of FaRP-immunoreactive elements as neuronal cells and a nerve net was performed by double immunohistochemical methods with the monoclonal anti-beta-tubulin antibody. A strong and widely distributed FaRPs immunoreactivity was detected: FaRPs-immunoreactive nerve cells were observed among and underlying gastrodermal epithelial cells, epidermal cells lining tentacles, muscular septs and gonophores. A diffuse FaRPs-immunoreactive nerve net was also found between epithelia and mesoglea and in the stalk of the gonophore. These results improve our knowledge of the gorgonian nervous system and demonstrate that most of the immunoreactive cells belong to neural elements. 相似文献
965.
Zagari RM Pozzato P Martuzzi C Fuccio L Martinelli G Roda E Bazzoli F 《Helicobacter》2005,10(6):615-619
BACKGROUND: Some authors have reported, using different protocols, that 13C-urea breath test (13C-UBT) is capable of assessing the intragastric Helicobacter pylori bacterial load, whereas others have not confirmed these data. Our aim is to evaluate the correlation between 13C-UBT values and H. pylori bacterial load. MATERIALS AND METHODS: One hundred ninety-two patients diagnosed H. pylori-positive by rapid urease test, histology, and 13C-UBT were enrolled. H. pylori bacterial load (H. pylori score) and gastritis activity (activity score) were evaluated according to the Updated Sydney System. 13C-UBT was performed according to the European Standard Protocol. Breath samples were obtained every 10 minutes for 60 minutes in 52 patients and at 30 minutes (T30) in 140 patients and analyzed by mass spectrometry. RESULTS: At T30, mean +/- SD excess delta 13CO2 excretion was 17.4 +/- 1.1, 29.9 +/- 2.2, and 48.7 +/- 4.8 in patients with H. pylori scores 1, 2, and 3, respectively. This difference was statistically significant: H. pylori score 1 versus 2, p < .005; score 1 versus 3, p < .05; score 2 versus 3, p < .05. A significant positive correlation (G = 0.59) was found between H. pylori score and activity score of chronic gastritis. At T40 and T50 significant correlation between mean excess delta 13CO2 excretion and bacterial load was achieved only in patients with H. pylori scores 1 and 3. CONCLUSIONS: 13C-UBT European Standard Protocol values correlate with H. pylori bacterial load and the activity of gastritis at T30 breath sampling time. 相似文献
966.
967.
Chicote E García AM Moreno DA Sarró MI Lorenzo PI Montero F 《Journal of industrial microbiology & biotechnology》2005,32(4):155-162
The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (, , and -Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters. 相似文献
968.
Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells 总被引:9,自引:0,他引:9
Mazzocca A Sciammetta SC Carloni V Cosmi L Annunziato F Harada T Abrignani S Pinzani M 《The Journal of biological chemistry》2005,280(12):11329-11339
The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage. 相似文献
969.
Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein 下载免费PDF全文
Di Matteo A Giovane A Raiola A Camardella L Bonivento D De Lorenzo G Cervone F Bellincampi D Tsernoglou D 《The Plant cell》2005,17(3):849-858
Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes. 相似文献
970.
The ytterbium complex [Yb((S)-THP)](3+) ((S)-THP = (1S,4S,7S,10S-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) is investigated in solution through NMR, near-IR absorption, and CD spectroscopy. Quantitative analysis of the paramagnetic pseudocontact NMR shift shows Lambda helicity of the ligand cage around the metal. The NIR CD spectrum recorded at acidic pH is found to be very similar to that of [Yb((R)-DOTMA)](-) ((R)-DOTMA = (1R,4R,7R,10R)-alpha,alpha',alpha',alpha'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which in solution assumes a twisted square antiprism (TSA) conformation. The similarity of the NIR CD spectra is discussed, and it is the first proof of the Lambda(lambda,lambda,lambda,lambda) conformation of [Yb((S)-THP)](3+). NIR CD spectra recorded in the pH range of 2-9 allow one to easily follow proton dissociation and to calculate the pK of this equilibrium in water (pK(A) = 6.4 +/- 0.1). This value agrees well with that determined for [Lu((S)-THP)](3+) using potentiometric methods. This demonstrates once again that NIR CD spectroscopy is a powerful technique for investigating the solution structure and dynamics of these complexes. 相似文献