JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005
Daichi Chikazu
Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source]

QSAR for Inhibition of Pseudomonas Species Lipase by 1-Acyloxy-3- N-n -octylcarbamyl-benzenes

MOLECULAR INFORMATICS, Issue 3 2009
Shyh-Ying Chiou
Abstract 1-Acyloxy-3- N-n -octylcarbamyl-benzenes (1,9) are synthesized to characterize the Quantitative Structure,Activity Relationship (QSAR) for the Third Acyl Group Binding Site (TACS) of Pseudomonas species lipase. Inhibitors 1,9 are characterized as pseudo or alternate substrate inhibitors of the enzyme. The inhibition constant (Ki) and carbamylation constant (k2) for the enzyme inhibitions by inhibitors 1,9 are determined. The carbamate carbons of the n -octylcarbamyl moieties of inhibitors 1,9 are nucleophilically attacked by the active site serine of the enzyme and the n -octylcarbamyl groups of inhibitors 1,9 are bound to the Acyl Group Binding Site (ACS) of the enzyme. Both pKi and log,k2 values are linearly corrected with the Hansch hydrophobicity , values of the substituents of the acyl moieties of inhibitors 1,7. The slopes for these corrections are 0.13 and 0.02, respectively. This result suggests that the enzyme inhibitions by inhibitors 1,7 have a common mechanism. Thus, all acyl moieties of inhibitors 1,7 should bind to the TACS of the enzyme since the acyl and carbamyl moieties of inhibitors 1,7 are meta to each other. This result also indicates that the major interaction between the acyl moiety of inhibitors 1,7 and the TACS of the enzyme is primarily the hydrophobic interaction. The more hydrophobic characters of inhibitors 1,7 are, the more tightly these inhibitors bind to the enzyme. In contrast, 1-triphenylacetoxy-3- N-n -octylcarbamyl-benzene (8) and 1-trimethylacetoxy-3- N-n -octylcarbamyl-benzene (9) do not bind to the TACS of the enzyme due to the fact that the inhibitions by both inhibitors are not linearly correlated with ,. It is possible that these two inhibitors are too bulky to fit into the TACS of the enzyme. [source]

An Artificial Metalloenzyme: Creation of a Designed Copper Binding Site in a Thermostable Protein,

ANGEWANDTE CHEMIE, Issue 30 2010
John Podtetenieff Dr.
Von der Natur inspiriert: Ortsspezifische Mutagenese ermöglichte den gezielten Aufbau einer Bindungsstelle mit dem His/His/Asp-Motiv zur CuII -Komplexierung in einem robusten Protein (siehe Bild). Das künstliche Metalloenzym katalysiert eine enantioselektive Diels-Alder-Reaktion. [source]

Cyclostreptin and Microtubules: Is a Low-Affinity Binding Site Required?

CHEMBIOCHEM, Issue 1 2010
Andrew J. Prussia Dr.
Abstract Cyclostreptin (CS) is a recently discovered natural product with cytotoxic activity caused by microtubule stabilization. It is the only known microtubule-stabilizing agent (MSA) that covalently binds to tubulin. It also exhibits the fast-binding kinetics seen for other MSAs. Through careful peptide digestion and mass spectrometry analysis, Buey et al. found that two amino acids are labeled by CS: Asn228, near the known taxane-binding site, and Thr220, in the type I microtubule pore. This led Buey et al. to propose Thr220 resides at the site previously predicted to be a way station or low-affinity site. By using molecular dynamics simulations and structural considerations of the microtubule pore and tubulin dimer, we conclude that postulation of a low-affinity site is unnecessary to explain the available experimental data. An alternative explanation views the microtubule pore as a structural entity that presents a substantial kinetic barrier to ligand passage to the known taxane-binding site,an entry point to the microtubule lumen that becomes completely blocked if cyclostreptin is bound at Thr220. Simulations of the free dimer also suggest a common mechanism of microtubule stabilization for taxane site MSAs through their conformational effect on the M-loop. Such an effect explains the low tubulin polymerization caused by cyclostreptin in vitro despite its covalent attachment. [source]

Tryptophan ,-Electron System Capping a Copper(I) Binding Site,A New Organometallic Bonding Mode in Proteins

CHEMBIOCHEM, Issue 11 2008
Olaf Kühl Dr.
,2 -Arene coordination (dotted lines) from the aromatic tryptophan side chain to CuI (red) in the prokaryotic CuI -transport protein CusF represents a new organometallic interaction in biology. [source]

Structural Investigation of a High-Affinity MnII Binding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations.

CHEMBIOCHEM, Issue 10 2003
Effects of Neomycin B on Metal-Ion Binding
Abstract Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity MnIIbinding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Kdof 4.4 ,M in buffer solutions containing 1,M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant,of 0.7 MHz, an asymmetry parameter,of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the MnIIligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by MnIIin crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the MnIIsite in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of,(14N)=+0.80 MHz, ,=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that MnIIis released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 ,M) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11,186). [source]

Infrared Spectroscopy of Water Cluster Anions, (H2O)n=3-24 - in the HOH Bending Region: Persistence of the Double H-Bond Acceptor (AA) Water Molecule in the Excess Electron Binding Site of the Class I Isomers.

CHEMINFORM, Issue 35 2006
Joseph R. Roscioli
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

Design and Synthesis of a New Class of Arginine Analogues with an Improved Anion Binding Site in the Side Chain.

CHEMINFORM, Issue 26 2005
Carsten Schmuck
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Enantiomerically Pure Tetrahydroquinoline Derivatives as in vivo Potent Antagonists of the Glycine Binding Site Associated to the NMDA Receptor.

CHEMINFORM, Issue 6 2004
Romano Di Fabio
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

CHEMINFORM, Issue 6 2004
Stefan Vogel
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

A Substituted Triaza Crown Ether as a Binding Site in DNA Conjugates.

CHEMINFORM, Issue 28 2003
Stefan Vogel
No abstract is available for this article. [source]

Dynamic Combinatorial Carbohydrate Libraries: Probing the Binding Site of the Concanavalin A Lectin

CHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2004
Olof Ramström Dr.
Abstract Dynamic combinatorial chemistry (DCC) has emerged as an efficient approach to receptor/ligand identification based on the generation of combinatorial libraries by reversible interconversion of the library constituents. In this study, the implementation of such libraries on carbohydrate,lectin interactions was examined with the plant lectin Concanavalin A as a target species. Dynamic carbohydrate libraries were generated from a pool of carbohydrate aldehydes and hydrazide linker/scaffold components through reversible acylhydrazone exchange, resulting in libraries containing up to 474 constituents. Dynamic deconvolution allowed the efficient identification of the structural features required for binding to Concanavalin A and the selection of a strong binder, a tritopic mannoside, showing an IC50 -value of 22,,M. [source]

Long-Term Modulation By Postnatal Oxytocin of the ,2 -Adrenoceptor Agonist Binding Sites in Central Autonomic Regions and the Role of Prenatal Stress

JOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2004
Z. Díaz-Cabiale
Abstract The aim of this work was to evaluate whether oxytocin administered in male rats subcutaneously early in life in the absence or presence of food restriction during pregnancy has life-long effects on the ,2 -agonist binding sites in the nucleus of the solitarii tract (NTS), in the hypothalamus and the amygdala, as evaluated by quantitative receptor autoradiography. Maternal food restriction alone increased the affinity of the ,2 -agonist [3H]UK14.304 binding sites exclusively in the NTS. In offspring from ad libitum fed dams, oxytocin treatment significantly increased the density of ,2 -agonist binding sites in the NTS and in the hypothalamus. The Kd value of the ,2 -agonist binding sites in the hypothalamus of these rats, but not in the other regions studied, was also significantly increased. In offspring from food-restricted dams, oxytocin treatment produced a significant increase of the Bmax values in the hypothalamus and the amygdala and the Kd value of the ,2 -agonist binding sites in the NTS of these rats also was selectively and significantly increased. These results suggest that a postnatal, oxytocin-induced increase of regional ,2 -adrenoceptor function can be seen in adulthood by a persistent, regionally selective increase in the density of central ,2 -adrenoceptor agonist binding sites, in the absence of an affinity change in the NTS. Such a regional increase of ,2 -adrenoceptor signalling in adulthood may contribute to the anti-stress action of postnatal oxytocin. By contrast, after prenatal stress, the potential increase in ,2 -adrenoceptor signalling takes place via selective increases of density with no changes of affinity of the ,2 -agonist binding sites in the hypothalamus and the amygdala. [source]

Structures of the Chromophore Binding Sites in BLUF Domains as Studied by Molecular Dynamics and Quantum Chemical Calculations,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Kazuya Obanayama
BLUF (blue-light sensing using FAD) domains constitute a new family of flavin-based blue light photoreceptors. The photocycle of BLUF is unique in the sense that a few hydrogen bond rearrangements are accompanied by only slight structural changes in the bound chromophore. The hydrogen bond rearrangements upon illumination have been inferred from spectral changes in the chromophore: ,10 nm redshift of the absorption maximum and ,16 cm,1 downshift of the C4=O stretching frequency. However, the exact features of the hydrogen bond network around the active site are still the subject of some controversy. In particular, the orientation of a conserved Gln (Gln63 in AppA) is presently one of the most questioned topics in the field. Here we perform molecular dynamics simulations for the wild-type AppA, AppA1-124C20S, BlrB and T110078 and furthermore quantum chemical calculations to investigate their spectroscopic properties in the dark and signaling states. On the basis of these results, we reveal the dynamic aspect of hydrogen bonding networks at the active site and propose theoretically reasonable models for the dark and signaling states of the BLUF domains. [source]

A Fragment-Based In,Situ Combinatorial Approach To Identify High-Affinity Ligands for Unknown Binding Sites,

ANGEWANDTE CHEMIE, Issue 33 2010
Sachin
In leitender Position: Die im Titel genannte Methode zur Identifizierung von Liganden ist besonders nützlich, wenn keine oder nur unvollständige strukturelle Informationen zur Bindungsstelle verfügbar sind. In einem Fragment-basierten Verfahren wird durch NMR-Experimente ein geeignetes Paar von Liganden ermittelt. Mit einem Rezeptor-vermittelten, kombinatorischen In-situ-Verfahren werden die Liganden anschließend verknüpft und so in eine neue hochaffine Leitstruktur überführt (siehe Bild). [source]

Location of Potential Substrate Water Binding Sites in the Water Oxidizing Complex of Photosystem,II,

ANGEWANDTE CHEMIE, Issue 25 2010
Simon Petrie Dr.
Auf der Suche nach Wasser: DFT-Rechnungen wurden auf einen Satz von Mn4Ca-Clustern als Modell des wasseroxidierenden Komplexes (WOC) im Photosystem,II angewendet. (siehe Bild; O rot, H weiß, Ca hellgrau, Mn dunkelgrau, C schwarz). Die Rechnungen liefern ein Modell des aktiven Zentrums, das jüngste experimentelle Daten zu Substratwechselwirkungen mit dem WOC erklärt und die wahrscheinlichsten Bindungsstellen für Substratwasser identifiziert. [source]

Structure-Based Synthetic Mimicry of Discontinuous Protein Binding Sites: Inhibitors of the Interaction of Mena EVH1 Domain with Proline-Rich Ligands

CHEMBIOCHEM, Issue 8 2006
Cornelia Hunke Dr.
Abstract The Mena EVH1 domain, a protein-interaction module involved in actin-based cell motility, recognizes proline-rich ligand motifs, which are also present in the sequence of the surface protein ActA of Listeria monocytogenes. The interaction of ActA with host Mena EVH1 enables the bacterium to actively recruit host actin in order to spread into neighboring cells. Based on the crystal structure of Mena EVH1 in complex with a polyproline peptide ligand, we have generated a range of assembled peptides presenting the Mena EVH1 fragments that make up its discontinuous binding site for proline-rich ligands. Some of these peptides were found to inhibit the interaction of Mena EVH1 with the ligand pGolemi. One of them was further characterized at the level of individual amino acid residues; this yielded information on the contribution of individual positions of the peptides to the interaction with the ligand and identified sites for future structure optimization. [source]

Site-Specific Modification of Epstein,Barr Virus-Encoded RNA 1 with N2 -Benzylguanosine Limits the Binding Sites Occupied by PKR

CHEMBIOCHEM, Issue 3 2004
Sujiet Puthenveetil
Examining viral decoys: Epstein,Barr virus (EBV) generates small RNA inhibitors of the human RNA-dependent protein kinase (PKR). We demonstrate that chemical synthesis of analogues of the EBV PKR inhibitor EBER1 bearing single N2 -benzylguanosine substitutions (BnG6 or BnG29) can be used to control the way PKR binds this RNA. [source]

Fentanyl Derivatives Bearing Aliphatic Alkaneguanidinium Moieties: A New Series of Hybrid Molecules with Significant Binding Affinity for ,-Opioid Receptors and I2 -Imidazoline Binding Sites.

CHEMINFORM, Issue 16 2004
Christophe Dardonville
No abstract is available for this article. [source]

Anion Receptors Containing -NH Binding Sites: Hydrogen-Bond Formation or Neat Proton Transfer?

CHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2005
Valeria Amendola Dr.
Abstract When the amide-containing receptor 1+ is in a solution of dimethyl sulfoxide (DMSO) in the presence of basic anions (CH3COO,, F,, H2PO4,), it undergoes deprotonation of the -NH fragment to give the corresponding zwitterion, which can be isolated as a crystalline solid. In the presence of less basic anions (Cl,, Br,, NO3,), 1+ establishes true hydrogen-bond interactions of decreasing intensity. The less acidic receptor 2+ undergoes neat proton transfer with only the more basic anions CH3COO, and F,, and establishes hydrogen-bond interactions with H2PO4,. An empirical criterion for discerning neutralisation and hydrogen bonding, based on UV/Vis and 1H NMR spectra, is proposed. [source]

Prediction of the Three-Dimensional Structure for the Rat Urotensin,II Receptor, and Comparison of the Antagonist Binding Sites and Binding Selectivity between Human and Rat Receptors from Atomistic Simulations

CHEMMEDCHEM, Issue 9 2010
Soo-Kyung Kim Dr.
Abstract Urotensin-II (U-II) has been shown to be the most potent mammalian vasoconstrictor known. Thus, a U-II antagonist might be of therapeutic value in a number of cardiovascular disorders. However, interspecies variability of several nonpeptidic ligands complicates the interpretation of in vivo studies of such antagonists in preclinical animal disease models. ACT058362 is a selective antagonist for the human U-II receptor (hUT2R) with a reported Kd value of ,4,nM in a molecular binding assay, but it is reported to bind weakly to rat UT2R (rUT2R), with a Kd value of ,1,500,nM. In contrast, the arylsulphonamide SB706375 is a selective antagonist against both hUT2R (Kd=,9,nM) and rUT2R (Kd=,21,nM). To understand the species selectivity of the UT2R, we investigated the binding site of ACT058362 and SB706375 in both hUT2R and rUT2R to explain the dramatically lower (,400-fold) affinity of ACT058362 for rUT2R and the similar affinity (,10,nM) of SB706375 for both UT2Rs. These studies used MembStruk and MSCDock to predict the UT2R structure and the binding site of ACT058362 and SB706375. Based on binding energies, we found two binding modes each with D1303.32 as the crucial anchoring point (Ballesteros,Weinstein numbering given in superscript). We predict that ACT058362 (an aryl,amine,aryl or ANA ligand) binds in the transmembrane (TM) 3456 region, while SB706375 (an aryl,aryl,amine or AAN ligand) binds in the TM 1237 region. These predicted sites explain the known differences in binding of the ANA ligand to rat and human receptors, while explaining the similar binding of the AAN compound to rat and human receptors. Moreover the predictions explain currently available structure,activity relationship (SAR) data. To further validate the predicted binding sites of these ligands in hUT2R and rUT2R, we propose several mutations that would help define the structural origins of differential responses between UT2R of different species, potentially indicating novel UT2R antagonists with cross-species high affinity. [source]

Identification of Putative Binding Sites of P-glycoprotein Based on its Homology Model

CHEMMEDCHEM, Issue 2 2008
Christoph Globisch
Abstract A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways. [source]

Tyrosine Hydrogen Bond Properties for the Two Binding Sites of Apoovotransferrin

CHINESE JOURNAL OF CHEMISTRY, Issue 10 2005
Ying-Qi Li
Abstract The interaction of gallium(III) with the ligands containing phenolic group(s), such as salicylic acid, 8-hydroxyquinoline, N,N' -bis(2-hydroxybenzyl)ethylenediamine- N,N, -diacetic acid (HBED), N,N, -ethylenebis[2-(o -hydroxyphenyl)glycine (EHPG), and ovotransferrin, was studied, respectively, by means of fluorescence in 0.01 mol/L Hepes at pH 7.4 and room temperature. Fluorescence intensity showed an increase when gallium(III) was bound to 8-hydroxyquinoline and HBED. In contrast, it was decreased with the interaction of gallium(III) with salicylic acid and EHPG. At pH 7.4, there was N···HO type intramolecular hydrogen bond in the former, and the latter existed O···HO type intramolecular hydrogen bond. Fluorescence titration of apoovotransferrin with gallium(III) displayed that the fluorescence intensity was decreased at the N-terminal binding site, while enhanced at the C-terminal binding site. It can account for the O···HO type intramolecular hydrogen bonds for the phenolic groups of Tyr92 and Tyr191 residues at the N-terminal binding site. And there are N···HO type intramolecular hydrogen bonds for Tyr431 and Tyr524 residues at the C-terminal binding site. In addition, under the same conditions, the conditional binding constant of gallium(III) with EHPG or HBED determined by fluorescence method is lg KGa-EHPG=19.18 or lg KGa-HBED=19.08. [source]

Binding site on human immunoglobulin G for the affinity ligand HWRGWV

JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2010
Haiou Yang
Abstract Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383,Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Gene-expression signature of adhesion/growth-regulatory tissue lectins (galectins) in transitional cell cancer and its prognostic relevance

HISTOPATHOLOGY, Issue 5 2007
S Langbein
Aims:, Lectins, and especially galectins, appear to be important in malignancy-associated processes. The aim was to analyse comprehensively the presence of galectins in urothelial tumours. Methods and results:, Non-cross-reactive antibodies against seven family members from the three subgroups (prototype: galectin-1, -2 and -7; chimera type: galectin-3; tandem-repeat type: galectin-4, -8 and -9) were used. Gene expression was monitored in specimens of normal urothelium, fresh tumour tissue and cell lines by real-time polymerase chain reaction (PCR). The presence and evidence of tumour-associated up-regulation were shown for galectin-1 and -3. This was less clear-cut for galectin-4 and -8. Galectin-7 was expressed in all cell lines; galectin-2 and -9 were detected at comparatively low levels. Galectin-2, -3 and -8 up-regulation was observed in superficial tumours, but not in muscle-invasive tumours (P < 0.05). Immunoreactivity correlated with tumour grading for galectin-1, -2 and -8, and disease-dependent mortality correlated with galectin-2 and -8 expression. Binding sites were visualized using labelled galectins. Conclusions:, The results demonstrate a complex expression pattern of the galectin network in urothelial carcinomas. Galectin-1, -2, -3 and -8 are both potential disease markers and also possible targets for bladder cancer therapy. [source]

Binding sites of [Ru(bpy)2(H2O)2](BF4)2 with sulfur- and histidine-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2005
Jin Hong
Abstract The composition and binding sites of cis -[Ru(II)(bpy)2]2+ -bound sulfur-containing peptides of Met-Arg-Phe-Ala, glutathione and oxidized glutathione, and also histidine-containing peptide of oxidized insulin B chain, were investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The composition of Ru(II)-containing peptides was precisely determined by ESI-MS, zoom scan and simulation of isotope distribution patterns. MS/MS analysis shows that, in sulfur-containing peptides, the Ru(II) complex prefers to anchor to a carboxyl group, although some other potential binding sites of thiol, thioether and N-terminal amino groups present in these peptides, and in oxidized insulin B chain, Ru(II) first anchors to His10, then either to the hydroxyl group of Thr27 or to the carboxyl group of Ala30. Its secondary structure and microenvironment surrounding the potential binding sites may affect the binding ability of cis -[Ru(II)(bpy)2]2+ to oxidized insulin B chain. Copyright © 2004 John Wiley & Sons, Ltd. [source]

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells,

THE JOURNAL OF PATHOLOGY, Issue 1 2010
Stéphanie Bougel
Abstract Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source]

No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+ -ATPase from short-term stimulated rat muscle

ACTA PHYSIOLOGICA, Issue 4 2009
T. Mishima
Abstract Aim:, We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+ -ATPase (SERCA) would account for contraction-induced changes in the catalytic activity of the enzyme as assessed in vitro. Methods:, Repetitive contractions were induced in rat gastrocnemius by electrical nerve stimulation. Measurements of sarcoplasmic reticulum properties were performed on control and stimulated muscles immediately after or at 30 min after the cessation of 5-min stimulation. In order to examine the properties at the ANBS, the binding capacity of SERCA to fluorescence isothiocyanate (FITC), a competitive inhibitor at the ANBS, was analysed in microsomes. Results:, Short-term electrical stimulation evoked a 23.9% and 32.6% decrease (P < 0.05) in SERCA activity and in the FITC binding capacity, respectively, in the superficial region of the muscle. Whereas SERCA activity reverted to normal levels during 30-min recovery, a restoration of the FITC binding capacity did not occur. Conclusion:, The discordant changes between the enzyme activity and the FITC binding suggest that, at least during recovery after exercise, changes in SERCA activity may not correlate closely with structural alterations to the ANBS within the enzyme. [source]

Close association of CD8+/CD38bright with HIV-1 replication and complex relationship with CD4+ T-cell count,

CYTOMETRY, Issue 4 2009
Edouard Tuaillon
Abstract Background: Measuring lymphocyte activation provides information in addition to CD4+ T-cell count for immune monitoring of HIV-1 infected patients. CD38 is a well-established activation marker that is generally analyzed on the whole population of CD8+ T-cells. Focusing specifically on CD38 high expression (CD8+/CD38bright) may be an interesting surrogate gating strategy because CD38bright characterizes principally activated memory cells. Methods: CD8+/CD38bright was investigated in 1,353 HIV-1 infected patients over a one-year period to establish relevant cutoff values and clarify the relationships of this marker with HIV-1 RNA viral load (VL) and CD4+ T-cell count. Results: The CD8+/CD38bright (>8,500 CD38 binding site per cells) is well correlated with HIV-1 VL (r = 0.87, P < 0.001) in this longitudinal follow-up of nonimmunodepressed patients that initiated antiviral therapy (ART). In aviremic patients on ART, the marker was highly predictive of VL rebound (sensitivity 93%, specificity 64% for a VL level of detection >200 copies/ml). While the CD8+/CD38bright moderately correlated with CD4+ T-cell count independently of the VL (r = ,0.37, P < 0.001), it increased dramatically in aviremic patient groups that exhibited profound CD4+ T-cell depletion (median 39% for CD4+ T-cell counts <50/mm3). This result indicates that other additional immunological and/or viral factors than readily detectable HIV-1 replication appears to be involved in T-cell activation of immunodepressed individuals. Conclusions: CD8+/CD38bright is an effective marker for monitoring T-cell activation, which is a central factor of HIV-1 pathogenesis. This gating strategy requires only a single additional staining in conventional four color CD4 protocols. © 2008 Clinical Cytometry Society [source]

Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

CYTOSKELETON, Issue 4 2004
Scott C. Mateer
Abstract IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, ,-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5,50 ,M Kd) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP12-210, which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP12-210 was found to be monomeric, to bind F-actin with a Kd of ,47 ,M, to saturate F-actin at a molar ratio of one IQGAP12-210 per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. Cell Motil. Cytoskeleton 58:231,241, 2004. © 2004 Wiley-Liss, Inc. [source]