Fluorescence analysis of G·C versus A·T binding of quinacrine to DNA

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10^?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.     

Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Albumin Rugby Park: a truncated albumin variant caused by a G → C splice-site mutation in nitro 13

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G → C mutation at oposition 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.     

Association between tumor necrosis factor-α promoter −308 A/G, −238 A/G, interleukin-6 −174 G/C and −572 G/C polymorphisms and periodontal disease: a meta-analysis

The aim of this study was to determine whether tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) promoter polymorphisms confer susceptibility to periodontitis in ethnically different populations. A literature search was performed using PubMed and Embase and a meta-analysis of the identified studies was conducted to explore the associations between TNF-α ?308 A/G, ?238 A/G, IL-6 promoter ?174 G/C and ?572 G/C polymorphisms and periodontitis. Seventeen comparison studies for the TNF-α ?308 A/G polymorphism and three studies for the TNF-α ?238 A/G polymorphism were included in the meta-analysis. And 16 separate studies for the IL-6 ?174 G/C polymorphism and 10 studies for the IL-6 ?572 G/C polymorphism were considered in our meta-analysis. Analysis after stratification by ethnicity indicated that the TNF-α ?308 A allele was associated with periodontitis in Brazilian, Asian, and Turkish populations (OR = 0.637, 95 % CI = 0.447–0.907, p = 0.013; OR = 0.403, 95 % CI = 0.204–0.707, p = 0.009; OR = 1.818, 95;  % CI = 1.036–3.189, p = 0.037). The meta-analysis showed no association between the TNF-α ?238 A/G polymorphism and periodontitis. The meta-analysis indicated an association of the IL-6 ?174 G/C polymorphisms with periodontitis in Brazilian populations (OR for GG + GC = 2.394, 95 % CI = 1.081–5.302, p = 0.031). Stratification by ethnicity and disease type indicated an association between the IL-6 ?572 G allele and chronic periodontitis (OR = 1.585, 95 % CI = 1.030–2.439, p = 0.036), and periodontitis in Europeans (OR = 2.118, 95 % CI = 1.254–3.577, p = 0.005). This meta-analysis demonstrates that the TNF-α ?308 A/G polymorphism confers susceptibility to periodontitis in Brazilian, Asian and Turkish populations. The IL-6 ?174 G/C polymorphism may confer susceptibility to periodontitis in Brazilians, and the IL-6 ?572 G/C polymorphism may be associated with susceptibility to periodontitis in Europeans, and chronic periodontitis.     

Competition and interplay between the lithium bonding and hydrogen bonding: R3C···HY···LiY and R3C···LiY···HY triads as a working model (R=H, CH3; Y=CN, NC)

UMP2 calculations with aug-cc-pVDZ basis set were used to analyze intermolecular interactions in R₃C···HY···LiY and R₃C···LiY···HY triads (R=H, CH₃; Y=CN, NC), which are connected via lithium and hydrogen bonds. To better understand the properties of these systems, the corresponding dyads were also studied. Molecular geometries and binding energies of dyads, and triads were investigated at the UMP2/aug-cc-pVDZ computational level. Particular attention was paid to parameters such as cooperative energies, and many-body interaction energies. All studied complexes, with the simultaneous presence of a lithium bond and a hydrogen bond, showed cooperativity with energy values ranging between ?1.71 and ?9.03 kJ mol^?1. The electronic properties of the complexes were analyzed using parameters derived from atoms in molecules (AIM) methodology. Energy decomposition analysis revealed that the electrostatic interactions are the major source of the attraction in the title complexes.     

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα_i free of Gβγ providing an unusual scaffold for the “G-switch” and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα_i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα_i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α₂-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα_i1-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα_i-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα_i and/or Gα_o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

The electronic structure of the C2H4O···2HF tri-molecular heterocyclic hydrogen-bonded complex: a theoretical study

The geometries of three isomers of the C₂H₄O···2HF tri-molecular heterocyclic hydrogen-bonded complex were examined through B3LYP/aug-cc-pVDZ calculations. Analysis of structural parameters, determination of CHELPG (charge electrostatic potential grid) intermolecular charge transfer, interpretation of infrared stretching modes, and Bader’s atoms in molecules (AIM) theory calculations was carried out in order to characterize the hydrogen bonds in each isomer of the C₂H₄O···2HF complex. The most stable structure was determined through the identification of hydrogen bonds between C₂H₄O and HF, (O···H), as well as in the hydrofluoric acid dimer, (HF^D–R···HF^D). However, the existence of a tertiary interaction (F^λ···H^α) between the fluoride of the second hydrofluoric acid and the axial hydrogen atoms of C₂H₄O was decisive in the identification of the preferred configuration of the C₂H₄O···2HF system. Figure Geometries of three isomers of the C₂H₄O···2HF tri-molecular heterocyclic hydrogen-bonded complex     

Why does the human factor IX gene have a G + C content of 40%?

The factor IX gene has a G + C content of approximately 40% in all mammalian species examined. In human factor IX, C----T and G----A transitions at the dinucleotide CpG are elevated at least 24-fold relative to other transitions. Can the G + C content be explained solely by this hot spot of mutation? Using our mathematical model, we show that the elevation of mutation at CpG cannot alone lower the G + C content below 45%. To search for other hot spots of mutation that might contribute to the reduction of G + C content, we assessed the relative rates of base substitution in our sample of 160 families with hemophilia B. Seventeen independent single-base substitutions are reported herein for a total of 96 independent point mutations in our sample. The following conclusions emerge from the analysis of our data and, where appropriate, the data of others: (1) Transversions at CpG are elevated an estimated 7.7-fold relative to other transversions. (2) The mutation rates at non-CpG dinucleotides are remarkably uniform; none of the observed rates are either more than twofold above the median for transitions or more than threefold above the median for transversions. (3) The pattern of recent mutation is compatible with the pattern during mammalian evolution that has maintained the G + C content of the factor IX gene at approximately 40%.     

C族G蛋白偶联受体激活过程

C族G蛋白偶联受体(G protein coupled receptor,GPCR)具有七螺旋跨膜域(heptahelical transmembranedomain,HD)、捕蝇夹域(venusflytrapdomain,VFT)和半胱氨酸富集域(cysteine-rich domain,CRD)等功能域,并在体内组成性形成二聚体。该文介绍C族G蛋白偶联受体激活进程中各功能域的构象变化,以及由此产生的构象学效应。     

Cα-H···O=C hydrogen bonds contribute to the specificity of RGD cell-adhesion interactions

Background  

The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.     

Survivin promoter ?31G/C (rs9904341) polymorphism and cancer susceptibility: a meta-analysis

This study aimed to perform a meta-analysis to assess the association of survivin −31 G/C promoter polymorphism and cancer risk. Thirteen case–control studies identified through PubMed and published between 2007 and 2011 with a total of 3329 cancer cases and 3979 controls were included in this meta-analysis. Odds ratio (OR) and 95% confidence interval (95% CI) were used to investigate the strength of the association. Overall, the pooled analysis showed that survivin −31C allele was associated with 1.27 fold increased risk of cancer compared with the −31G allele (95% CI = 1.091–1.479; random model). Subgroup analyses based on type of cancer and ethnicity were also performed, and results indicated that survivin −31G/C polymorphism was not associated with risk of gastric cancer [OR = 2.879; 95% CI = 0.553–15.004) for CC vs.GG] and esophageal cancer [OR = 1.352; 95% CI = 0.494–3.699) for CC vs.GG]. Stratification on the basis of ethnicity showed that the risk due to −31C allele was significant only in Asian population [OR = 1.894; 95% CI = 1.206–2.974 for CC vs.GG]. The present meta-analysis suggests an important role of survivin −31 G/C polymorphism with cancer risk especially in Asian population. However, further studies with larger sample size are required to draw more comprehensive conclusions and provide more precise evidence in individual cancers.     

Intramolecular C---H···O hydrogen-bond mediated stabilization of a cis-DPro imide-bond in a stereocontrolled heterochiral model peptide

The X-ray diffraction analysis of a stereocontrolled heterochiral designed model peptide Boc-(D) Pro-Thr-OMe (1) revealed the existence of an unusual folded molecular structure, stabilized via an effective unconventional C---H…O type intramolecular hydrogen-bond, encompassing a noncovalent 12-membered ring-motif. Together with an uncommon type a disposition of the urethane moiety, the tightly folded topology is compounded with a cis-(D) Pro imide-bond. The overall conformation is suggested to be the reminiscent of specific type VI β-turn structures, hitherto, characterized across the Aaa-cis-Pro peptide-bonds in globular proteins and polypeptides. The (13) C NMR spectrum of 1 in an apolar CDCl(3) environment revealed the presence of approximately an equal population of cis and trans isomers unexpectedly, analogous to Pro side-chain, the (13) C NMR chemical-shifts of Thr C(β) -resonance is observed to be sensitive toward cis-trans isomerization. In conjunction with solid-state FT-IR spectral data, we established that a network of complex intermolecular hydrogen-bonds stabilize a self-complementary noncovalent helical hexagonal self-assembly and crystallographic supramolecular aggregate. The results incline us to highlight that the stabilization of cis-(D) Pro peptide-bond in crystalline state may be driven by the favorable energy of formation of an unconventional weak C---H…O intramolecular hydrogen-bond.     

水牛染色体G、C带核型的观察

七十年代以来,由于染色体分带技术的发展,大大地促进了对哺乳动物染色体的研究。在水牛方面,近年来国外的研究时有报道,而国内甚少,尚无应用分带技术对染色体作进一步的分析。为了积累我国家畜染色体资料,探讨水牛种质以及为开展水牛遗传病的检查提供依据,我们对水牛的染色体核型和G、C带作了研究,现报告如下。     

A selection system to study C5a–C5a-receptor interactions: Phage display of a novel C5a anaphylatoxin,Fos-C5aAla27

Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a–C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFo-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt₂cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5a^Ala27. Both the purified and the phage displayed Fos-C5a^Ala27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5a^Ala27 exhibited the same binding profile as compared to rhC5a. Fos-C5a^Ala27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10⁶. After four successive rounds of panning on differentiated U937 cells Fos-C5a^Ala27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a–C5a-receptor interactions, i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.     

Hydrogen bonds determine the structures of the ternary heterocyclic complexes C₂H₄O···2HF, C₂H₅N···2HF and C₂H₄S···2HF: density functional theory and topological calculations

A theoretical study of structural, electronic, topological and vibrational parameters of the ternary hydrogen-bonded complexes C₂H₄O···2HF, C₂H₅N···2HF and C₂H₄S···2HF is presented here. Different from binary systems with a single proton donor, the tricomplexes have the property of forming multiple hydrogen bonds, which are analyzed from a structural and vibrational point of view, but verified only by means of the quantum theory of atoms in molecules (QTAIM). As traditionally done in the hydrogen bond theory, the charge transfer between proton donors and acceptors was computed using the CHELPG calculations, which also revealed agreement with dipole moment variation and a cooperative effect on the tricomplexes. Furthermore, redshift events on proton donor bonds were satisfactorily identified, although, in this case, an absence of experimental data led to the use of a theoretical argument to interpret these spectroscopic shifts. It was therefore the use of the QTAIM parameters that enabled all intermolecular vibrational modes to be validated. The most stable tricomplex in terms of energy was identified via the strength of the hydrogen bonds, which were modeled as directional and bifurcated.     

A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.     

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on G�� Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Gα subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Gα subunit is feasible and should constitute a new strategy for therapeutic intervention.G protein-coupled receptors (GPCRs)³ represent the largest superfamily of signaling proteins with a very high impact on drug discovery (1). Approximately 30% of the current drug targets are indeed GPCRs and these latter are involved in all major disease areas (2). The classical drug discovery process selects and optimizes compounds that interact selectively with a specific receptor (1), but recent reports show that certain critical conditions such as cancer (3) or pain (4) are driven by the concomitant activation of many different GPCRs (5). Novel therapeutic strategies could therefore emerge from the simultaneous blockade of the various GPCRs involved in such pathologies. The GPCR signaling downstream cascade triggers several protein/protein interactions that may be blocked or modulated by small molecules (6). Such protein/protein interactions involve the GPCR transmembrane domain and the heterotrimeric G protein complex, composed of an α subunit (Gα) and a βγ dimer (Gβγ), which interact sequentially with several partners (e.g. guanine nucleotides, effectors, and regulatory proteins) (7). This offers multiple possibilities to develop small molecules controlling heterotrimeric G protein signaling (6, 8, 9). For example, Higashijima et al. (10, 11) showed that Mastoparan, a peptide toxin from wasp venom, directly acts on G proteins to mimic the role played by the activated receptors. The anti-helminthic drug Suramin and some analogs represent a second class of compounds that directly interact with G proteins and interfere with nucleotide exchange (12–14). Small molecules modulating regulator of G protein signaling proteins have also been proposed for drug development (15). More recently, Bonacci et al. (16) have described fluorescein analogs that display central pain relief activity via binding to the Gβγ subunits. From our own group, we have reported in vivo inhibition of the GPCR signaling pathway by two closely related imidazopirazine containing small molecules, displaying potent antiproliferative activity (BIM-46174) (17) and potent pain relief activity (BIM-46187) (18).Here, we examined the molecular mechanisms underlying the biological activity of BIM-46187 with the various constituents of the GPCR signaling pathways. We report that this small molecule prevents GPCR-G protein signaling through a selective binding to the Gα protein subunit. Our results support the concept of targeting and inhibiting the heterotrimeric G protein complex as an approach to treat certain pathologies involving simultaneous activation of several GPCRs and/or heterotrimeric G proteins.     

Parallel-Stranded Oligonucleotides with Alternating d(A-isoG)/d(T·C) and d(A·G)/d(T·m5isoC) Sequences

Abstract

Parallel-stranded (ps) DNA hairpins with alternating d(A-isoG)/d(T·C) (designated as ps-t1) and d(A·G)/d(T·m⁵isoC) (ps-t2) sequences were studied by means of UV, CD and fluorescence spectroscopy. The thermostability of d(A·G)/d(T·m⁵isoC) sequence was close to that of aps d(G·A)/d(T·C). The stability of the ps d(A·isoG)/d(T·C) sequence was even higher than that of a related anti-parallel-stranded (aps) d(G·A)/d(T·C) sequence, being unique for ps DNAs studied so far.