The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

IV型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

[背景] 部分细菌的DNA骨架会发生磷硫酰化修饰,硫结合结构域（Sulfur Binding Domain,SBD）可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同,ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶（5mC）修饰DNA的SET and RING-Associated （SRA）结构域。晶体结构显示,单独的SBD是单体,而SBD-SRA是双体。[目的] 探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。[方法] 凝胶迁移实验（Electrophoresis Mobility Shift Assay,EMSA）比较SBD、SBD-SRA对硫修饰DNA结合力的差异;对参与SBD-SRA二聚体化的关键氨基酸残基突变,并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。[结果] 相较于SBD结构域,SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合,当二聚体化界面连续的L₂₆₁LGET₂₆₅突变成A₂₆₁AAAA₂₆₅时,突变体对硫修饰DNA的结合力下降到与SBD相似的水平。[结论] 根据EMSA实验结果可以初步判断,SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力;L₂₆₁LGET₂₆₅是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。     

Impacts of hydrophobicity and ionicity of phendione-based cobalt(II)/(III) complexes on binding with bovine serum albumin

Abstract

For efficient designing of metallodrugs, it is imperative to analyse the binding affinity of those drugs with drug-carrying serum albumins to comprehend their structure–activity correlation for biomedical applications. Here, cobalt(II) and cobalt(III) complexes comprising three phendione ligands, [Co(phendione)₃]Cl₂ (1) and [Co(phendione)₃]Cl₃ (2), where, phendione = 1,10-phenanthroline-5,6-dione, has been chosen to contrast the impact of their hydrophobicity and ionicity on binding with bovine serum albumin (BSA) through spectrophotometric titrations. The attained hydrophobicity values using octanol/water partition coefficient method manifested that complex 1 is more hydrophobic than complex 2, which could be attributed to lesser charge on its coordination sphere. The interaction of complexes 1 and 2 with BSA using steady state fluorescence studies revealed that these complexes quench the intrinsic fluorescence of BSA through static mechanism, and the extent of quenching and binding parameters are higher for complex 2. Further thermodynamics of BSA-binding studies revealed that complexes 1 and 2 interact with BSA through hydrophobic and hydrogen bonding/van der Waals interactions, respectively. Further, UV–visible absorption, circular dichroism and synchronous fluorescence studies confirmed the occurrence of conformational and microenvironmental changes in BSA upon binding with complexes 1 and 2. Molecular docking studies have also shown that complex 2 has a higher binding affinity towards BSA as compared to complex 1. This sort of modification of ionicity and hydrophobicity of metal complexes for getting desirable binding mode/strength with drug transporting serum albumins will be a promising pathway for designing active and new kind of metallodrugs for various biomedical applications.

Communicated by Ramaswamy H. Sarma     

Cyclic DGR-peptidomimetic containing a bicyclic reverse turn inducer as a selective αvβ5 integrin ligand

3-Aza-6,8-dioxabicyclo[3.2.1]octane-based amino acids as reverse turn inducers have been introduced into cyclic peptidomimetics containing the RGD or DGR retro-sequence, in order to achieve a stereochemical scanning of the binding capability of the resulting molecules towards α_vβ₃ and α_vβ₅ integrins, resulting in retro-inverso DGR peptides as micromolar ligands. A comparative analysis between the conformational preferences of 4 and of its isomer 3, having the opposite RGD sequence, was reported with respect to the binding activity, giving insight into the factors affecting the preferential binding of 4 to the α_vβ₅ integrin.     

Synthesis of 3′-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists

On the basis of high binding affinity of 3′-aminoadenosine derivatives 2b at the human A₃ adenosine receptor (AR), 3′-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A₃AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A₃AR.     

Pharmacological evaluation of ocular β-adrenoceptors in rabbit by tissue segment binding method

AimsThis study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) β-adrenoceptors in rabbit to characterize the plasma membrane β-adrenoceptors and binding affinities of β-adrenoceptor antagonists.Main methodsThe tissue segment binding method with a hydrophilic radioligand (?)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-³H]benzimidazol-2-one ([³H]-CGP12177) was employed.Key findingsSpecific and saturable binding of [³H]-CGP12177 to intact tissue segments was detected by using (±)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective β₁- and β₂-adrenoceptor antagonists revealed a single population of β₂-adrenoceptors in ocular tissues and of β₁-adrenoceptors in atria, but mixed populations of β₁- and β₂-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for β₂-adrenoceptors was approximately 158-fold higher than for β₁-adrenoceptors, indicating the β₂-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (?)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [³H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.SignificanceThis study with tissue segment binding method suggests that the binding affinity of (?)-enantiomers of β-adrenoceptor antagonists for plasma membrane β-adrenoceptors (β₁-adrenoceptors of atria, β₂-adrenoceptors of ocular tissues, and mixed β₁-/β₂-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between β-adrenoceptor antagonists.     

New dinuclear copper(II) and zinc(II) complexes for the investigation of sugar–metal ion interactions

We have studied the binding interactions of biologically important carbohydrates (d-glucose, d-xylose and d-mannose) with the newly synthesized five-coordinate dinuclear copper(II) complex, [Cu₂(hpnbpda)(μ-OAc)] (1) and zinc(II) complex, [Zn₂(hpnbpda)(μ-OAc)] (2) [H₃hpnbpda = N,N′-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N′-diacetic acid] in aqueous alkaline solution. The complexes 1 and 2 are fully characterized both in solid and solution using different analytical techniques. A geometrical optimization was made of the ligand H₃hpnbpda and the complexes 1 and 2 by molecular mechanics (MM+) method in order to establish the stable conformations. All carbohydrates bind to the metal complexes in a 1:1 molar ratio. The binding events have been investigated by a combined approach of FTIR, UV–vis and ¹³C NMR spectroscopic techniques. UV–vis spectra indicate a significant blue shift of the absorption maximum of complex 1 during carbohydrate coordination highlighting the sugar binding ability of complex 1. The apparent binding constants of the substrate-bound copper(II) complexes have been determined from the UV–vis titration experiments. The binding ability and mode of binding of these sugar substrates with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in ¹³C NMR spectra for carbon atoms C1, C2, and C3 of sugar substrates.     

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

New binary copper(II) complexes containing intercalating ligands: DNA interactions,an unusual static quenching mechanism of BSA and cytotoxic activities

New binary copper(II) complexes – [Cu(4-mphen)₂(NO₃)]NO₃·H₂O (1), [Cu(5-mphen)₂ (NO₃)]NO₃·H₂O (2), the known complex [Cu(dmphen)₂(NO₃)]NO₃ (3) and [Cu(tmphen)₂ (NO₃)]NO₃·H₂O (4) - (4-mphen: 4-methyl-1,10-phenanthroline, 5-mphen: 5-methyl-1,10-phenanthroline, dmphen: 4,7-dimethyl-1,10-phenanthroline, tmphen: 3,4,7,8-tetramethyl-1,10-phenanthroline), have been synthesized and characterized by CHN analysis, ESI-MS, FTIR and single-crystal X-ray diffraction techniques. Interaction of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectral titration, ethidium bromide (EB) and Hoechst 33,258 displacement assay and thermal denaturation measurement. These complexes cleaved pUC19 plasmid DNA in the absence and presence of an external agent. Notably, in the presence of H₂O₂ as an activator, the cleavage abilities of these complexes are obviously enhanced at low concentration. Addition of hydroxyl radical scavengers like DMSO shows significant inhibition of the DNA cleavage activity of these complexes. BSA quenching mechanism was investigated with regard to the type of quenching, binding constant, number of binding locations and the thermodynamic parameters. The experimental results suggested that the probable quenching mechanism was an unusual static process and hydrophobic forces play a dominant role. The CT-DNA and BSA binding efficiencies of these complexes follow the order: 4 > 3 > 1 > 2. Furthermore, in vitro cytotoxicities of these complexes on tumor cells lines (Caco-2, MCF-7 and A549) and healthy cell line (BEAS-2B) showed that these complexes exhibited anticancer activity with low IC₅₀ values. The effect of hydrophobicity of the methyl-substituted phenanthrolines on DNA and protein binding activities of these complexes is discussed.     

Honokiol and Magnolol Increase the Number of [3H]Muscimol Binding Sites Three-Fold in Rat Forebrain Membranes In Vitro Using a Filtration Assay,by Allosterically Increasing the Affinities of Low-Affinity Sites

1. The bark of the root and stem of various Magnolia species has been used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. The biphenolic compounds honokiol (H) and magnolol (M), the main components of the Chinese medicinal plant Magnolia officinalis, interact with GABA_A receptors in rat brain in vitro. We compared the effects of H and M on [³H]muscimol (MUS) and [³H]flunitrazepam (FNM) binding using EDTA/water dialyzed rat brain membranes in a buffer containing 150 mM NaCl plus 5 mM Tris-HCl, pH 7.5 as well as [³⁵S]t-butylbicyclophosphorothionate (TBPS) in 200 mM KBr plus 5 mM Tris-HCl, pH 7.5. H and M had similar enhancing effects on [³H]MUS as well as on [³H]FNM binding to rat brain membrane preparations, but H was 2.5 to 5.2 times more potent than M. 2. [ ³ H]FNM binding. GABA alone almost doubled [³H]FNM binding with EC₅₀ = 450 nM and 200 nM using forebrain and cerebellar membranes, respectively. In the presence of 5 M H or M the EC₅₀ values for GABA were decreased to 79 and 89 nM, respectively, using forebrain, and 39 and 78 nM, using cerebellar membranes. H and M potently enhanced the potentiating effect of 200 nM GABA on [³H]FNM binding with EC₅₀ values of 0.61 M and 1.6 M using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 M, and 22 and 34%. 3. [ ³ H]MUS binding. H and M increased [³H]MUS binding to whole forebrain membranes about 3-fold with EC₅₀ values of 6.0 and 15 M. Using cerebellar membranes, H and M increased [³H]MUS binding ~68% with EC₅₀ values of 2.3 and 12 M, respectively. Scatchard analysis revealed that the enhancements of [³H]MUS binding were due primarily to increases in the number of binding sites (B_max values) with no effect on the high affinity binding constants (K_d values). The enhancing effect of H and M were not additive. 4. [ ³⁵ S]TBPS binding. H and M displaced [³⁵S]TBPS binding from sites on whole rat forebrain membranes with IC₅₀ values of 7.8 and 6.0 M, respectively. Using cerebellar membranes, the corresponding IC₅₀ values were 5.3 and 4.8 M. These inhibitory effects were reversed by the potent GABA_A receptor blocker R5135 (10 nM), suggesting that H and M allosterically increase the affinity of GABA_A receptors for GABA and MUS by binding to sites in GABA_A receptor complexes. 5. Two monophenols, the anesthetic propofol (2,6-diisopropylphenol, P) and the anti-inflammatory diflunisal (2,4-difluoro-4-hydroxy-3-biphenyl carboxylic acid, D) also enhanced [³H]MUS binding, decreased the EC₅₀ values for GABA in enhancing [³H]FNM binding and potentiated the enhancing effect of 200 nM GABA on [³H]FNM binding, although enhancements of [³H]MUS binding for these monophenols were smaller than those for H and M, using forebrain and cerebellar membranes. The enhancing effect of P and D on [³H]MUS binding were almost completely additive. 2,2-biphenol was inactive on [³H]MUS and [³H]FNM binding. These, and other preliminary experiments, suggest that appropriate ortho (C2) and para (C4) substitution increases the GABA-potentiating activity of phenols. 6. The potentiation of GABAergic neurotransmission by H and M is probably involved in their previously reported anxiolytic and central depressant effects.     

Synthesis,DNA Binding,and Oxidative Cleavage Studies of Fe(II) and Co(III) Complexes Containing Bioactive Ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF₆)₂ (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L₁L₂)] (PF₆)₃ (2) (where L₁L₂ = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K_b has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10⁵ M^?1 for (1) and 4.8 × 10⁵ M^?1 for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Structure-activity relationship on DNA binding and anticancer activities of a family of mixed-ligand oxidovanadium(V) hydrazone complexes

The title family of mixed-ligand oxidovanadium(V) hydrazone complexes are [V^VO(HL¹)(hq)] (1) and [V^VO(HL²)(hq)] (2), where (HL¹)^2? and (HL²)^2? are the dinegative form of 2-hydroxybenzoylhydrazone of acetylacetone (H₃L¹) and benzoylacetone (H₃L²), respectively, and hq^? is the mononegative form of 8-hydroxyquinoline (Hhq). Complexes were used to determine their binding constant with CT DNA using various spectroscopic techniques namely, electronic absorption, fluorescence and circular dichroism spectroscopy. The binding constant values suggest the intercalative mode of binding with the CT DNA and follow the order: 2 > 1. The bulky size as well as electron withdrawing property of the phenyl group (which is present in the β-diketone part of the hydrazone moiety in complex 2 in place of a CH₃ group in complex 1) is responsible for the higher activity of 2 than 1. Complexes were screened for cytotoxic activity on cervical cancer cells and were found to be potentially active (IC₅₀ value for 1 and 2 is 33 and 29 μM, respectively), even better than the widely used cis-platin (IC₅₀ = 63.5 μM) and carboplatin (IC₅₀ = > 200 μM) which is evident from the respective IC₅₀ value. Nuclear staining experiment suggests that these complexes kill the SiHa cancer cells through apoptotic mode. The molecular docking study also suggested the intercalative mode of binding of these complexes with CT DNA and HPV 18 DNA.     

Effect of DNA Local Conformation on the Affinity and Binding Specificity of bis-Netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked tail-to-tail via cis-diammineplatinum group (Nt–Pt(NH ₃ )–Nt) or aliphatic pentamethylene chain (Nt–(CH ₂ ) ₅ –Nt), has been studied. Both bis-netropsins have been shown to bind to DNA oligomer 5"-CCTATATCC-3" (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5"-CCXCC-3" [where X = TTATT (II), TTAT (III), TTTTT (IV), and AATTT (V)] along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of Nt–(CH ₂ ) ₅ –Nt with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin ( ₂) to monodentate binding constant ( ₁) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for Nt–Pt(NH ₃ )–Nt and Nt–(CH ₂ ) ₅ –Nt binding as a hairpin has been found to be DNA duplex 5"-CGTATACG-3".     

Synthesis and binding assays of novel 3,3-dimethylpiperidine derivatives with various lipophilicities as σ1 receptor ligands

Starting from two carbocyclic analogs, a series of 3,3-dimethylpiperidine derivatives was prepared and tested in radioligand binding assays at σ₁ and σ₂ receptors, and at Δ₈–Δ₇ sterol isomerase (SI) site. The novel compounds mostly bear heterocyclic rings or bicyclic nucleus of differing lipophilicities. Compounds 18a and 19a,b demonstrated the highest σ₁ affinity (K_i = 0.14–0.38 nM) with a good selectivity versus σ₂ binding. Among them, 18a had the lowest C log D value (3.01) and only 19b was selective versus SI too. Generally, it was observed that more planar and hydrophilic heteronuclei conferred a decrease in affinity for both σ receptor subtypes.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Molecular docking,a tool to determine interaction of CuO and TiO2 nanoparticles with human serum albumin

BackgroundWe study the human serum albumin (HSA) protein-CuO nanoparticle interaction to identify the specific binding site of protein with CuO nanoparticles by molecular docking and compared it with HSA-TiO₂ nanoparticle interaction.MethodsThe protein structural data that was obtained using Autodock 4.2.ResultsIn case of CuO np-HSA interaction, the distances from the centre of Subdomain IIIA to Arg-472 is 2.113 Å and Lys 475, Glu 492, Ala 490, Cys 487, Ala 490 are the bound neighbouring residues with Lys 475, Glu 492 at aliphatic region. The binding energy generated was ?1.64 kcal mol^?1. However, for TiO₂ nanoparticle, the binding region is surrounded by Arg 257, Ala 258, Ser 287, His 288, Leu 283, Ala 254, Tyr 150 (subdomain II A) as neighbouring residue. Moreover, Glu 285, Lys 286 forms aliphatic grove for TiO₂-HSA, Ser-287 at the centre region form hydrogen bond with nanoparticle and Leu 283, Leu 284 forming hydrophopobic grove for TiO₂ nanoparticle-HSA interaction. The binding energy generated was ?2.47 kcal mol^?1.ConclusionsAnalysis suggests that CuO bind to suldow site II i.e subdomain III A of HSA protein where as TiO₂ nanoparticle bind to suldow site I i.e subdomain IIA of HSA protein.General significanceThe structural information that derives from this study for CuO and TiO₂ nanoparticles may be useful in terms of both high and low-affinity binding sites when designing these nanoparticles based drugs delivery system.     

Synthesis,characterization, DNA and HSA binding studies of isomeric Pd (II) antitumor complexes using spectrophotometry techniques

Two new Palladium(II) isomeric complexes, [Pd (Gly)(Leu)](I) and [Pd (Gly)(Ile)](II), where Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT-IR, ¹H NMR, and UV–Vis. The complexes have been tested for their In vitro cytotoxicity against cancer cell line K₅₆₂ and their binding properties to calf thymus DNA (CT-DNA) and human serum albumin (HSA) have also been investigated by multispectroscopic techniques. Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The energy transfer from HSA to these complexes and the binding distance between HSA and the complexes (r) were calculated. The results obtained from these studies indicated that at very low concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant (K_b), apparent biomolecular quenching constant (k_q), and number of binding sites (n) for CT-DNA and HSA were calculated using Stern–Volmer equation. The calculated thermodynamic parameters indicated that the hydrogen binding and vander Waals forces might play a major role in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of I > II with DNA- and II > I with HSA-binding.     

Antibacterial combination therapy using Co3+, Cu2+, Zn2+ and Pd2+ complexes: Their calf thymus DNA binding studies

Four Co(III)-, Cu(II)-, Zn(II)-, and Pd(II)-based potent antibacterial complexes of formula K₃[Co(ox)₃].3H₂O (I), [Cu(bpy)₂Cl]Cl.5H₂O (II), [Zn(bpy)₃]Cl₂ (III), and [Pd(bpy)₂](NO₃)₂ (IV) (where ox is oxalate and bpy is 2,2′-bipyridine) were synthesized. They were characterized by elemental analyses, molar conductance measurements, UV–Vis, FTIR, ¹H NMR, and ¹³C NMR spectra. These metal complexes were ordered in three combination series of I + II, I + II + III, and I + II + III + IV. Antibacterial activity was tested for each of these four metal complexes and their combinations against Gram-positive and Gram-negative bacteria. All compounds were more potent antibacterial agents against the Gram-negative than those of the Gram-positive bacteria. The four metal complexes showed antibacterial activity in the order I > II > III > IV and the activity of their combinations followed the order of I + II + III + IV > I + II + III > I + II. CT-DNA binding studies of complex I and its three combinations were carried out using UV–vis spectral titration, displacement of ethidium bromide (EB), and electrophoretic mobility assay. The results obtained from UV–vis studies indicated that all series interact effectively with CT-DNA. Fluorescence titration revealed that the complexes quench DNA-EB strongly through the static quenching procedures. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) were determined at different temperatures of 293, 300, and 310 K, respectively. The calculated thermodynamic parameters supported that hydrogen binding and Van der Waals forces play a major role in association of each series of metal complexes with CT-DNA and follow the above-binding affinity order for the series.     

谷氨酸棒杆菌anti-σ因子CseE及其突变体与σ因子SigE的相互作用分析

[目的]谷氨酸棒杆菌是重要的氨基酸生产菌株,本研究针对SigE与ZAS家族蛋白CseE相互作用机制进行探索研究,重点分析CseE突变体影响与SigE结合能力的机制。[方法]本研究选择谷氨酸棒杆菌ATCC 13032来源的SigE和CseE蛋白为研究目标,利用遗传学方法获得过表达的重组谷氨酸棒杆菌,通过RT-qPCR研究SigE调控sigE和cseE的转录情况。同时,利用ITC和His pull-down实验验证ZAS家族的CseE蛋白与Zn²⁺及SigE的结合情况。之后对CseE蛋白进行功能域分析、多序列比对,研究功能域关键氨基酸位点对SigE结合能力的影响。其次对SigE和CseE蛋白进行分子对接和动力学模拟,分析关键氨基酸影响其结合的机制。[结果]谷氨酸棒杆菌SigE调控基因sigE和cseE的转录并且其活性受CseE蛋白控制。CseE蛋白为ZAS家族蛋白,具有Zn²⁺结合能力。CseE_His83A、CseE_cys87A和CseE_cys90A突变体不会影响与SigE的结合能力,而CseE_C87A-C90A和CseE_{His83A-C87A-C90A}突变体与SigE的结合能力略有下降。分子动力学模拟发现SigE-CseE_C87A-C90A和SigE-CseE_{His83A-C87A-C90A}之间的结合能量为-17.23 kcal/mol和-14.06 kcal/mol,分别比未突变体系结合能量降低22.8%及36.9%。[结论]谷氨酸棒杆菌SigE通过聚集RNA聚合酶来调控基因sigE和cseE的表达。CseE蛋白属于ZAS家族,具有Zn²⁺结合能力同时通过与SigE蛋白互作来抑制SigE活性。CseE_C87A-C90A及CseE_{His83A-C87A-C90A}突变体能影响与SigE结合的能力,减弱对SigE活性的控制。本研究产生的三维结构和确定的氨基酸关键位点为后续探索谷氨酸棒杆菌SigE和CseE响应环境压力机制提供了理论基础。     

Dynamic Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Evidence of Intercalator Creeping

Abstract

We have used one and two dimensional exchange ¹H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1′-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)₂ ( 1 ) and d(CGCTAGCTAGCG)₂ ( 2 ). TOTO binds preferentially to the (5′-CTAG-3′)₂ sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1 -TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2 -TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process (“creeping”) along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.