Structure of the complex of heptakis(2,6-di-O-methyl)-beta-cyclodextrin with (2,4-dichlorophenoxy)acetic acid

The structure of the complex formed by heptakis(2,6-di-O-methyl)-beta-cyclodextrin and (2,4-dichlorophenoxy)acetic acid was studied by X-ray diffraction. The dichlorophenyl moiety of the guest molecule was found outside the host hydrophobic cavity in the primary methoxy groups region whereas the oxyacetic acid chain penetrates the cavity from the primary face. The host molecules stacks along the a crystal axis forming a column. In the space between three successive hosts of the column, a guest molecule is accommodated.     

Amphiphilic chitosan nanosphere: studies on formation, toxicity, and guest molecule incorporation

Amphiphilic chitosan developed by conjugating hydrophobic phthalimido groups and hydrophilic poly (ethylene glycol) chains gives a well-dispersed colloidal solution in polar solvents and shows a regular nano-sized spherical structure ( approximately 200 nm) with negatively charged surface (-31.24+/-4.85 mV). An in vivo acute oral toxicity test confirms the non-toxicity of the chitosan nanosphere with LD(50) higher than 2000 mg/kg body weight. A success of incorporating amine molecules into the nanosphere declares that the heterogeneous incorporation system is more effective than the homogeneous ones. The study on guest molecule incorporation using alkylamines and carboxylic acid as models suggests requirements that the guest be hydrophobic and positively charged. TGA study clarifies that the weight loss at 250-300 degrees C relating to the amount of guest incorporation via heterogeneous system is as high as 42.3%, whereas those of homogeneous systems are about 16.86-28.27%. The nanosphere size is significantly changed after guest incorporation, that is, from 200 nm to approximately 500-1000 nm.     

Host-guest properties of the trinuclear arene-ruthenium cluster cation [H3Ru3(C6H6)(C6Me6)2(O)]

The trinuclear arene-ruthenium cluster cation [H₃Ru₃(C₆H₆)(C₆Me₆)₂(O)]⁺, containing a μ₃-oxo cap and three arene ligands that span a hydrophobic pocket above the metal skeleton, has been crystallised as tetrafluoroborate salt in the presence of various guest molecules. The host-guest complexes have been characterised by single-crystal X-ray structure analysis. With chloroform as the guest molecule, a CHCl₃ molecule sits perfectly in the hydrophobic pocket, the hydrogen atom being encapsulated inside the cavity. When dioxane is added during the crystallisation process, the cluster forms infinite chains which are connected by a complex network of hydrogen bonds involving the μ₃-oxo ligand, water and dioxane molecules. Interestingly, in the presence of phenol, a water molecule is hydrogen-bonded between the μ₃-oxo ligand and the phenol molecule, forming a one-dimensional μ₃-O ? H₂O ? HO hydrogen-bonded chain. Finally, with benzoic acid, a head-to-tail host-guest chain is obtained, the phenyl ring being incorporated in the hydrophobic pocket, while the acid group is hydrogen-bonded to the μ₃-oxo ligand.     

The crystal structure of the 1:1 complex of beta-cyclodextrin with trans-cinnamic acid

The crystal structure of the 1:1 complex of beta-cyclodextrin (cyclomaltoheptaose) with trans-cinnamic acid was studied by X-ray diffraction. Two beta-cyclodextrin molecules related by a twofold crystal axis form dimers in the hydrophobic cavity of which, two guest molecules are entirely buried. The complex crystallizes in the monoclinic C2 space group with channel-type molecular packing. The oxygen atoms of the carboxylate group of the trans-cinnamic acid molecule form strong hydrogen bonds with two water molecules lying in the interdimeric space of the hydrophobic channel.     

Crystal structure of the dimeric beta-cyclodextrin complex with 1,12-dodecanediol

The structure of the complex of beta-cyclodextrin (beta-CD) with 1,12-dodecanediol has been determined at 173 K and refined to a final R=0.0615 based on 22,386 independent reflections. The complex crystallizes in the triclinic space group P1; with a=17.926(4), b=15.399(3), c=15.416(3) A, alpha=103.425(4), beta=113.404(4), gamma=98.858(4) degrees, D(c)=1.362 Mg cm(-3) and V=3651.4(13) A(3) for Z=1. One molecule of the diol is located as a guest in the hydrophobic cavity of a beta-CD-dimer, forming a [3]pseudorotaxane. The guest molecule shows a disorder over two positions. The hydroxyl groups of the diol emerge from the primary faces of the beta-CD dimer and form several hydrogen bonds with water molecules lying in the interstitial space, similarly to dimeric complexes of beta-CD with other alpha,omega-bifunctional guests.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Aggregate formation and radiolytic degradation of amphiphilic DNA block copolymer possessing disulfide bond

We have designed a novel aggregate of DNA block copolymer (DBC) that is sensitive to hypoxic X-irradiation. The DBC consists of tetrahydropyrane-protected 2-hydroxyethyl methacrylate as a hydrophobic unit and oligodeoxynucleotides as a hydrophilic unit, which are linked to a radiation-sensitive disulfide bond. The DBC self-assembled efficiently to form aggregates that encapsulated small molecules such as nile red and pyrene. Hypoxic X-irradiation could then induce reductive degradation of the DBC aggregates via an exchange reaction of the disulfide bond to release guest molecules.     

A label-free electrochemical DNA sensor based on exonuclease III-aided target recycling strategy for sequence-specific detection of femtomolar DNA

The present work demonstrates a rapid, single-step and ultrasensitive label-free and signal-off electrochemical sensor for specific DNA detection with excellent discrimination ability for single-nucleotide polymorphisms, taking advantage of Exonuclease III (Exo III)-aided target recycling strategy to achieve signal amplification. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity on the duplex DNAs with 3'-overhang and single-strand DNA is limited. In response to the specific features of Exo III, the proposed E-DNA sensor is designed such that, in the presence of target DNA, the electrode self-assembled signaling probe hybridizes with the target DNA to form a duplex in the form of a 3'-blunt end at signaling probe and a 3'-overhang end at target DNA. In this way, Exo III specifically recognizes this structure and selectively digests the signaling probe. As a result, the target DNA dissociates from the duplex and recycles to hybridize with a new signaling probe, leading to the digestion of a large amount of signaling probes gradually. A redox mediator, Ru(NH(3))(6)(3+) (RuHex) is employed to electrostatically adsorbed onto signaling probes, which is directly related to the amount and the length of the signaling probes remaining in the electrode, and provides a quantitative measure of sequence-specific DNA with the experimentally measured (not extrapolated) detection limit as low as 20 fM. Moreover, this E-DNA sensor has an excellent differentiation ability for single mismatches with fairly good stability.     

Tuning the binding of coumarin 6 with DNA by molecular encapsulators: effect of β‐cyclodextrin and C‐hexylpyrogallol[4]arene

We report in this paper that the binding of coumarin 6 (C6) to DNA can be tuned by complexing it with host structures, viz. β‐cyclodextrin (β‐CD) and C‐hexylpyrogallol‐4‐arene (C‐HPA). Because host molecules are used as carriers of small molecules onto target sites, the exposed part of the guest molecule needs to be found out, and the relationship between the host : guest ratio and the mode of binding with the target macromolecule, that is, the DNA needs to be analyzed, in order to comprehend the preferred binding moiety and tune the binding. In this paper, the formation of the inclusion complex of C6 with β‐CD and with C‐HPA is studied by UV‐visible, fluorescence, 2D rotating‐frame nuclear Overhauser effect correlation spectroscopy and diffusion‐ordered spectroscopy nuclear magnetic resonance spectra and molecular modeling. C6 forms a 1:1 complex with β‐CD and a 1:2 complex with C‐HPA. The studies on the protonation of C6 in the presence and the absence of the host molecules suggest that the chromone part of C6 is outside the β‐CD molecule, whereas it is fully covered by C‐HPA. The binding of C6 with calf thymus DNA (ctDNA) occurs through intercalation and hydrogen bonding, and the host–guest structures remain intact on binding with ctDNA. The oxygens of the C6 molecules are exposed when inside the host molecules and aid in the hydrogen bonding with DNA. Copyright © 2014 John Wiley & Sons, Ltd.     

Theoretical tests of the mechanical protection strategy in protein nanomechanics

We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single‐molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single‐molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force‐extension patterns of the guest protein. We study examples of such systems within a coarse‐grained structure‐based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force‐displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single‐molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure. Proteins 2014; 82:717–726. © 2014 Wiley Periodicals, Inc.     

Identification of a novel small molecule targeting UQCRB of mitochondrial complex III and its anti-angiogenic activity

Our recent study has shown that ubiquinol-cytochrome c reductase binding protein (UQCRB), the 13.4-kDa subunit of mitochondrial complex III, plays a crucial role in hypoxia-induced angiogenesis via mitochondrial reactive oxygen species (ROS)-mediated signaling. Here we report a new synthetic small molecule targeting the mitochondrial oxygen sensor UQCRB that was identified by pharmacophore-based virtual screening and in vitro and in vivo competition binding analyses. 6-((1-Hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-one (HDNT) binds to the hydrophobic pocket of UQCRB and potently inhibits in vitro angiogenesis of human umbilical vein endothelial cells without cytotoxicity. Furthermore, the binding of HDNT to UQCRB suppressed mitochondrial ROS-mediated hypoxic signal transduction. These results demonstrated that HDNT is a novel synthetic small molecule targeting UQCRB and exhibits anti-angiogenic activity by modulating the oxygen-sensing function of UQCRB.     

NMR diffusion as a novel tool for measuring the association constant between cyclodextrin and guest molecules

In this paper we introduce the use of diffusion measurements by nuclear magnetic resonance (NMR) spectroscopy for determining association constants of weak and very weak interactions between cyclodextrin and guest molecules, as long as both the free and complexed guest molecules are soluble to an extent that allows good sensitivity in the NMR experiment. The experimental setup and data analysis is discussed for three different guest molecules: L-phenylalanine, L-leucine and L-valine, representing different strengths of interaction. The underlying assumptions are discussed and the scope of the method (range of K(a) values, requirements to the guest molecule) are discussed. The method's main advantage is its general applicability independent of chromogenic or electrochemical properties of the guest molecule. Whereas calorimetric methods that exhibit a similar generality, are applicable mainly to strong interactions, NMR diffusion measurements are applicable to weaker interactions down to the theoretical limit of 1 M(-1), the upper limit for K(a) values to be determined by it is approximately 200. A further advantage of the method is the low amount of sample needed. The method is in principle applicable to any case of molecular recognition between a host and guest molecule leading to weak interactions.     

DNA疫苗抗原提呈的传感器分子研究新进展

DNA疫苗进入细胞后,除了转译成蛋白质抗原,通过MHC分子进行内源性或外源性抗原提呈外,近年来还发现可直接与相应的被称为核酸传感器分子,如TLR9、DAI、AIM2、STING、DDX41解旋酶和RNA聚合酶Ⅲ等结合,继而激活不同的免疫信号通路.基于DNA疫苗的传感器分子和信号通路研制免疫佐剂,可有效增强DNA疫苗的免疫原性.     

Nitric oxide as a developmental and metabolic signal in filamentous fungi

The short-lived hydrophobic gas nitric oxide (NO) is a broadly conserved signaling molecule in all domains of life, including the ubiquitous and versatile filamentous fungi (molds). Several studies have suggested that NO plays a vast and diverse signaling role in molds. In this review, we summarize NO-mediated signaling and the biosynthesis and degradation of NO in molds, and highlight the recent advances in understanding the NO-mediated regulation of morphological and physiological processes throughout the fungal life cycle. In particular, we describe the role of NO in molds as a signaling molecule that modulates asexual and sexual development, the formation of infection body appressorium, and the production of secondary metabolites (SMs). In addition, we also summarize NO detoxification and protective mechanisms against nitrooxidative stress.     

A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

Potential of enzyme mimics in biomimetic sensors: a modified-cyclodextrin as a dehydrogenase enzyme mimic

This paper reports the application of a dehydrogenase enzyme mimic as a biomimetic sensor. The model compound investigated was a beta-cyclodextrin (beta-CD) derivative with a nicotinamide group attached to the secondary face of a beta-CD (Fig. 1g). It was envisaged that the nicotinamide group would act as the electron transfer agent and that the cyclodextrin would provide a suitable hydrophobic cavity for the reaction to take place in. Ethanol, propranalol, dopamine and acetone were used as substrates in backgrounds of hydrophilic and hydrophobic anions. Electrochemical and fluorescence techniques were used to study the catalytic effects in solution. It was found that the size of the analyte and the hydrophobicity of the anion affected the catalytic activity of the dehydrogenase mimic. Catalytic effects were most enhanced with ethanol and dopamine in presence of larger and more strongly solvated anions, SO4(2-) and H2PO4- which are excluded from the cavity. The molecule was also immobilised in a sol-gel matrix and investigated as a sol-gel electrochemical biomimetic sensor. Concentration dependence with increasing aliquots of ethanol was observed. These results indicated that a re-usable biomimetic sensor is indeed feasible.     

The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity

The enhancement of a single strain DNA probe linked to the sensor surface is of crucial importance in DNA molecule recognition. By means of nanogold modification of the sensor surface in addition to the nanogold amplifier, DNA detection sensitivity higher than 10(-16)mol/L was obtained in a Quartz Crystal microbalance (QCM) system, much higher than the ordinary QCM sensor without surface modification by nanogold.     

A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe

This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modi?ed "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modi?ed "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase.     

One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface

We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. lambda DNA molecules were ligated with 3'-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified lambda DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified lambda DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.     

Insight into the denaturation transition of DNA

DNA undergoes a helix-to-coil transition (also called denaturation transition) upon heating. This transition can also be facilitated by using solvent mixtures (for example water–alcohol). An increase in the hydrophobic tail of the second solvent molecule first decreases then increases the melting temperature appreciably. Measurement on 4% DNA in a series of water–alcohol mixtures shows that the helix-to-coil melting transition is driven by the solvent ability to cross the hydrophobic sugar-rich region. DNA is behaving like a cylindrical micelle.