Interplay of mechanical and binding properties of Fibronectin type I

Fibronectins (FNs) are a major component of the extracellular matrix (ECM), and provide important binding sites for a variety of ligands outside and on the surface of the cell. Similar to other ECM proteins, FNs are consistently subject to mechanical stress in the ECM. Therefore, it is important to study their structure and binding properties under mechanical stress and understand how their binding and mechanical properties might affect each other. Although certain FN modules have been extensively investigated, no simulation studies have been reported for the FN type I (Fn1) domains, despite their prominent role in binding of various protein modules to FN polymers in the ECM. Using equilibrium and steered molecular dynamics simulations, we have studied mechanical properties of Fn1 modules in the presence or the absence of a specific FN-binding peptide (FnBP). We have also investigated how the binding of the FnBP peptide to Fn1 might be affected by tensile force. Despite the presence of disulfide bonds within individual Fn1 modules that are presumed to prevent their extension, it is found that significant internal structural changes within individual modules are induced by the forces applied in our simulations. These internal structural changes result in significant variations in the accessibility of different residues of the Fn1 modules, which affect their exposure, and, thus, the binding properties of the Fn1 modules. Binding of the FnBP appears to reduce the flexibility of the linker region connecting individual Fn1 modules (exhibited in the form of reduced fluctuation and motion of the linker region), both with regard to bending and stretching motions, and hence stabilizes the inter-domain configuration under force. Under large tensile forces, the FnBP peptide unbinds from Fn1. The results suggest that Fn1 modules in FN polymers do contribute to the overall extension caused by force-induced stretching of the polymer in the ECM, and that binding properties of Fn1 modules can be affected by mechanically induced internal protein conformational changes in spite of the presence of disulfide bonds which were presumed to completely abolish the capacity of Fn1 modules to undergo extension in response to external forces.     

Sensitive determination of amide herbicides in environmental water samples by a combination of solid‐phase extraction and dispersive liquid–liquid microextraction prior to GC–MS

In this paper, solid‐phase extraction (SPE) in combination with dispersive liquid–liquid microextraction (DLLME) has been developed as a sample pretreatment method with high enrichment factors for the sensitive determination of amide herbicides in water samples. In SPE–DLLME, amide herbicides were adsorbed quantitatively from a large volume of aqueous samples (100 mL) onto a multiwalled carbon nanotube adsorbent (100 mg). After elution of the target compounds from the adsorbent with acetone, the DLLME technique was performed on the resulting solution. Finally, the analytes in the extraction solvent were determined by gas chromatography–mass spectrometry. Some important extraction parameters, such as flow rate of sample, breakthrough volume, sample pH, type and volume of the elution solvent, as well as salt addition, were studied and optimized in detail. Under optimum conditions, high enrichment factors ranging from 6593 to 7873 were achieved in less than 10 min. There was linearity over the range of 0.01–10 μg/L with relative standard deviations of 2.6–8.7%. The limits of detection ranged from 0.002 to 0.006 μg/L. The proposed method was used for the analysis of water samples, and satisfactory results were achieved.     

Non‐B DNA Structures Show Diverse Conformations and Complex Transition Kinetics Comparable to RNA or Proteins—A Perspective from Mechanical Unfolding and Refolding Experiments

With the firm demonstration of the in vivo presence and biological functions of many non‐B DNA structures, it is of great significance to understand their physiological roles from the perspective of structural conformation, stability, and transition kinetics. Although relatively simple in primary sequences compared to proteins, non‐B DNA species show rather versatile conformations and dynamic transitions. As the most‐studied non‐B DNA species, the G‐quadruplex displays a myriad of conformations that can interconvert between each other in different solutions. These features impose challenges for ensemble‐average techniques, such as X‐ray crystallography, NMR spectroscopy, and circular dichroism (CD), but leave room for single‐molecular approaches to illustrate the structure, stability, and transition kinetics of individual non‐B DNA species in a solution mixture. Deconvolution of the mixture can be further facilitated by statistical data treatment, such as iPoDNano (i ntegrated po pulation d econvolution with nano meter resolution), which resolves populations with subnanometer size differences. This Personal Account summarizes current mechanical unfolding and refolding methods to interrogate single non‐B DNA species, with an emphasis on DNA G‐quadruplexes and i‐motifs. These single‐molecule studies start to demonstrate that structures and transitions in non‐B DNA species can approach the complexity of those in RNA or proteins, which provides solid justification for the biological functions carried out by non‐B DNA species.     

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.     

喜树碱与胰蛋白酶的相互作用

喜树碱(camptothecin,CPT)是从珙桐科乔木喜树中分离得到的一类重要抗癌药物,对动物肿瘤及白血病均有明显的抑制作用[1]。CPT分子为五环结构,含有一个吡咯[3,4-b]喹啉环,一个共轭吡啶环和一个α-羟基六元内脂环[2]。CPT通过嵌合抑制DNA拓扑异构酶Ⅰ的活性,对卵巢癌、结肠直肠癌     

Radiolabeling of EGCG with 125I and its biodistribution in mice

The aim of the present study was to label EGCG with ¹²⁵I and to determine its radiopharmaceutical potential in mice. EGCG was labeled with ¹²⁵I using the iodogen method. The labeling yield and the radiochemical purity of ¹²⁵I–EGCG were determined by radio thin-layer chromatography (RTLC). The Labeling yield was approximately 89.4 %. The radiochemical purity was approximately 96.4 %. The biodistribution studies of the labeled compound (specific activity; 0.47 TBq/μg) were performed in male Kunming mice. The uptakes of ¹²⁵I–EGCG in some organs were determined at different time after injection to the mice. The radioactivity in each organ was counted and the percentage of injected activity per gram of tissue weight (%ID/g) for each organ and blood was calculated. Incorporation of radioactivity in the various tissue/organ was confirmed by microautoradiography. ¹²⁵I–EGCG uptake in the stomach and salivary gland was higher than other organ/tissue. The black silver grains was concentrated in the nucleus, cytoplasm, intercellular substance and capillaries of that various organs, and its unevenly distributed. Thus, ¹²⁵I–EGCG may be radiopharmaceutical for the imaging of the stomach and salivary gland.     

Fractional coverage of defects in self-assembled thiol monolayers on gold

Au electrodes are alkylated by self-assembled organic monolayers of octadecanethiol from alcohol solution. The electron tunnelling resistance of a monolayer-coated gold electrode has been investigated by ac impedance. The relation between the fractional coverage of different defects and the corresponding film thickness at these ‘collapsed’ sites has been deduced from electron tunnelling theory. By using the concepts of average film thickness at defect (d_a) and average fractional coverage of defect (θ_a), we have obtained the θ_ad_a plot. The influence of the apparent standard rate constant on the shape of the θ_ad_a plot has been discussed. In our experiments, Fe(CN)₆^3−/4− is used as a redox probe to study the θ_ad_a plot of an octadecanethiol monolayer. The θ_a versus d_a plot indicates that the defects with d_a<6 methylene groups and θ_a<0.1 can increase the apparent standard rate constant from 1.9×10⁻¹⁰ cm s⁻¹, which is the theoretical value calculated from electron tunnelling theory, to 2.9×10⁻⁷ cm s⁻¹. The average thickness of the whole monolayer (ATWM), which is obtained from the θ_a versus d_a plot and which can indicate the blocking property of the monolayer, is 11 methylene groups.     

Cationic recognition by tert-butylcalix[4]arene-functionalized nanoprobes

A nanoparticle-based strategy has been demonstrated using structurally-tailored tert-butylcalixarenes immobilized on gold nanoparticles to tune the guest access to the calixarene cone cavity for cationic recognition. This strategy exploits the interparticle charge-induced aggregation upon selective capture of metal cations into the nanoparticle-immobilized tert-butylcalixarenes, which produces calorimetric changes for the detection. A possible pathway for the binding of M(n+) into the t-BCA structure and the interparticle interaction is proposed for the formation of an electric double layer inducing the interparticle association responsible for the red-shifted surface plasmon resonance band of the nanoparticles. The value of this class of calorimetric nanoprobes will be in the area of designing advanced host-guest probes using a variety of calixarene ligands for ionic recognition in a simplistic detection format.     

Complexation thermodynamics between butyl rhodamine B and calix[n]arenesulfonates (n = 4, 6, 8)

The characteristics of host–guest complexation between water-soluble calix[n]arenesulfonates (CnS, n = 4, 6, 8) and butyl rhodamine B (BRB) were investigated by fluorescence spectrometry. Fluorescence spectroscopy experiments were performed in pH 8.0 Tris(3-aminomethane)–HCl buffer solution at different temperature to calculate the stability constants (K_S) for the stoichiometric 1:1 inclusion complexes of C4S, C6S, and C8S with BRB. The complex stability constant monotonically increased with the number of phenolic units in the calixarene ring. The thermodynamic parameters at T = 298 K for the inclusion complexes were calculated through Van’t Hoff analysis. The inclusion complexes of CnS with BRB were driven by the favorable enthalpic changes, accompanying negative entropy changes. The stability constants were affected by the acidity of the solution. When pH was 8.0, the stability constants reached the maximum. The complex interaction was mainly attributed to the weak forces including electrostatic interaction and hydrogen bonding.