Unusual monomolecular DNA quadruplex structures using bunch-oligonucleotides

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I-IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.     

Monomolecular condensation of lambda-DNA induced by cobalt hexamine

Measurements of static and dynamic light scattering have been used to distinguish between monomolecular DNA condensation and aggregation of condensed molecules. In low salt, using Co³⁺(NH₃)₆ as the condensing agent, and at λ-DNA concentrations below 0.2 μg/mL, the transition curves for monomolecular condensation and aggregation are well separated for times of 16 h. In these conditions, the intensity of scattered light (90°) and also the diffusion coefficient of the condensed DNA show reasonable values for monomolecular condensation that are independent of DNA concentration and also of Na⁺ Co³⁺(NH₃)₆ concentrations for which monomolecular condensation is complete. At higher Co³⁺(NH₃)₆ concentrations, which produce aggregation (as judged by the intensity of scattered light), the diffusion coefficient decreases sharply. The transition curve for monomolecular condensation is independent of DNA concentration but shows a hysteresis loop. The kinetics of condensation are slow in the forward direction and fast in the reverse direction, indicating that the actual transition curve is measured closely by reversal experiments. Aggregation is blocked kinetically in both the forward and reverse directions when Co³⁺(NH₃)₆ is the condensing agent at low Na⁺ concentrations. When spermine or spermidine is the condensing agent and observations are made at 16 h, it is not possible to separate the transition curves for monomolecular condensation and for aggregation in conditions that are successful with Co³⁺(NH₃)₆. Some interesting properties of monomolecular condensation are noted. (1) The transition is not a two-state reaction, as judged by measurements of the diffusion coefficient through the transition zone. (2) The transition for monomolecular condensation is diffuse. (3) The dimensions of the monomolecular condensates have been calculated from the translational diffusion coefficient for an assumed toroidal shape by the formula derived by Allison and coworkers [(1981) Biopolymers 20 , 469–488]. These dimensions are in reasonable agreement with ones deduced from electron microscopy by Chattoraj and coworkers [(1978) J. Mol. Biol. 121 , 327–337]. (4) The phase diagram relating the Na⁺ to the Co³⁺(NH₃)₆ concentrations needed for condensation has a slope of 0.6 in a log–log plot. According to numerical solutions of Manning's theory for the atmospheric binding of competing cations to DNA, this means that condensation occurs at a late stage in the replacement of Na⁺ by Co³⁺(NH₃)₆ around the DNA. The fraction of DNA phosphate charge neutralized at condensation is computed to be in the neighborhood of 0.9, as found by Wilson and Bloomfield [(1979) Biochemistry 18 , 2192–2196], but to vary with the Na⁺ concentration.     

趋化因子CCL11-糖胺聚糖-趋化因子受体CCR3的相互作用研究

目的 探究趋化因子CCL11与受体CCR3、糖胺聚糖（glycosaminoglycans，GAGs）相互作用过程及机制，为深入阐明CCL11-GAGs-CCR3相互作用关系提供理论参考。方法 利用基因工程技术，构建筛选了CCR3-EGFP单分子表达水平的CHO稳转细胞系，利用全内反射荧光成像（total internal reflection fluorescence，TIRF）与等温滴定量热（isothermal titration calorimetry，ITC）技术研究了不同体外溶液条件下GAGs与CCL11的相互作用，并利用趋化实验及活细胞单分子成像实验考察了GAGs-CCL11对CCR3-EGFP稳转细胞趋化行为的调控及CCR3-EGFP在细胞膜上聚集状态的影响。结果 随着硫酸软骨素链长度的增加，其与CCL11结合放热增多，表明其相互作用力增强，其促进CCL11聚集作用增强。单分子荧光成像技术结合趋化试验研究发现，不同种类及不同比例的GAGs均会影响CCL11与CCR3的相互作用，GAGs的加入，抑制了CCL11对CCR3-EGFP稳转细胞的趋化效应及促CCR3-EGFP聚集的能力，且随着硫酸软骨素分子质量的增加，抑制作用显著增强。结论 GAGs的存在可以显著调控CCL11的聚集状态，进而影响其与受体CCR3的相互作用，本研究为进一步阐明CCL11-GAGs-CCR3相互作用关系提供了一定的实验基础。     

Synthesis and characterization of tetra-end linked oligonucleotides capable of forming monomolecular G-quadruplexes

The chemical synthesis of two new G-rich Tetra-End-Linked-oligodeoxyribonucleotides (TEL-ODNs) as well as (1)H-NMR and CD spectra of the corresponding monomolecular quadruplexes (IVa and IVb) has been reported. The new TEL-ODNs, characterized by the presence of short branches in the linker moiety, could be very useful for the achievement of monomolecular quadruplexes with predetermined strand orientation.     

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles,monomolecular films and UV-absorbing surface-coated substrate

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10?ng of enzymes, against 100?ng to 10?μg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.     

THE RATE OF GROWTH OF THE DAIRY COW : IV. GROWTH AND SENESCENCE AS MEASURED BY THE RISE AND FALL OF MILK SECRETION WITH AGE.

Data are presented on the effect of age on milk secretion in the dairy cow. From the age when milk secretion usually begins (2 years) to the age when maximum body weight is reached (about 8 years) increase of milk secretion and increase of body weight with age follow the same exponential course, which is the course of a monomolecular reaction of chemistry. After this age, unlike body weight which remains practically constant, milk secretion declines exponentially, that is, the course of decline follows the course of decline of a monomolecular reaction. The whole course of milk secretion with age was therefore found to follow approximately the course of two simultaneous, consecutive, monomolecular reactions. This is taken to mean that growth and senescence go on simultaneously from the beginning to the end of life, and that each follows an exponential law with age; and therefore perhaps that the course of the two processes are limited by two consecutive chemical reactions.     

Exact Probability Distributions of Selected Species in Stochastic Chemical Reaction Networks

Chemical reactions are discrete, stochastic events. As such, the species’ molecular numbers can be described by an associated master equation. However, handling such an equation may become difficult due to the large size of reaction networks. A commonly used approach to forecast the behaviour of reaction networks is to perform computational simulations of such systems and analyse their outcome statistically. This approach, however, might require high computational costs to provide accurate results. In this paper we opt for an analytical approach to obtain the time-dependent solution of the Chemical Master Equation for selected species in a general reaction network. When the reaction networks are composed exclusively of zeroth and first-order reactions, this analytical approach significantly alleviates the computational burden required by simulation-based methods. By building upon these analytical solutions, we analyse a general monomolecular reaction network with an arbitrary number of species to obtain the exact marginal probability distribution for selected species. Additionally, we study two particular topologies of monomolecular reaction networks, namely (i) an unbranched chain of monomolecular reactions with and without synthesis and degradation reactions and (ii) a circular chain of monomolecular reactions. We illustrate our methodology and alternative ways to use it for non-linear systems by analysing a protein autoactivation mechanism. Later, we compare the computational load required for the implementation of our results and a pure computational approach to analyse an unbranched chain of monomolecular reactions. Finally, we study calcium ions gates in the sarco/endoplasmic reticulum mediated by ryanodine receptors.     

A comparative kinetic study on human pancreatic and Thermomyces lanuginosa lipases: Inhibitory effects of tetrahydrolipstatin in the presence of lipid substrates

The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400 000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.     

Monomolecular surface film and tubular myelin figures of the pulmonary surfactant in hamster lung

Summary Perfusion fixation via pulmonary trunk was applied to the alveolar lining layer in situ at different lung volumes using a fixative containing tannic acid-ferrocyanide osmium. The monomolecular surface film and hypophasic tubular myelin figures were enhanced. In the range of transpulmonary pressure (1–10 cmH₂O), the surface film appeared in the form of a single, electron-dense leaflet, 2.7±0.6 nm (M±SD) in thickness while trilaminar membrane structure was retained in all parts of the tubular myelin figures of the hypophase. The surface film was attached underneath at right angles with trilaminar membranes which formed the outermost parts of the tubular myelin. Such structural continuity was taken to support a view that the phospholipid unit membrane of the tubular myelin figure would be transformed at the hydrophobic phase into a pair of monomolecular leaflets, eventually forming the surface film.     

New perfluorinated polycationic dimerizable detergents for the formulation of monomolecular DNA nanoparticles and their in vitro transfection efficiency

We describe the synthesis of new perfluorinated dimerizable detergents which contain a tricationic or tetracationic (linear or branched spermine, respectively) polar head, and report on their cmc, their ability to condense DNA into cationic monomolecular DNA nanoparticles as well as on the in vitro transfection efficiency of these nanoparticles. Such cationic nanoparticles were prone to display efficient cell transfection properties as a result of increased contact to the anionic cell surface and internalization by endocytosis, low size compatible with improved intracellular diffusion and nuclear pore crossing, and the presence of amine function of low pK(a) for their endosomal escape. The challenge was to design polymerizable polycationic detergents that display a cmc high enough for the monomer to perform monomolecular DNA condensation (as cationic particles) and low enough for the dimer to form stable nanoparticles capable of efficient cell transfection. Although we succeeded in formulating small-sized cationic monomolecular DNA nanoparticles (<40 nm) with these dimerizable perfluorinated spermine-based detergents for N/P ratios of up to 5 (N=number of detergent amine equivalents/P=number of DNA phosphate equivalents), these small-sized cationic nanoparticles proved to be poor non-specific transfection agents in vitro, even in the presence of chloroquine. Their poor transfection potential could be due more likely to Brownian motion which prevents these very small-sized particles from sedimentation and adsorption onto the adherent cell monolayer, and, consequently, from proteoglycan-triggered endocytosis.     

Anomalous pH dependence of the coexistence pressure of the polymerizable two-chain N-lipid methyl-bis(pentacosadiinoyl-oxyethyl)-amine

Molecular films from polymeric materials play an important role in basic research as well as in technology. We have synthesized the double chain diacetylenic ammonium lipid N-bis-(10, 12-pentacosadiinoyl)-oxy-ethyl)-N-methyl-amine (ONCO). We have characterized monomolecular films at the air/water interface by means of microfluorescence filmbalance techniques and by atomic force microscopy (AFM). ONCO forms stable monomolecular films that exhibit a fluid-solid phase transition with a transition enthalpy of 90 kJ/mol at 10 °C and neutral pH. The coexistence pressure was found to decrease with decreasing protonation, which is in contrast to the commonly found Coulomb mechanism. A change in the chain packing due to a different nitrogen bond angle is discussed as a possible mechanism. This model is cor-roborated by the finding that crystals at high and at low pH differ in their polymerization properties as measured by microfluorescence. Received: 9 October 1996 / Accepted: 13 March 1997     

Effect of gamma-hydroxypropano deoxyguanosine, the major acrolein-derived adduct, on monomolecular quadruplex structure of telomeric repeat d(TTAGGG)(4)

The three oligodeoxyribonucleotides (ODNs) a-c, having the telomeric repeat d(TTAGGG)(4) sequence and incorporating gamma-hydroxypropano deoxyguanosine at different positions, were synthesized. Gel electrophoresis and CD analyses indicated that the ODNs assume monomolecular quadruplex structures in Na+ and in K+ buffers. The T(m) values, obtained by CD melting experiments, showed that the presence of the acrolein-dG adduct into the ODN b decreases the thermal stability of the monomolecular quadruplex structure in Na+ solution, whereas for a and c no significant effect could be detected in the same experimental conditions. On the contrary, all ODNs a-d show the same behaviour in K+ buffer. These findings are briefly discussed.     

Glycolipid-cholesterol monolayers: towards a better understanding of the interaction between the membrane components

In this work, the interaction between a synthetic analog of archaeal lipids and cholesterol was studied using Langmuir technique. The lipid, β-Mal(3)O(C(16+4))(2), contained phytanyl chains attached via two ether bonds to the sn-2 carbon of the glycerol backbone. The preliminary studies showed that monolayers formed with the pure lipid have a liquid-like character; here, a hypothesis that admixing cholesterol to β-Mal(3)O(C(16+4))(2) could confer a higher rigidity on the films was tested. To check this proposal, two-dimensional miscibility of cholesterol and β-Mal(3)O(C(16+4))(2) in monomolecular films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy and polarization-modulation infrared reflection absorption spectroscopy. The stability of the monomolecular films was evaluated based on thermodynamics of mixing of cholesterol and β-Mal(3)O(C(16+4))(2). Atomic level information concerning the orientation of molecules and the degree of hydration of polar headgroups was obtained from molecular dynamics simulations.     

Fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface

The fluorescence properties of chlorophyll $\text{a}$ and $\text{b}$ monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorophyll molecules. Over the range of the mean distances from 27 to 21 Å, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll $\text{a}$ were observed. In the case of chlorophyll $\text{b}$, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 Å. When the mean distance was 18 Å, the short wavelength component of chlorophyll $\text{b}$ disappeared, and only the long wavelength component was observed.     

重组细菌多药外排泵AcrAB-TolC的转运功能和动态组装

【背景】多药外排泵多以膜蛋白复合体形式存在,是导致细菌耐药性的重要原因。外排泵的转运功能和组装过程对于细菌耐药性和药物研发具有重要意义。【目的】以多药外排泵耐药结节细胞分化家族(resistance-nodulation-division family, RND)的重要成员AcrAB-TolC复合体为对象,研究其转运活性和体外组装特性。【方法】基于大肠杆菌AcrAB-TolC复合体基因序列,分别构建含有acrA、acrB、tolC基因的重组质粒,表达和纯化复合体各亚基,利用荧光光谱、等温滴定量热法(isothermal titration calorimetry,ITC)等技术分析复合体及亚基的转运功能、亚基与底物的相互作用,以及亚基间的相互作用和动态装配。【结果】实现了AcrAB-TolC复合体各组分的表达和纯化(纯度>98%),证实表达有各组分的活细胞提高了对于溴化乙锭(ethidium bromide,EB)的转运活性,并发现群体感应效应信号分子N-hexanoyl-L-homoserine lactone (C6-HSL)能够抑制AcrB、TolC对于EB的转运活性。ITC结果进一步证实了C6-HSL与AcrB、TolC的相互作用。ITC结果还显示AcrA分别与AcrB、TolC之间存在明显的相互作用,而AcrB与TolC之间无明显的相互作用。在体外装配实验中观测到AcrAB-TolC亚基的单分子荧光强度随时间增加,证实了复合体亚基在膜上的动态组装过程。【结论】实现了AcrAB-TolC外排泵及亚基的表达和纯化,证实了AcrAB-TolC对底物的转运活性及与底物的相互作用,观察到AcrAB-TolC的动态组装过程。以上结果为研究多药外排泵导致的细菌耐药性及抗菌策略具有重要意义。     

Letter: Electron microscopic observation of DNA condensates at low pH values

Coliphage T2 DNA in sufficiently low concentration forms a monomolecular conderisate in acidic (pH 1.9) saline (0.3 m) solution. Electron microscopic observations of acidic monolayered DNA-cytochrome c films confirm previous indirect evidence concerning the dimensions of the in vitro condensation products and their ability to increase and compose confluent aggregates. The phenomena described here suggest that hydrophobic interactions may play a role in both the condensation and aggregation processes.     

Fluroescence properties of chlorophyll a and b monomolecular films at the air-water interface.

The fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorphyll molecules. Over the range of the mean distances from 27 to 21 A, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll a were observed. In the case of chlorophyll beta, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 A. When the mean distance was 18 A, the short wavelength component of chlorophyll beta disappeared, and only the long avelength component was observed.     

THE KINETICS OF SENESCENCE

The course of decline of vitality with age due to the process of senescence, when not complicated by the process of growth, follows a simple exponential law; that is the degree of vitality or of senescence (defining vitality as the reciprocal of senescence) at any moment is, regardless of age, a constant percentage of the degree of vitality or senescence of the preceding moment. This exponential law is the same as the law of monomolecular change in chemistry. During the actively growing period of life the index of vitality rises, due to the process of growth and the course of vitality in the case when the growing period is included in the vitality curve, follows a rising and falling course. This rising and falling course may often be represented by an equation containing two exponential terms which is practically the equation used to represent the course of accumulation and disappearance of a substance as the result of two simultaneous consecutive monomolecular chemical reactions.     

Spatiotemporal Dynamics of Zucchini Squash Lethal Chlorosis and its Relationship with the Frankliniella zucchini Population

Lethal chlorosis of cucurbits, caused by the tospovirus Zucchini lethal chlorosis virus (ZLCV), is an important disease in the Brazilian zucchini squash crop. The virus is transmitted by the thrips Frankliniella zucchini. Progress of the disease in time and space was studied in zucchini squash experimental fields to better understand disease epidemiology. Nine independent experiments were carried out between December 2006 and September 2010. The effects of the disease were assessed every 2–7 days, depending on the experiment. The thrips population was monitored in five of these experiments. For disease progress over time, four models (exponential, monomolecular, logistic and Gompertz) were tested. Disease progress in space analysis included both the index of dispersion and Taylor's power law. The monomolecular model was the best fit to the disease incidence data, and spatial analysis indicated aggregated diseased plants at the end of the season in most experiments. A correlation was detected between the number of collected thrips and the incidence of zucchini squash lethal chlorosis. The results indicate that the thrips population significantly contributed to the primary spread of disease incidence. We propose that disease management should focus mainly on the elimination of the source of the inoculum.     

Kinetic Properties of a Novel Fusarium solani (phospho)lipase: A Monolayer Study

Using the monomolecular film technique, we studied interfacial properties of Fusarium solani lipase (FSL). This lipolytic enzyme was found to be unique among the fungal lipases possessing not only a lipase activity but also a high phospholipase one.The FSL was able to hydrolyze dicaprin films at various surface pressures. The surface pressure dependency, the stereospecificity, and the regioselectivity of FSL were performed using optically pure stereoisomers of diglyceride (1,2‐sn‐ dicaprin and 2,3‐sn‐dicaprin) and a prochiral isomer (1,3‐sn‐dicaprin) spread as monomolecular films at the air–water interface. The FSL prefers adjacent ester groups of the diglyceride isomers (1,2‐sn‐dicaprin and 2,3‐sn‐dicaprin) at low and high surface pressures. Furthermore, FSL was found to be markedly stereospecific for the sn‐1 position of the 1,2‐sn‐enantiomer of dicaprin at both low and high surface pressures.Moreover, FSL shows high activities on phospholipids monolayers. However, this enzyme displays high preference to zwitterionic phospholipids compared to the negatively charged ones. Chirality, 25:35‐38, 2012. © 2012 Wiley Periodicals, Inc.