Cholesterol Modulates the Interaction of β-Amyloid Peptide with Lipid Bilayers

The interaction of an amphiphilic, 40-amino acid β-amyloid (Aβ) peptide with liposomal membranes as a function of sterol mole fraction (X_sterol) was studied based on the fluorescence anisotropy of a site-specific membrane sterol probe, dehydroergosterol (DHE), and fluorescence resonance energy transfer (FRET) from the native Tyr-10 residue of Aβ to DHE. Without Aβ, peaks or kinks in the DHE anisotropy versus X_sterol plot were detected at X_sterol ≈ 0.25, 0.33, and 0.53. Monomeric Aβ preserved these peaks/kinks, but oligomeric Aβ suppressed them and created a new DHE anisotropy peak at X_sterol ≈ 0.38. The above critical X_sterol values coincide favorably with the superlattice compositions predicted by the cholesterol superlattice model, suggesting that membrane cholesterol tends to adopt a regular lateral arrangement, or domain formation, in the lipid bilayers. For FRET, a peak was also detected at X_sterol ≈ 0.38 for both monomeric and oligomeric Aβ, implying increased penetration of Aβ into the lipid bilayer at this sterol mole fraction. We conclude that the interaction of Aβ with membranes is affected by the lateral organization of cholesterol, and hypothesize that the formation of an oligomeric Aβ/cholesterol domain complex may be linked to the toxicity of Aβ in neuronal membranes.     

Dynamics of kinks in inhomogeneous polynucleotide chains

In the present paper the dynamics of the nonlinear conformational excitations — kinks, in inhomogeneous polynucleotide DNA chains is investigated. To calculate the kink rest energy E₀ and its size d the method of dynamical interval is used. This makes it possible to take into account that all coefficients of the model dynamic equation — the sine-Gordon equation depend on the sequence of bases. It is shown that the method gives an opportunity to calculate dynamical characteristics of any artificial and real sequences that is important for implementing tasks associated with the search and analysis of functionally important DNA sites.     

Synthesis,characterization, theoretical,anti-bacterial and molecular docking studies of quinoline based chalcones as a DNA gyrase inhibitor

A series of fourteen (A₁–A₁₄) new qunioline based chalcones were synthesized by condensing 2,7-dichloro-8-methyl-3-formyl quinoline with acetophenone and acetylthiophenes, and subsequently characterized by IR, NMR and Mass spectroscopy. All the compounds were screened for antibacterial activities and found potentially active antibacterial agents. Bioassay, theoretical and dockings studies with DNA gyrase (the enzyme required for super coiling of DNA of bacteria) results showed that the type and positions of the substituents seemed to be critical for their antibacterial activities. The bromo and chloro substituted chalcone displayed high anti-bacterial activity. The A₄ and A₆ showed high interaction with DNA gyrase, contributing high free binding energy (ΔG −8.18 and −8.88 kcal).     

Theoretical Analysis of Disruptions in DNA Minicircles

Under sufficient bending stress, which appears in DNA minicircles and small DNA loops, the double helix experiences local disruptions of its regular structure. We developed a statistical-mechanical treatment of the disruptions in DNA minicircles, studied experimentally by Du et al. The model of disruptions used in our Monte Carlo simulation of minicircle conformations specifies these conformations by three parameters: DNA bend angle at the disruption, θ_d; local DNA unwinding caused by the disruption; and the free energy associated with the disruption in the unstressed double helix, G_d. The model is applicable to any structural type of disruption, kinks or opening of single basepairs. The simulation shows that accounting for both torsional and bending deformation associated with the disruptions is very important for proper analysis. We obtained a relationship between values of G_d and θ_d under which the simulation results are compatible with the experimental data. The relationship suggests that the free energy of basepair opening, which includes flipping out both bases, is significantly higher than the generally accepted value. The model is also applied to the analysis of j-factors of very short DNA fragments.     

Kink dynamics in inhomogeneous DNA

A mathematical model for studies of peculiar features of the dynamics of local conformational distortions (kinks) has been constructed. General formulas determining the dependence of the main dynamic characteristics of kinks: size, energy, density of energy, and velocity on the composition of inhomogeneous DNA have been obtained. Quantitative estimations of the characteristics have been made for kinks activated in inhomogeneous polynucleotide chains with sequences analogous to promoter sequences A1, A2, and A3 of bacteriophage T7 genome.     

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A ₂₆₀/A ₂₈₀ between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A ₂₆₀/A ₂₈₀ ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A ₂₆₀/A ₂₈₀ measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin^® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.     

Comparisons of direct extraction methods of microbial DNA from different paddy soils

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.     

Energy supply of the okapi in captivity: intake and digestion trials

In the nutrition of browsing ruminants in captivity, adequate nutrient digestibility and energy content of diet is debated. Problems related to energy‐provision and low forage intake have been reported for the okapi and other browsers like the giraffe, particularly during winter. High‐fiber concentrates like unmolassed beet pulp have some potential to improve the nutritional management of these species. Using a total of six okapis in captivity, seven feeding trials were carried out at two facilities (A+B) on a structured but opportunistic base. Three trials (A₁, A₂, B₁) were conducted when animals were fed their regular diet including grain based energy concentrates, fruits and vegetables, and alfalfa (Medicago sativa) hay. Two trials (A₅, B₂) examined the effect of unmolassed beet pulp, and two (A₃,₄) examined the effect of unmolassed beet pulp+fresh browse. Daily intake and feces production were quantified over 8–12 days. Samples were analyzed for dry matter, crude ash, neutral detergent fiber (NDF)/acid detergent fiber (ADF)/acid detergent lignin (ADL), crude protein, and gross energy. Metabolizable energy content of diets was estimated via a factor (0.83) from digestible energy. The proportion of beet pulp in diets was 13% (A₃), 24% (A₄), 20% (A₅), and 21% (B₂). Browse proportion was 11% (A₃) and 32% (A₄). Daily feed intake ranged between 1.5–1.7% of body weight (BW), digestibility of organic matter between 61–74%. Digestibility of fiber (NDF) was higher in beet pulp diets (A₃=39%, A₄=60%, A₅=54%, B₂=61%) than in the others (A₁=48%, A₂=33%, B₁=48%). Supply of metabolizable energy (ME) ranged between 0.50–0.70 MJ ME/(kg BW^0.75*day), meeting energy requirements of okapis of 0.50–0.53 MJ ME/(kg BW^0.75*day) in general. Diets with beet pulp+browse were not found to be highest, but in the upper level of the range of forage proportions of this study. Palatable browse species were preferred over all other feedstuff offered. The use of unmolassed beet pulp as energy‐concentrate for browsing ruminants like the okapi can be recommended because diets high in this high‐fibre feedstuff resulted in adequate energy intakes. Zoo Biol 0:1–14, 2006. © 2006 Wiley‐Liss, Inc.     

CC/GG Contacts Facilitate the B to A Transition of DMA in Solution

Abstract

Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTG- CAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts.

First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents.

A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex. The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA. The B-phility value was estimated to be 3 kcal/mol of 10-mer.

The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex. The designed 10-mers made it possible to estimate quantitatively the A- phility of CC/GG contact as compared with an average DNA: (F_A-F_B)CC=0.2 Kcal/mol, (F_A-F_B)DNA=0.7 Kcal/mol.     

A fast, simple, and reliable high-yielding method for DNA extraction from different plant species

Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.     

Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)

The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 μg g^?1 of B. maxima fresh weight. This DNA was 100–200 kb in length, and its A₂₆₀/A₂₈₀ and A₂₃₀/A₂₆₀ ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 10³ independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high‐stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.     

Structural Basis of Stable Bending in DNA Containing An Tracts. Different Types of Bending

Abstract

Structural determinants of DNA bending of different types have been studied by theoretical conformational analysis of duplexes. Their terminal parts were fixed either in an ordinary low-energy B-like conformation or in “anomalous” conformations with a narrowed minor groove typical of A_n tracts. The anomalous conformations had different negative tilt angles (up to about zero), different propeller twists and minor groove widths. Calculations have been performed for DNA fragments A_nT_m, T_nA_m, A_nGCT_m, A_nCGT_m, T_mGCA_n, T_mCGA_n which are the models of the junction of two anomalous structures on A_n and T_m tracts. On the AT step of the A_nT_m fragment the minor groove can be easily narrowed so that a whole unbent fragment of anomalous structure is formed on A_n T_m. According to our energy estimates, there should not be any reliable bending on A_nT_m. In contrast, in all other cases there was a pronounced roll-like bending into the major groove in the chemical symmetry region. Calculations of the junction between the anomalous and ordinary B-like structure for G_nT_m and C_nA_m have shown that there is an equilibrium bending with a tilt component towards the chain having the anomalous structure at the 5′-end. From our calculations it is impossible to determine precisely the direction of bending, though it can be suggested that the roll component of bending might be directed towards the major groove. The anomalous structure is the main reason of bending; alternations of pyrimidines and purines can modulate the value and the direction of equilibrium bending (only the value in the case of self-complemantary fragments).

The results are consistent with the experimental data and promote a better understanding of the problem of DNA bending.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura-2/AM标记药物敏感的肺腺癌细胞A₅₄₉和抗顺铂药物的肺腺癌细胞A₅₄₉/DDP两种细胞胞内游离Ca²⁺,用碘化丙锭(PI)标记细胞DNA,检测其胞内Ca²⁺的变化及两种细胞增殖能力和细胞周期.实验结果表明,抗药性细胞株A₅₄₉/DDP胞浆内游离Ca²⁺的浓度仅为药物敏感细胞株A₅₄₉的1/3左右,同时前者的细胞增殖能力较后者明显增强,而且细胞周期也明显缩短.当用BAPTA-AM和EGTA或A₂₃₁₈₇和Thapsigargin处理细胞以降低或升高其胞内自由Ca²⁺浓度时可改变细胞的生长周期,二者也呈现明显差别.这些结果表明,对顺铂产生耐药性的人肺腺癌A₅₄₉/DDP细胞胞内Ca²⁺浓度的降低,可能影响细胞的增殖,缩短细胞的生长周期,特别是影响起决定作用的G1期,从而有利于肿瘤细胞多药耐药特性的维持.     

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12

Four hitherto undescribed endodeoxyribonucleases, temporarily designated A₁, A₂, A₃, and B, have been isolated from E. coli K-12. Each requires Mg⁺⁺ and is not stimulated by ATP or S-adenosylmethionine. A₃ is strongly inhibited by Fe⁺⁺⁺ and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3′-phosphatase. A₁ (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3′-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A₂ (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3′-hydroxyl- and 5′-phosphoryl termini. A₃ (molecular weight approximately 38,000) cleaves fd DNA to form 3′-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A₃. B appears to form 3′-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.     

A Theoretical Model for the Binding of Cis-Pt(NH3)2 +2 to DNA

Abstract

The binding of cis-Pt(NH₃)₂B₁B2 to the bases B₁ and B₂, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB,pdB₂ sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cw-Pt(NH₃)₂ ⁺² (cis-PDA) binds to N7(G), N3(C), 02(C), 06(G), N3(A), N7(A), 04(T) and 02(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of ?70° and ?40° at the receptor site to orient the base pairs for ligand binding to B, and B₂ to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG·cis-PDA chelate from the dCpdG·cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate.

The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti ? syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

OPTIMIZATION OF DNA EXTRACTION FROM BROWN ALGAE (PHAEOPHYCEAE) BASED ON A COMMERCIAL KIT1

Large‐scale DNA molecular studies require reliable and efficient tools for DNA extractions. However, for some plant species and brown algae, isolation of high‐quality DNA is difficult. We developed a novel method for isolating high‐quality DNA from the polysaccharide‐rich and polyphenol‐rich brown algae based on a commercial kit and protocol (Qiagen) by optimizing the lysis step and including a chloroform/isoamyl alcohol supplementary purification step. DNAs from 24 brown algal species extracted using the original and the modified Qiagen protocol were compared for yield, quality, and effectiveness in PCR amplification. There was no significant difference in the yields between protocols. However, a statistically significant increase in DNA purity was obtained with the modified protocol, for which the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ absorbance ratios averaged 1.66 ± 0.05 and 1.31 ± 0.01, respectively, compared to 1.37 ± 0.04 and 0.52 ± 0.04 with the original protocol. DNAs extracted by the modified procedure were more successfully amplified by PCR (nuclear, mitochondrial, and chloroplastic regions) than DNAs extracted using the original commercial kit and protocol. Importantly, the modified protocol can be applied in a high‐throughput (e.g., 96‐well plate) format, allowing a higher efficiency for downstream molecular analysis. In addition, improved DNA quality could increase its stability for long‐term storage.     

Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 g g^-1 dry mycelia and the A₂₆₀/A₂₈₀ ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A₂₆₀/A₂₈₀ ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.Revisions requested 8 September 2004; Revisions received 1 November 2004     

Rapid,High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique

A protocol for extracting high quality DNA from cypress (Cupressus sempervirens L.), a gymnosperm of economic and ecological importance to the Mediterranean, is presented using the commercially supplied DNeasy^TM Plant Mini Kit (QIAGEN GmbH, Germany). Additional steps were introduced in the QIAGEN protocol to significantly enhance DNA quantity and quality. For one sample, the procedure can be completed in less than one hour, and more than 10 samples can be processed in a day. DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The average DNA yield obtained from 100 mg starting material was around 22 g, which compares very favorably with numbers indicated by the manufacturer. Additionally, restriction and PCR analyses of the extracted DNA showed its compatibility with downstream applications. Using this DNA, the parameters for the randomly amplified polymorphic DNA (RAPD) protocol were optimized based on the use of (1) an AmpliTaq^® DNA Polymerase, Stoffel fragment (Perkin-Elmer), and (2) a high initial denaturation temperature (97.5 °C). Reproducible amplification products were achieved in all PCR reactions. Seven to eight bands per primer were obtained with most individuals. This represents a number at the high end of published results with other plant species.     

A measure of bending in nucleic acids structures applied to A-tract DNA

A method is proposed to measure global bending in DNA and RNA structures. It relies on a properly defined averaging of base-fixed coordinate frames, computes mean frames of suitably chosen groups of bases and uses these mean frames to evaluate bending. The method is applied to DNA A-tracts, known to induce considerable bend to the double helix. We performed atomistic molecular dynamics simulations of sequences containing the A₄T₄ and T₄A₄ tracts, in a single copy and in two copies phased with the helical repeat. Various temperature and salt conditions were investigated. Our simulations indicate bending by roughly 10° per A₄T₄ tract into the minor groove, and an essentially straight structure containing T₄A₄, in agreement with electrophoretic mobility data. In contrast, we show that the published NMR structures of analogous sequences containing A₄T₄ and T₄A₄ tracts are significantly bent into the minor groove for both sequences, although bending is less pronounced for the T₄A₄ containing sequence. The bending magnitudes obtained by frame averaging are confirmed by the analysis of superhelices composed of repeated tract monomers.