Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues

Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar-inverse hexagonal (L_α-H_II) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.     

Bilayer Mixing,Fusion, and Lysis Following the Interaction of Populations of Cationic and Anionic Phospholipid Bilayer Vesicles

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.     

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillumcells with plants is discussed.     

Cationic amphiphiles induce fusion of acidic liposomes

Decylamine, dodecylamine and tetradecylamine induced aggregation and fusion of acidic liposomes at concentrations of about 1 mM, 75 μM and 75 μM, respectively. Aggregation was assayed as increase in turbidity. Fusion was assayed as intermixing of membranes and contents, and was observed in the electron-microscope to form large liposomes. Only at higher concentrations did these amphiphiles induce massive leakage of the liposomes' contents. Similar effects were caused by hexadecylpyridinium bromide (CP) and hexadecyltrimethylammonium bromide (CTAB). The trivalent cation 4-dodecyldiethylenetriamine and the more hydrophobic amphiphile, trioctylmethylammonium chloride, induced fusion at concentrations of about 10–20 μM. Octylamine and heptylamine induced size increase at mM concentrations. They induced membrane intermixing but little or no content intermixing. Thus, these amphiphiles seem to promote size increase either by transfer of lipid or mainly by ‘cracking and annealing’.     

Effect of Cationic Comb-Type Copolymer on Quadruplex Folding of Human Telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA) _n in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA) ₃ GGG) and d((GGGTTA)₇GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA) ₃ GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

Thermodynamic features of the chemical and thermal denaturations of human serum albumin

The unfolding process of human serum albumin between pH 5.4 and 9.9 was studied by chemical and thermal denaturations. The experimental results showed that there is no correlation between the stability of albumin at different pH values determined by both methods. The free energy change of unfolding versus concentration of guanidine showed a close dependence on the pH, suggesting that the variation of the electrical charge of albumin influences the final state of the unfolded form of the protein. Spectroscopic techniques, such as native fluorescence of the protein and circular dichroism, demonstrated that the unfolded state of the protein obtained from both methods possesses a different helical content. The solvophobic effect and the entropy of the chains have no influence on the final unfolding state when the protein is unfolded by thermal treatment, while, when the protein is unfolded by chemical denaturants, both effects depend on the medium pH. The results indicate that guanidine and urea interact with albumin by electrostatic forces, yielding a randomly coiled conformation in its unfolded state, while thermal denaturation produces a molten globule state and the aggregation of the protein; therefore, both methods yield different structurally unfolded states of the albumin.     

Cationic polyacrylamides promote binding of cellulase and amylase

Cationic polyacrylamides (c-PAMs) increase the binding of cellulase to cellulose and of α-amylase to cornstarch. The c-PAMs increase the rate of hydrolysis of both substrates by about the same amount, which suggests that the increased binding is responsible for the enhanced rates. These results support our proposed mechanism where the c-PAM neutralizes the charge of the substrate through a “patching” mechanism, which reduces the charge repulsion between fiber and enzyme.     

Interaction of Isoquinoline Alkaloids with Polymorphic DNA Structures

The interaction of berberine, palmatine, and coralyne with the B, Z, and H^L form of poly[d(G‐C)] was studied. Berberine and palmatine showed moderate binding to the B form, while coralyne showed higher binding, as revealed from spectroscopic and thermodynamic data. Berberine and coralyne binding to the B form was exothermic and enthalpy‐driven, while palmatine showed exothermic binding which was favored by both negative enthalpy and negative entropy changes. Berberine and palmatine neither bind nor converted the Z‐form structure to B form. Coralyne, on the other hand, exhibited a strong binding affinity to Z DNA structure that was enthalpy‐driven. Berberine binding to the H^L form was cooperative, exothermic, and favored by both negative enthalpy and negative entropy changes with the formation of an induced CD band. Palmatine showed weak binding, while coralyne showed a strong binding with the H^L form. The structural differences in the isoquinoline alkaloids appear to influence the affinity and mode of interactions with these polymorphic DNA structures.     

天然沸石的交换性阳离子类型与农业适用性

天然沸石有良好的离子交换和吸附特性,因此,它在农业上的应用也越来越受到人们的重视,但据我们发现,不同产地沸石,占主导地位的交换性阳离子种类大不相同,如果盲目应用,就会产生相反的结果。通过实验证实,天然沸石的阳离子类型,特别是含交换性Na~ 较高的一类沸石与土壤、肥料相互作用后,对植物生长有很大影响。     

Fluorometrical Analysis of Guanidino Compounds in Human Cerebrospinal Fluid

Abstract: Guanidino compounds in CSF of 57 human subjects were determined fluorometrically after reaction with phenanthrenequinone in alkali solution, using HPLC. Creatinine (65.2 ± 13.4 nmol/ml), arginine (24.7 ± 6.4 nmol/ml), and homoarginine (0.7 ± 0.3 nmol/ml) were found in all subjects. Trace amounts of guanidinosuccinic acid and guanidinoacetic acid were detected in some of the subjects. Brain guanidino compounds, taurocyamine, N -acetylarginine, and methylguanidine were not detected in CSF.     

Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

源于大型真菌的小分子抗肿瘤化合物研究进展

大型真菌是一类药用资源极为丰富的真菌,许多大型真菌具有显著的抗肿瘤活性。本文综述了源于大型真菌的具有抗肿瘤活性的小分子物质,如萜类、酚类、苯并吡喃酮类和甾类等。以期对大型真菌的活性筛选、化学分析和开发利用提供帮助。     

Cationic lipids and polymers are able to enhance adenoviral infection of cultured mouse myotubes

Adenovirus-mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down-regulation of the adenovirus internalisation receptors, alpha(v)/beta3 and alpha(v)beta5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirus-mediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.     

Cationic liposome-mediated delivery of siRNAs in adult mice

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.     

疏水修饰聚阳离子高分子SP-Chol 在COS-7 细胞中转染和毒性的研究

目的:研究以精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以胆固醇氯甲酸酯作为疏水基团连接剂合成的疏水修饰聚阳离子高分子SP-Chol对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法:以荧光素酶质粒为报告基因,研究SP-Chol与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究SP-Chol对COS-7细胞的毒性。结果:COS-7细胞实验显示,SP-Chol具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论:SP-Chol是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

Interaction Specificity of Benzimidazol Group Compounds with AT-Containing Polynucleotides

Abstract

The interaction between polynucleotides: poly(dA)-poly(dT), poly(dA-dT), poly(am2dA- dT), and the AT-specific compounds of benzimidazol group has been studied. It is been shown that these compounds bind to poly(dA)-poly(dT) and poly(dA-dT) at low and high salt concentration in solution. Poly(am2dA-dT) interacts with AT-specific compounds only at low salt, where this polynucleotide is in a B-form, but not at high salt when the polynucleotide converts to another conformation. Thus, the interaction specificity of the groove-binding ligands is influenced not only by the minor groove substituents, but the peculiarities of the secondary structure of polynucleotides.     

Interaction of radiolabeled endotoxin molecules with human monocyte membranes

Radiolabeled and biologically active endotoxin molecules were prepared, and their binding to monocyte plasma membranes was studied. The binding of ³H-endotoxin and ⁵¹Cr-lipid A to isolated membranes was found to be less specific and of lower apparent affinity than that observed using whole cells. Plasma membranes isolated from intact, viable ⁵¹Cr-lipid A-pretreated monocytes were found to contain a significant portion of the cell-associated ⁵¹Cr-lipid A following Percoll density gradient fractionation of post-nuclear homogenates. When monocytes were pretreated with ³H-endotoxin under the same experimental conditions, all of the label was recovered in the extracellular medium, and subcellular fractionation revealed no fractions which contained tritium. Taken together, our results suggest that specific and high affinity interactions between monocyte membranes and endotoxin molecules are likely to depend on plasma membrane structures which are assembled in intact monocytes but which are disrupted when plasma membranes are isolated from these cells.     

疏水修饰聚阳离子高分子SP-Chol在COS-7细胞中转染和毒性的研究

目的：研究以精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以胆固醇氯甲酸酯作为疏水基团连接剂合成的疏水修饰聚阳离子高分子SP-Chol对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：以荧光素酶质粒为报告基因,研究SP-Chol与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究SP-Chol对COS-7细胞的毒性。结果：COS-7细胞实验显示,SP-Chol具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：SP-Chol是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。