Atomic force microscopy studies of the adhesive properties of DPPC vesicles containing β-carotene

A role of carotenoids as modulators of physical properties of model and biological membranes has been already postulated. However, there is a lack of information on the influence of these pigments on interactions between the lipids which form such membranes. This paper applies atomic force microscopy (AFM) in to study the effects of β-carotene on the adhesion properties of DPPC multilamellar liposomes. This allowed us to gain, for the first time, a direct insight into the interactions between the components in model systems on a molecular level. We observe that the adhesive forces in DPPC multilamellar liposomes containing 1mol% of β-carotene decrease exponentially with increasing temperature, and that at about 37°C they diminish. In the case of pure liposomes the decline in adhesion is of a different nature and the adhesive forces disappear at 34°C. The adhesive forces are about 5 times higher at 31°C in the presence of β-carotene than in its absence. However, measurements using differential scanning calorimetry (DSC) showed a shift of the lamellar-to-undulled-lamellar phase transition toward lower temperatures by about 0.8 ± 0.2°C in a system containing β-carotene. The enthalpy changes (ΔH) of this transition are similar for both systems. For the main transition, gel-to-liquid crystalline, the peak is shifted by about 0.5 ± 0.1°C, and ΔH decreases by about 30% in liposomes treated with β-carotene in comparison to pure liposomes. Our results suggest increased cooperation between liposome components in a system with enriched β-carotene, which cause a change in phase transition temperatures. Moreover, these interactions are very sensitive to temperature.     

Thermotropic phase behavior of DPPC liposome systems in the presence of the anti-cancer agent ‘Ellipticine’

This letter presents our first results on the structural changes in DPPC multilamellar vesicles dispersed in water in the presence of the anti-cancer agent Ellipticine. The thermotropic phase transitions of the lamellar packing inside lipid vesicles were characterized in situ by small angle X ray diffraction. The results lead to the determination of a critical concentration value for drug loading on the vesicle system around 4% molar fraction of Ellipticine, an indication of the localization of the drug in the alkyl chains and the influence of the drug on the decreasing rate of the bilayer period after the main phase transition.     

Interaction of cationic surfactants with DPPC membranes: effect of a novel Nα-benzoylated arginine-based compound

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving N^α-benzoyl-arginine decyl amide (Bz-Arg-NHC₁₀), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC₁₀was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m⁻¹. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC₁₀ increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC₁₀. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC₁₀ solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC₁₀ adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids’ removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants.

     

Lateral pressure profiles in cholesterol–DPPC bilayers

By means of atomistic molecular dynamics simulations, we study cholesterol–DPPC (dipalmitoyl phosphatidylcholine) bilayers of different composition, from pure DPPC bilayers to a 1:1 mixture of DPPC and cholesterol. The lateral pressure profiles through the bilayers are computed and separated into contributions from the different components. We find that the pressure inside the bilayer changes qualitatively for cholesterol concentrations of about 20% or higher. The pressure profile in the inside of the bilayer then turns from a rather flat shape into an alternating sequence of regions with large positive and negative lateral pressure. The changes in the lateral pressure profile are so characteristic that specific interaction between cholesterol and molecules such as membrane proteins mediated solely via the lateral pressure profile might become possible.     

反向DPPC脂囊泡中短杆菌肽S的二维氢核磁共振研究

本文用300MHz核磁共振仪对反向DPPC脂囊泡中短杆菌肽S进行了研究.用二维相关谱(COSY)和二维NOE谱(NOESY)对反向脂囊泡中短杆菌肽S的共振峰进行了识别,短杆菌肽S所有的共振峰都被指定.并在此基础上,通过NOESY及一维自旋回波谱对短杆菌肽S在反向脂囊泡中的构象进行了分析.结果表明,短杆菌肽S在反向脂囊泡中不再为平面环状结构,而是有某种程度的折叠.     

山莨菪碱诱导DPPG／DPPC混合物形成交插凝胶相──荧光偏振研究

山莨菪碱诱导ＤＰＰＧ脂质体交插结构,其脂酰链末端插到对面分子层脂酰链第五个碳原子的位置,而生物膜中普遍存在的ＤＰＰＣ不能被山莨菪碱诱导形成交插相,但ＤＰＰＧ／ＤＰＰＣ混合物则能形成交插相,即伴随ＤＰＰＧ的交插,ＤＰＰＣ分子也发生交插。当ＤＰＰＧ／ＤＰＰＣ摩尔比为２：１或１：１时,其脂酰链末端插到对面分子层第八个碳原子的位置。当ＤＰＰＧ／ＤＰＰＣ摩尔比为１：２时,就不能发生交插而呈完全的非交插状态。同时,发现当体系中钠离子浓度达到４００ｍｍｏｌ／Ｌ时,山莨菪碱就不能再诱导ＤＰＰＧ形成交插凝胶相。     

Mg~2+与DPPC脂质体和脂酶体相互作用的拉曼光谱研究

本文用激光拉曼光谱研究了Mg~(2 )对DPPC脂质体及其与H~ —ATP酶复合体重组的脂酶体的影响。由实验结果得出,在20℃温度下,脂质体和脂酶体磷脂碳氢键都未处于全反式构型,仍具有一定的无序度,而且,链间的侧向耦合较弱,因而碳氢链具有较大的绕主轴转动自由度。Mg~(2 )与脂质体和脂酶体磷脂头部的相互作用对脂质体和脂酶体中脂分子碳氢链C—C骨架的排列状态影响不大。但是,在脂酶体中Mg~(2 )的作用加大了碳氢链的转动运动自由度。     

胆固醇对二棕榈酰磷脂酰胆碱（DPPC）多层膜结构的影响

用小角Ｘ射线衍射（ＳＡＸＤ）和差示扫描量热（ＤＳＣ）方法研究胆固醇对二棕榈酸磷脂酰胆碱多层膜结构的影响。在不同胆因醇浓度及不同温度下的衍射实验和热吸收曲线实验显示纯磷脂的尖锐的凝胶－液晶相变随胆固醇含量增加而逐渐消失。当胆固醇含量约为４０（ｍｏｌ）％或更多时,衍射曲线和热吸收曲线均显示存在分相现象。     

牛胰多肽（BPP）与DPPC脂质体相互作用的付立叶变换红外光谱研究

本文详细报导用付立叶变换红外光谱法研究去氧胆盐(DOC)和牛胰多肽(BPP)与DPPC脂质体的相互作用.发现DOC和脂质作作用后,DPPC的CH:对称和反对称伸展振动频率向高波数方向移动,相交曲线变宽,相变温度降低;BPP和DPPC作用后,红外光谱无明显变化,但它却能抑制由DOC对DPPC脂质体的上述作用.从而对BPP具有细胞保护作用的分子机制作了进一步的阐述.     

Three dimensional (temperature–tension–composition) phase map of mixed DOPC–DPPC vesicles: Two solid phases and a fluid phase coexist on three intersecting planes

Mapping the phase behavior of multicomponent phospholipid membranes has been an ongoing pursuit, motivated by interest in both fundamental physics and cell function. Prior investigations addressed temperature-composition space and the features of the associated domains. The current study additionally considers membrane tension, analogous to pressure in bulk materials. Focusing on model mixed 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC and DPPC respectively) membranes, we examine the thermodynamic impact of tension on fluid-solid coexistence and the nature of phase-separated domains. Reported here is the 3 dimensional composition-temperature-tension phase map containing three intersecting curved surfaces. Depending on the system’s position in this 3D space, giant unilamellar vesicles containing DOPC and DPPC may exhibit, in addition to a 2-component fluid Lα phase, two different types of solid DPPC-rich domains: tracer-excluding hexagonal patches or tracer-selective stripes. The fluid phase occurs at high temperatures. At cool temperatures striped solid DPPC-rich domains coexist with the fluid at elevated tensions. These stripes occur independent of tension, at the coolest temperatures. At low tensions and intermediate temperatures, patchy solid DPPC-rich domains coexist with the Lα fluid and may persist, if kinetically trapped, at lower temperatures. We associate the striped DPPC domains with a tilt-gel (Lβ’) morphology and the hexagonal DPPC patches with a dense corrugated ripple phase (Pβ’). These assignments, based on the reported areal densities of the corrugated and tilt solids, enabled first principles estimates of the coexistence boundaries that match the experiments well, including the tension sensitivity of coexistence curves and triple-point-like features for fixed composition.     

荧光光谱法研究铈离子与二棕榈酰磷脂酰胆碱（DPPC）脂质体的相互作用

通过荧光光谱法研究了稀土Ｃｅ＾３＋与二棕榈酰磷脂酰胆碱（ＤＰＰＣ）脂质体的相互作用,结果表明,Ｃｅ＾３＋－ＤＰＰＣ体系的激发波长在２４７ｎｍ,２９４ｎｍ,发射波长在３４４ｎｍ,与水合Ｃｅ＾３＋的荧光光谱完全不同。这表明Ｃｅ＾３＋与ＤＰＰＣ形成了复合物,该复合物的荧光光谱同Ｃｅ＾３＋－ＤＨＰ复合物的荧光光谱一致,可以认为Ｃｅ＾３＋与ＤＰＰＣ分子上的磷酸基团相作用。     

荧光光谱法研究铈离子与二棕榈酰磷脂酰胆碱（DPPC）脂质体的相互作用

通过荧光光谱法研究了稀土元素Ｃｅ３＋与二棕榈酰磷脂酰胆碱（ＤＰＰＣ）脂质体的相互作用,结果表明,Ｃｅ３＋－ＤＰＰＣ体系的激发波长在２４７ｎｍ,２９４ｎｍ,发射波长在３４４ｎｍ,与水合Ｃｅ３＋的荧光光谱完全不同。这表明Ｃｅ３＋与ＤＰＰＣ形成了复合物,该复合物的荧光光谱同Ｃｅ３＋－ＤＨＰ复合物的荧光光谱一致。可以认为Ｃｅ３＋与ＤＰＰＣ分子上的磷酸基团相作用。     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.