DNA-Protein Complex in Circular DNA from Bacillus Bacteriophage GA-1

DNA prepared from bacteriophage GA-1 contains circular DNA molecules, which are converted to linear molecules by treatment with trypsin.     

THE TIME-COURSE OF POLAR MOVEMENT OF GIBBERELLIN THROUGH ZEA ROOTS

The polarity of movement of gibberellin through sections cut from near the root tips of Zea mays L. was studied, using methods like those we previously used in roots for auxin and in petioles for auxins, cytokinins, and gibberellic acid (GA-3). One μg GA-3 was added in a donor agar block and gibberellin activity in the receiver agar at the opposite end of the section was measured directly with a modified barley endosperm bioassay. The movement of gibberellin was away from the root tip (basipetal) and thus opposite in direction to the polarity of auxin through such root sections. The time-course of basipetal movement was dissimilar to that for gibberellin or auxin movement through petiole sections. It took 14-18 hr for gibberellin activity equivalent to 6 ng GA-3 to collect in the basal receivers on roots. Apical receivers showed activity equivalent to 1.6 ng GA-3 at 14-18 hr. Less than 0.01 ng equivalent GA-3 was collected from sections to which GA-3 was not added, so the 6 and 1.6 ng were almost entirely due to the added GA-3. These general conclusions were confirmed with an experiment using ¹⁴C-GA-3. A decline in activity in receivers was found in some experiments at 18 hr, paralleling earlier results with GA-3, IAA, and adenine in petioles and IAA in roots.     

Protease-Sensitive Transfection of Bacillus subtilis with Bacteriophage GA-1 DNA: a Probable Case of Heterologous Transfection

The host bacterium of bacteriophage GA-1, Bacillus sp. G1R, was compared with respect to its taxonomic relationship to Bacillus subtilis, B. licheniformis, and B. pumilis. The physiological-biochemical properties of Bacillus sp. G1R are equal to those of B. licheniformis, but the thermal denaturation midpoint of G1R DNA differs by 3 C and the buoyant density by 0.005 g/cm(3) from that of B. licheniformis. Transformation with G1R donor DNA was neither observed in B. licheniformis nor in B. subtilis-competent recipients. Bacteriophage GA-1 shows neither infectivity on B. licheniformis nor on B. subtilis. However, infection of competent B. subtilis cultures with phenol-extracted GA-1 DNA results in the production of infective GA-1 particles. The transfecting activity of GA-1 DNA is destroyed by treatment with proteolytic enzymes. Resistance of transfecting DNA to inactivation by trypsin develops earlier than that to inactivation by DNase. Protease-treated GA-1 DNA competes with transforming DNA to approximately the same extent as does untreated GA-1 DNA, suggesting that uptake of GA-1 DNA is not affected by protease treatment. CsCl density gradient centrifugation reveals that the density of trypsinized GA-1 DNA is 0.004 g/cm(3) greater than that of untreated DNA.     

Proton NMR bandshape studies of lamellar liquid crystals and gel phases containing lecithins and cholesterol.

Proton NMR spectra for gel and liquid crystalline samples, composed of dimyristoyl and/or dipalmitoyl lecithin, cholesterol and water, can be consistently interpreted in terms of mesophase symmetry and molecular diffusion according to a model proposed by Wennerstrom (Wennerstrom, H. (1973) Chem. Phys. Lett. 18, 41-44). It is shown by computer simulation that the characteristic "super-lorentzian" bandshape of the lamellar mesophase can be described by the superposition of three gaussian curves. The NMR signal of the gel phase can be simulated by the superposition of two gaussian curves with widths at half height of 2.5 kHz and 19 kHz. An upper limit of the lateral diffusion coefficient of the lecithin molecules in the gel phase is calculated to be about 5-10(-15) m-2/s. It is therefore concluded that the static intermolecular dipolar couplings average to zero in the lamellar mesophase. An estimation of the order parameter of the liquid crystalline phase is made from experimental data and a calculated "rigid lattice" linewidth. A two phase system is shown to exist in the temperature range 28-34 degrees C for a mesophase of a mixture of dimyristoyl and dipalmitoyl lecithin. The presence of cholesterol results in enhanced lateral diffusion of the lecithin molecules at temperatures below the Chapman transition point.     

Nuclear magnetic resonance studies of the aggregation of dihexanoyllecithin and of diheptanolyllecithin in aqueous solutions.

Aggregation of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (dihexanoyllecithin) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (diheptanoyllecithin) in aqueous solutions has been investigated by 1H nuclear magnetic resonance spectroscopy. The chemical shifts and line widths of the NMR signals of the lecithins are dependent on the total concentration of lecithin above the critical micelle concentration. Signals for both lecithins in the aggregated state exhibit line widths which are appreciably smaller than the dipolar line width calculated using the overall rotational correlation time of the micelle. Signals of the alpha-methylene protons of the carboxylic acid side chains of dihexanoyllecithin and diheptanoyllecithin undergo the greatest change in chemical shift on aggregation. A single averaged spectrum of the alpha-methylene protons is observed in lecithin solutions of concentrations ranging from one to four times the critical micelle concentration demonstrating that individual lecithin molecules are in rapid exchange, with respect to a frequency of 18 Hz, between the monomeric and the aggregated states. Plots of the chemical shift of the alpha-methylene protons versus concentration of lecithin approximate a micelle formation curve. At about five times the critical micelle concentration for both dihexanoyllecithin and diheptanoyllecithin the alpha-methylene pattern indicates that there are at least two magnetic environments for lecithin molecules in the aggregated state. Furthermore, individual lecithin molecules are in slow exchange between the two environments which are distinguished by a chemical shift difference of about 2 Hz.     

Nuclear magnetic resonance and light scattering studies of the aggregation of dipalmitoyl-phosphatidylcholine-Benzene systems

Magnetic resonance, light scattering measurements and visual observations have been performed on lecithin-benzene systems as a function of temperature from 80 to ?60°C and concentration from $\text{n = n}{}_{\mathrm{<mtext>benzene</mtext>}}\text{/n}{}_{\mathrm{<mtext>DPPC</mtext>}}\text{= 3?200}$.From these measurements the transition curves from micellar solution to the crystalline state were deduced. More than 5 benzene molecules per lecithin molecule are necessary to get a micellar solution.Both the size of crystallites and the distribution of pores in the crystalline state depend on the rate of cooling and history.For the crystal structure a biporous model is suggested.At about 0°C the bulk of the benzene freezes, but a small amount of about 3 benzene molecules per lecithin molecule remains unfrozen to about ?50°C.The systems studied show considerable temperature hysteresis and ageing effects.Small amounts of water of one to two water molecules per lecithin molecule drastically change the properties of the lecithin systems.     

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.     

Effect of valinomycin on the structure of water-lecithin liposomes]

It has been established that water--lecithin liposomes in the heptane phase are formed in the course of equilibration of lecithin solution in heptane with water phase containing Na, K-picrate. The salts penetrate both the water nucleus of liposomes and their lecithin shell. The addition of valinomycin to this system does not change the water content in liposomes, but considerably increases the salt absorbed by the lipid shell of liposomes. A characteristic S-shape curve showing the relation between picrate extraction and the valinomycin concentration might be explained as an indication of a cooperative change of the structure of lecithin shell. This phase transition is induced by one valinomycin molecule per 10(3)--10(4) lecithin molecules.     

Idiotypic analysis of anti-GAT antibodies. VII. Common idiotype on hybridoma antibodies to poly(Glu60 Ala40)

A single DBA/2 mouse, immunized with L-glutamic acid60-L-alanine40 (GA), was used to produce hybridoma cell lines. Seven hybridoma anti-GA antibodies were obtained for idiotypic analyses. Two hybridoma anti-L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) antibodies, preferentially reactive to GA, were studied in parallel. Anti-idiotypic antisera to purified anti-GAT and anti-GA serum antibodies and to hybridoma anti-GA antibodies were analyzed by idiotype binding and inhibition of idiotype binding assays. Five of the nine hybridoma antibodies exhibited common GA-1 idiotypic specificities previously demonstrated on the majority of anti-GA antibodies of inbred mouse strains of differing immunoglobulin heavy chain linkage groups; these hybridoma antibodies also possessed private idiotypic determinants. Two GA-1 negative hybridoma anti-GA antibodies appeared identical by immunochemical criteria, arguing that somatic hybridization does not artifactually generate private idiotypic determinants. The results demonstrate that the common GA-1 idiotype system is associated with a family of nonidentical but idiotypically related antibody molecules present in a single DBA/2 mouse, and these antibodies are part of the "GA-1 idiotypic family".     

Regulation of phospholipase A2 activity by the lipid-water interface: a monolayer approach

Interfacial regulation of phospholipase A2 activity on lecithin monolayers was investigated by using radioactively labeled enzyme. Labeling of the protein with 125I did not produce a change of the enzyme and protein properties as compared to the 3H fully amidinated phospholipase A2. The induction time observed during pre-steady-state kinetics reflects the rate-limiting step of the penetration of the enzyme in the interface. This penetration is reversible. However, in the surface pressure range where the enzyme is able to hydrolyze the lecithin films, the desorption of the protein from the film is slow as compared to the adsorption. Below a surface pressure of 10 dyn/cm nonspecific adsorption occurs. Using lecithins with fatty acids of different chain lengths, we have shown that the kinetics of the penetration process is governed by the packing density of the substrate molecules independent of the surface pressure. However, the steady-state surface concentration of the enzyme increases with the fatty acyl chain length of the lecithin, indicating that hydrophobic interaction occurs between phospholipase A2 and the lipid molecules at the interface. From the lecithins used pancreatic phospholipase A2 preferentially splits substrate molecules with nine carbon atoms in the acyl chain.     

NMR Investigations of the interaction of water with lecithin in benzene solutions

NMR investigations of ¹H (chemical shifts, line widths) and of ³¹P (relaxation times, T₁) performed on the three-component system of lecithin-benzene-water show that there is an interaction of water with the phosphate group in two regions of different mobility and structure. A fast exchange of the water molecules takes place between both regions. The region of strong interaction involves about 2 and that of the weaker interaction about 5 water molecules per lecithin molecule. When the water concentration is increased a third region is formed which is assigned to the water molecules that are located beyond the two regions of interaction with the phosphate group, but within the micelle. This water has a different structure from that of the second region of interaction with the phosphate group and may also have a different mobility.Addition of water increases the motion of the head groups of the lecithin molecules. This is due to a loosening of the packing of lecithin molecules.     

The binary phase diagram of lecithin and cholesteryl linolenate

The condensed binary phase diagram of cholesteryl linolenate-egg yolk lecithin has been determined by polarizing light microscopy, differential scanning calorimetry and X-ray diffraction. On increasing the temperature lecithin forms rectangular, cubic and hexagonal liquid-crystalline structures into which varying amounts of cholesteryl linolenate are incorporated. As more cholesteryl linolenate is incorporated, the transition temperatures between different phases are lowered. The rectangular and cubic structures incorporate only small amounts of cholesteryl linolenate; the molar ratios, lecithin to cholesteryl linolenate, being 11:1 and 16:1, respectively. However, the hexagonal phase, in which the phosphorylcholine groups of the lecithin molecules form the core of the rod-like assembly of molecules, incorporates up to approximately 25% cholesteryl linolenate by weight, corresponding to a molar ratio 3:1. At higher concentrations, cholesteryl linolenate forms an excess phase and may be present as crystals, smectic or cholesteric liquid crystals, or as liquid oil, depending on the temperature. At higher temperatures, a large zone of a single isotropic liquid phase exists in which large amounts of lecithin are solubilized by the cholesterol ester. Up to 40% cholesteryl linolenate by weight, the transition temperatures between different phases are influenced by approximately 1% water (by weight) associated with egg lecithin.It is probable that the incorporated apolar cholesterol ester molecules are associated primarily with the apolar hydrocarbon chain region of the different lecithin structures. The resultant decrease in the observed transition temperatures would suggest an overall chain-disordering role for the incorporated cholesteryl linolenate molecules. The influence of cholesteryl linolenate on the thermodynamic stability of the different lecithin structures, together with the models suggested for the molecular orientations of cholesterol esters in the different liquid crystalline structures, may be relevant to the role of these lipids in more complex biological systems, particularly serum lipoproteins.     

Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.     

2D- and 3D-QSAR modelling,molecular docking and in vitro evaluation studies on 18β-glycyrrhetinic acid derivatives against triple-negative breast cancer cell line

Abstract

Triple-negative breast cancers (TNBCs) are one of the most aggressive and complex forms of cancers in women. TNBCs are commonly known for their complex heterogeneity and poor prognosis. The present work aimed to develop a predictive 2D and 3D quantitative structure–activity relationship (QSAR) models against metastatic TNBC cell line. The 2D-QSAR was based on multiple linear regression analysis and validated by Leave-One-Out (LOO) and external test set prediction approach. QSAR model presented regression coefficient values for training set (r²), LOO-based internal regression (q²) and external test set regression (pred_r²) which are 0.84, 0.82 and 0.75, respectively. Five properties, Epsilon4 (electronegativity), ChiV3cluster (valence molecular connectivity index), chi3chain (retention index for three-membered ring), TNN5 (nitrogen atoms separated through 5 bond distance) and nitrogen counts, were identified as important structural features responsible for anticancer activity of MDA-MB-231 inhibitors. Five novel derivatives of glycyrrhetinic acid (GA) named GA-1, GA-2, GA-3, GA-4 and GA-5 were semi-synthesised and screened through the QSAR model. Further, in vitro activities of the derivatives were analysed against human TNBC cell line, MDA-MB-231. The result showed that GA-1 exhibits improved cytotoxic activity to that of parent compound (GA). Further, atomic property field (APF)-based 3D-QSAR and scoring recognise C-30 carboxylic group of GA-1 as major influential factor for its anticancer activity. The significance of C-30 carboxylic group in GA derivatives was also confirmed by molecular docking study against cancer target glyoxalase-I. Finally, the oral bioavailability and toxicity of GA-1 were assessed by computational ADMET studies.

Communicated by Ramaswamy H. Sarma     

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.     

A spin probe study of the influence of cholesterol on motion and orientation of phospholipids in oriented multibilayers and vesicles

Spin probes have been used to study at the molecular level the influence of cholesterol on bilayers of egg lecithin and dipalmitoyl lecithin. Distinct differences between the two lecithin systems were revealed. Increasing amounts of cholesterol result in extension of the fatty acid chains and decreased amplitude of motion of the long axes of the fatty acids in egg lecithin. In dipalmitoyl lecithin cholesterol causes an increase in the mobility and amplitude of motion of the fatty acid side chains, presumably due to alteration of the molecular interactions between phospholipids by relaxing the close packing of these molecules. These data provide an explanation for the condensing and fluidizing effects of cholesterol in water-containing phases and monolayers of egg lecithin and dipalmitoyl lecithin, respectively, and for the permeability behavior of egg lecithin and dipalmitoyl lecithin liposomes in the presence and absence of cholesterol. Differences are revealed between the spin bilayer environments in hydrated phospholipid films and vesicles.     

Effect of calcium ions on the density of lecithin and its effective molecular volume in lecithin-water dispersions

As the packing structure of lipid molecules in the liposomes will vary in the presence of ions, it is expected that the density of lipid and the effective volume of lipid molecules in the dispersions will also vary, albeit minutely. Density measurements of lipid-water dispersions with the addition of Ca(2+) ions were determined accurately. The effect of Ca(2+) ions on the molecular packing structure of the liposomes was elucidated from the results obtained. The results for the density of the lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.0782 and 1.0579 g cm(-3) at 25 degrees C; and 1.0048 and 0.9961 g cm(-3) at 50 degrees C. The average values of the effective molecular volume of lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.131E-21 and 1.152E-21 cm(3) at 25 degrees C; and 1.213E-21 and 1.224E-21 cm(3) at 50 degrees C.     

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin:cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.