Anomalies of nanosecond ultrasonic relaxation in the lipid bilayer transition.

The frequency dependence of ultrasonic velocity as well as absorption in a suspension of sonicated dipalmitoylphosphatidylcholine vesicles was measured by a differential ultrasonic resonator. The frequency was scanned between 1.3 and 13 MHz and the temperature was varied from 25 to 47 degrees C. A pronounced relaxation was observed in the time range of 10 ns. The data were analyzed assuming a single relaxation which appeared to be a good approximation. The relaxation time as well as relaxation strength increased anomalously in the vicinity of the gel-to-liquid crystal transition of 41.5 degrees C. This result represents the first definite evidence of the critical slowing down in the lipid bilayer and is discussed in terms of the Landau theory of phase transition. The possible biological significance of the mechanical relaxation is also presented.

Degree of dissociation of apohemoglobin studied by nano-second fluorescence-polarization technique.

A fluorescent dye 1-anilino-8-naphthalene sulfonate was complexed with human apohemoglobin and sperm whale apomyoglobin. Nanosecond fluorescence-polarization kinetics were measured for each of these complexes in KC1 solutions to obtain their fluorescence lifetimes and rotational correlation times. The rotational correlation time of apohemoglobin-dye complex was found to be 21 ns, which was about twice that of apomyoglobin-dye complex, 11 ns. These values were constant over an ionic strength range from 0 to 1.7. Circular dichroism spectra (215-300 nm) and fluorescence lifetimes of the complexes were also found to be independent of the ionic strength, indicating that no gross conformational change occurs with the change in the salt concentration, These results suggest that apohemoglobin remains dimeric over the ionic-strength range examined.

Localization of the galactoside binding site in the lactose carrier of Escherichia coli.

The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers. The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules. The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids. By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined. The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion. The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore.

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Glöckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.

Synthesis and cytotoxic and antitumor activity of esters in the 1,2-dihydroxy-1,2-dihydroacronycine series.

Seven 1,2-dihydroxy-1,2-dihydroacronycine and 1,2-dihydroxy-1,2-dihydro-6-demethoxyacronycine esters and diesters were synthesized via osmic oxidation of acronycine or 6-demethoxyacronycine followed by acylation. The 6-demethoxyacronycine derivatives were found to be inactive, whereas in contrast, all of the acronycine derivatives were more potent than acronycine itself when tested against L1210 cells in vitro. Four selected acronycine derivatives (17,19, 21, and 22) were evaluated in vivo against murine P388 leukemia and colon 38 adenocarcinoma implanted in mice. All compounds were markedly active against P388 at doses 4-16-fold lower than acronycine itself. Against the colon 38 adenocarcinoma, the three compounds 17, 21, and 22 were highly efficient. 1,2-Diacetoxy-1,2-dihydroacronycine (17) was the most active, all the treated mice being tumor-free on day 23.

Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy.

A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported. These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase. As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region. The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines. The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues. After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials.

CYTOTOXIC ACTIVITY OF KAEMPFEROL GLYCOSIDES AGAINST HUMAN LEUKAEMIC CELL LINES IN VITRO.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control. 2000 Academic Press@p$hr Copyright 2000 Academic Press.

Megistosarcimine and megistosarconine, two alkaloids from Sarcomelicope megistophylla.

Two new alkaloids, megistosarcimine and megistosarconine, were isolated from the aerial parts of Sarcomelicope megistophylla. Their structures have been elucidated on the basis of their spectral data and molecular modeling.

Furomegistines I and II, two furanopyridine alkaloids from the bark of Sarcomelicope megistophylla.

Two alkaloids, furomegistine I (1) and furomegistine II (2), were isolated from the bark of Sarcomelicope megistophylla. Their structures have been elucidated on the basis of MS and NMR data. Both belong to the category of furanopyridine alkaloids and should be considered as oxidation products of a furo[2,3-b]quinoline precursor. The two alkaloids exhibited moderate cytotoxic activity.

Ultrasonic measurements of two-component lipid bilayer suspensions.

Two-component lipid bilayers of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine were studied by measuring ultrasonic velocity and absorption at 3 MHz. The phase diagram of the two-component lipid bilayers is discussed based upon the transition anomalies of the ultrasonic velocity as well as absorption, and it is suggested that this binary system has two critical points. The bulk modulus of lipid bilayers was determined from the ultrasonic velocity to be (2.2-3.0) x 10(10) dyne/cm2, whereas the bulk viscosity calculated from the absorption was 10-20 P except for the transition regions.

A theoretical method for distinguishing between soluble and membrane proteins.

A method for distinguishing between membrane and soluble proteins in an amino acid sequence was developed, using only two parameters associated with the hydrophobicity: the average hydrophobicity and the power spectral density of period longer than 30 residues. The power spectral density was calculated by a maximum entropy method of Fourier transformation. Membrane proteins could be distinguished from soluble proteins with a distinction rate as high as 97%. This fact strongly suggests that the morphology of proteins, i.e., membrane or soluble forms, is determined thermodynamically through the hydrophobicity of polypeptides.

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes.

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. I. Transition anomalies of ultrasonic properties.

The effect of Ca2+ on a gel-to-liquid crystal transition as well as the mechanical properties of dipalmitoylphosphatidylcholine bilayers was studied by an ultrasonic technique. Transition temperature increased with increase in Ca2+ concentration, whereas the variation of ultrasonic anomalies indicated that dipalmitoylphosphatidylcholine bilayers exhibited maximum pseudocritical fluctuation at a Ca2+ concentration of about 10 mM. Hardening of dipalmitoylphosphatidylcholine membranes due to the Ca2+ binding was observed above 10 mM CaCl2, suggesting the lateral compression of the lipid bilayer by bound Ca2+. Long-range attraction between bound Ca2+ and the head groups of surrounding lipid molecules was proposed from these calcium effects.

Dynamic properties of concentrated suspensions of charged polystyrene spheres.

Ordered structure formation of charged polystyrene spheres was studied by measuring the order-disorder phase diagrams as well as the mechanical properties. The phase diagrams indicate that the ordering of polystyrene spheres obeys Lindemann's law of crystal melting, in which the Lindemann's parameter is about 5%. The rigidity of about 10(3) dyn/cm(2) was observed in the ordered suspension of polystyrene spheres as measured by a torsional quartz crystal method. The steady flow properties of suspensions of polystyrene spheres showed a remarkable change from a Bingham body to a Newtonian liquid at the transition point. The limit of elasticity in the ordered phase was about 1 dyn/cm(2). The viscosity in the disordered phase was well explained by the free volume theory of liquids. It is concluded from these facts that the ordered phase of polystyrene spheres is a real "crystal" whereas the disordered phase is a "liquid". Properties of ordered structures in biological systems are also discussed.

Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system.

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378-379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15-20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. II. Fluorescence anisotropy study.

The effect of Ca2+ on the molecular mobility in dipalmitoylphosphatidylcholine membranes was studied by steady-state and time-resolved measurements of fluorescence anisotropy. The fluorescence anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene in the hydrocarbon region indicated that the free volume of molecular rotation became more restricted when the Ca2+ concentration was increased. The decrease of the molecular mobility was observed from 1 mM Ca2+, at which the number of bound Ca2+ is much less than that of the total lipid molecules. A distinct difference between Ca2+ and Mg2+ effects suggested that the change in various membrane properties was induced by the binding of these ions. From these results we propose a long-range attractive interaction between bound Ca2+ and the polar head groups of distant phosphatidylcholine molecules.

Hydrophobic core of molten-globule state of bovine carbonic anhydrase B.

The interaction which stabilizes the intermediate state of the protein folding and/or unfolding is important for understanding the structure formation mechanism of proteins. The partitioning of a hydrophobic fluorescence probe, pyrene, into the core of a 'molten globule' structure of bovine carbonic anhydrase B was measured, revealing a partition coefficient of about 10(4). The result leads to the conclusion that the compact structure of the molten-globule state is formed by the hydrophobic interaction, as detergent micelles are formed by the same interaction.

Denaturation of bacteriorhodopsin by organic solvents.

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.

Cytotoxic activity and antiproliferative effects of a new semi-synthetic derivative of Ent-3 beta-hydroxy-13-epi-manoyl oxide on human leukemic cell lines.

Ent-3 beta-hydroxy-13-epi-manoyl oxide (1), was converted to its thiomidazolide derivative (2) which was tested for its cytotoxic activity against a panel of established human leukemic cell lines. Compound 2, exhibited cytotoxic activity against 13 of the cell lines tested. Additionally, compound 2 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis and on cell proliferation. The morphology of the cells and the kind of death induced, was investigated. Flow cytometry experiments on a leukemic cell line was also performed. The results show that the semi-synthetic compound, showed a significant antiproliferative effect and kills cells through the process of apoptosis. The appearance of the apoptotic signs was time and dose dependent. From the flow cytometry experiments, a synchronisation through a delay of the cells in G0/1, phase seems to take place.

Cytotoxicity against human leukemic cell lines, and the activity on the expression of resistance genes of flavonoids from Platanus orientalis.

The cytotoxic activity of three flavonoids, belonging to the kaempherol series, was evaluated against 15 human leukemic cell lines. Flavonoids bearing acyl substituants, 2 and 3, were found to be the most active compounds. A further compound, 1, was examined for its ability to modulate the expression of MDR-1 and GST-pi resistance genes and compounds 2 and 3 for their effect on the uptake of [3H]-thymidine as a marker of DNA synthesis.