Nuclear magnetic resonance and thermal studies of drug doped dipalmitoyl phosphatidyl choline-H2O systems

The influence of the sulfone drugs, diamino diphenyl sulfone and diamino monophenyl sulfone on the phase transitions and dynamics of dipalmitoyl phosphatidyl choline-H₂O/D₂O vesicles have been investigated using differential scanning calorimetry and nuclear magnetic resonance. Our results show that diamino diphenyl sulfone interacts quite strongly with the headgroups of dipalmitoyl phosphatidyl choline whereas the diamino monophenyl sulfone-dipalmitoyl phosphatidyl choline interaction is quite weak. This is attributed to the difference in the structure and hydrophobic character of the two drugs.     

Differential scanning calorimetry of dipalmitoyl phosphatidylcholine analogues and of their interaction products with basic polypeptides

The thermotropic behaviour of dipalmitoyl phosphatidylcholine analogues with a varying number (n) of CH₂ groups between the phosphate and the quaternary ammonium has been investigated. The temperature (T_m) and the enthalphy (ΔH) of the phase transition are non-monotonous functions of the number of CH₂ groups. T_m oscillates between 40 and 45°C and ΔH between 7 and 13 kcal/mol for a variation of n between 2 and 11.It is concluded that the hydrocarbon chains in the head groups do not penetrate the hydrocarbon region and do not contribute directly to the melting of the acyl chains. It is suggested that their length may affect the critical ballance between the attractive and the repulsive forces within the bidimensional lattice of the head groups.Copolypeptides of lysine with phenylalanine do not appreciably affect the T_m but have a pronounced effect on ΔH of the lipid phase transition, which depends strongly on the ratio of the two amino acids in the polypeptide. The effect of copolypeptide of any defined composition on ΔH is also a non-monotonous function of the number of CH₂ groups in the phosphatidylcholine head group, but it does not parallel completely the oscilations in the T_m and ΔH of the pure lipids.     

Physicochemical properties of dipalmitoyl phosphatidylcholine after interaction with an apolipoprotein of pulmonary surfactant

We studied the interaction between an apolipoprotein of pulmonary surfactant and the principal lipid found in this material, dipalmitoyl phosphatidylcholine. The apolipoprotein was extracted from canine surfactant and purified to greater than 90% homogeneity. The apolipoprotein was mixed for 16 h at room temperature with dipalmitoyl phosphatidylcholine dispersed in a buffer containing 0.1 M NaCl and 3mM CaCl2. Unbound lipid, unbound protein, and recombinants of lipid and protein were separated by density gradient centrifugation. 71% of the apolipoprotein was found associated with dipalmitoyl phosphatidylcholine. In comparable experiments using bovine plasma albumin about 13% of the albumin was recovered with the lipid. The physicochemical state of the lipid in the apolipoprotein-lipid complex was modified after binding of the protein. A distinct phase transition at 42 degrees C could no longer be detected, and the rate of adsorption to an air-liquid interface of the apolipoprotein-lipid complex was greater than that of the lipid alone. Surface tension vs. surface area isotherms of the dipalmitoyl phosphatidylcholine-apolipoprotein materials, however, were similar to those exhibited by pure dipalmitoyl phosphatidylcholine. The results suggest a physiological role for this apolipoprotein. It may bind to dipalmitoyl phosphatidylcholine under conditions expected in vivo, and may modify the physical properties of the aggregated dipalmitoyl phosphatidylcholine to form domains of lipid in a liquid-crystalline array. The complex dipalmitoyl phosphatidylcholine and apolipoprotein would have the physical properties necessary for its physiological function, allowing it to absorb to the alveolar interface and reduce its surface tension to less than 10 dynes/cm. Dipalmitoyl phosphatidylcholine, by itself, is in a gel-crystalline array below its phase transition temperature (42 degrees C) and would be incapable of effecting these actions.     

Sarcoplasmic reticulum. XVI. The permeability of phosphatidyl choline vesicles for calcium

The Ca permeability of phosphatidyl choline vesicles of diverse fatty acid composition was measured. The rate of ⁴⁵Ca release from liposomes equilibrated with 1 mm⁴⁵CaCl₂ was found to be about 8 × 10⁻¹⁸ moles of Ca/cm²/sec for egg lecithin and about 5.3 × 10⁻¹⁷ moles of Ca/cm²/sec for dioleyllecithin at 30 °. Incorporation of cholesterol into dioleyllecithin micelles reduced the rate of Ca release. The Ca permeability of the phosphatidyl choline micelles was insensitive to changes in the pH, calcium or sodium concentration of the medium but increased with increasing temperature. The effect of temperature was most marked with dioleyl lecithin dispersions, but was clearly apparent with dipalmitoyl, plant, bovine, and egg lecithins as well. The activation energy of Ca release fell in the range of 4.2–9.6 kcal/mole. Macrocyclic antibiotics (valinomycin, tyrocidin, and gramicidin) at relatively high concentration increased the rate of Ca release similarly to their effects on fragmented sarcoplasmic reticulum membranes.     

Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (L_o)/liquid-disordered (L_d) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale L_o/L_d miscibility is higher for PDPC than for POPC ternary mixtures. At 37 °C, no micron-scale L_o/L_d phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the L_o domains observed in PDPC mixtures is assumed to result from the higher difference in L_o/L_d order parameter rather than hydrophobic mismatch.     

γ-Glutamyl 16-diaminopropane derivative of vasoactive intestinal peptide: a potent anti-oxidative agent for human epidermoid cancer cells

We previously demonstrated that the γ-glutamyl 16 amine derivative of vasoactive intestinal peptide (VIP) acts as structural VIP agonist with affinity and potency higher than VIP. Herein, we have evaluated the effects of VIP and γ-Gln16-diaminopropane derivative of VIP (VIP-DAP3) on the proliferation and protection from oxidative stress induced by hydrogen peroxide (H₂O₂) on epidermoid carcinoma cell lines. We have found that 10⁻¹¹ M VIP-DAP3 completely antagonized the inhibition induced by H₂O₂ on both cell proliferation and S-phase distribution while these effects were only partially antagonized by equimolar concentrations of VIP. Moreover, both oxidative stress and intracellular lipid oxidation induced by H₂O₂ were reduced by VIP and completely antagonized by VIP-DAP3. Thereafter, we have found that H₂O₂ increased p38 kinase activity and both HSP70 and HSP27 expression. VIP and VIP-DAP3 again antagonized these effects partially or totally, respectively. H₂O₂ reduced the activity of extracellular signal-regulated kinases Erk-1/2 and Akt, signalling proteins involved in proliferation/survival pathways. Again VIP restored the activity of both kinases while VIP-DAP3 caused indeed an increase of their activity as compared to untreated cells. These data suggest that VIP-DAP3 has a stronger anti-oxidative activity as compared to VIP likely based on its super-agonistic binding on the putative receptor.     

Stimulation and conformational change of G<Subscript>o</Subscript>α induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oα was highly purified from Escherichia coli cotransformed with pQE60 G_oα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G_oα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of G_oα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×10⁵ to (4.1±0.43) × 10⁵ (M⁻¹). However, no obvious changes of fluorescence spectra of G_oα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

Bradykinin-stimulated release of arachidonate from phosphatidyl inositol in mouse fibrosarcoma cells

We recently proposed a new pathway by which arachidonate is released from platelet phosphatidyl inositol after stimulation by either thrombin or calcium ionophore A23187. The initial step in arachidonate liberation involves hydrolysis of phosphatidyl inositol to form 1,2-diacylglycerol which is subsequently hydrolyzed by a diacylglycerol lipase to liberate arachidonate for the prostaglandin and lipoxygenase pathways. Whether this pathway is unique to platelets or accounts for arachidonate release from other tissues has not been previously studied. Thus we have now investigated arachidonate metabolism in mouse fibrosarcoma cells (HSDM₁C₁) grown in culture. These cells contain approximately 7.6% of their total phospholipid as phosphatidyl inositol in the resting state (range 6.5–8.3%). When bradykinin (12 μM) is added to the fibrosarcoma cells, there is a rapid depletion of membrane phosphatidyl inositol reaching 62 ± 8% S.D. of baseline values by 15 seconds, falling to 36 ± 6% by 15 minutes. The drop in membrane phosphatidyl inositol is accompanied by release of arachidonate and PGE₂ into the culture medium. The time course of phosphatidyl inositol breakdown and PGE₂ formation supports the idea that phosphatidyl inositol breakdown provides the arachidonate for prostaglandin synthesis in mouse fibrosarcoma cells. Crude extracts of HSDM₁C₁ cells contained sufficient phosphatidyl inositol-specific phospholipase C activity and diacylglycerol lipase activity to account for arachidonate release in these cells.     

Is Vitamin E Toxic to Neuron Cells?

Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of α-tocopherol (αT) in PKC pathway and the anti-cancer properties of γ-tocotrienol (γT3). This study aims to elucidate whether protective effects shown by αT and γT3 in H₂O₂-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H₂O₂ is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of αT or γT3 at concentrations ≤10 μM. Similar to our previous work, γT3 was found to be neurotoxic at concentrations ≥100 μM, whereas αT showed no neurotoxicity. Cellular uptake of γT3 was higher than that of αT. Treating cells simultaneously with either γT3 or αT and with then H₂O₂ led to higher expression of Bax and Bcl-2 than in neurons exposed to H₂O₂ alone. Analysis of Bcl-2/Bax ratio as ‘survival index’ showed that both pretreatment of γT3 and αT followed by H₂O₂ increase the ‘survival index’ of Bcl-2/Bax ratio compared to H₂O₂-treated cells, while treatment of γT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with αT alone. Similar treatment of γT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas αT did not alter its expression compared to H₂O₂-treated cells. Treating neurons with only γT3 or αT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with γT3 exerting stronger expression for proteins involved than αT. In conclusion, low doses of γT3 and αT confer neuroprotection to H₂O₂-treated neurons via their antioxidant mechanism but γT3 has stronger pro-apoptosis tendency than αT by activating molecules involved in the neuronal apoptotic pathway in the absence of H₂O₂.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Magnetic resonance studies on the binding of etomidate to lipid bilayers

Physicochemical studies on the binding of etomidate, a fast acting anaesthetic, with lipid bilayers have been carried out. ESR spin labeling studies indicate that the gel to liquid crystalline phase transition of dipalmitoyl phosphatidyl choline (DPPC) vesicles retains its cooperative nature on incorporation of the anaesthetic. For a 5:1 lipid to drug molar ratio, the phase transition occurs at an unusually lower temperature than those observed with other drug-DPPC systems. Results of 13C NMR and 1H NOE experiments suggest that the drug molecules reside in the close proximity of the terminal of hydrocarbon chains of the lipid molecules. 31P NMR and Electron Microscopic experiments indicate that the presence of etomidate alters the normal lamellar structure of DPPC vesicles into hexagonal (HII) type. Based on these observations, a model for drug-lipid binding has been proposed.     

Influence of surfactant protein C on the interfacial behavior of phosphatidylethanolamine monolayers

In the current work we study with monolayer tensiometry and Brewster angle microscopy (BAM) the surface properties of Dipalmitoleoylphosphatidylethanolamine (DPoPE) films at the air/water interface in presence and absence of specific surfactant protein C (SP-C). DPoPE is used, as it readily forms both lamellar (L_α) and non-lamellar inverted hexagonal (H_II) phases and appears as a suitable model phospholipid for probing the interfacial properties of distinct lipid phases. At pure air/water interface L_α shows faster adsorption and better surface disintegration than H_II phase. The interaction of DPoPE molecules with SP-C (predeposited at the interface) results in equalizing of the interfacial disintegration of the both phases (reaching approximately the same equilibrium surface tension) although the adsorption kinetics of the lamellar phase remains much faster. Monolayer compression/decompression cycling revealed that the effect of SP-C on dynamic surface tensions (γ _max and γ _min) of mixed films is remarkably different for the two phases. If γ _max for L_α decreased from the first to the third cycle, the opposite effect is registered for H_II where γ _max increases during cycling. Also the significant decrease of γ _min for L_α in SP-C presence is not observed for H_II phase. BAM studies reveal the formation of more uniform and homogeneously packed DPoPE monolayers in the presence of SP-C.     

Cooperative unit size in the gel-liquid crytalline phase transition of dipalmitoyl phosphatidylcholine-water multilayers: An estimate from raman spectroscopy

The temperature dependence of the Raman spectral transitions assigned to the acyl chain C-C stretching modes of dipalmitoyl phosphatidylcholine was determined for the gel, phase transition and liquid crystalline states of the lipid multilayers. The van't Hoff enthalpy differences $\text{ΔH}{}_{\mathrm{<mtext>VH</mtext>}}$ between trans and gauche rotational isomers were obtained from the Raman spectral data for the temperature region characteristics of each bilayer state. An average size for the cooperative unit undergoing the chain melting process during the phase transition was estimated from the ratio of the appropriate van't Hoff enthalpy to an adjusted calorimetric enthalpy.     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

Quantification of dipalmitoyl lecithin in lung wash and extracts

A quantitative method for the measurement of dipalmitoyl lecithin in lung wash and extracts has been described. A known amount of tritiated dipalmitoyl lecithin is added to lung wash or tissues prior to homogenization and extraction. The lipid extract is subjected to a sequence of chromatographic separations, and after each separation the amount of radioactivity and phosphorus is determined. Specific activity is calculated in terms of disintegrations per minute per micromole of phosphorus. Constant specific activity is achieved in the last two thin-layer separations. The losses incurred in the procedure are determined by the recovery of radioactivity in the final purification step; the total amount of dipalmitoyl lecithin present in the pulmonary extract is calculated after correcting for losses. The accuracy, sensitivity, and precision of the method have been evaluated by using adult and fetal lung extracts as well as fetal lung wash. The sensitivity was found to be 0.24 mg and the coefficient of variance ranged between 3.0 and 6.9% with an average of 5%. Changes in dipalmitoyl lecithin levels as well as wet lung weight, dry lung weight, protein, and DNA have been reported.     

Pulse NMR study of phase transitions in dipalmitoyl phosphatidylcholine multilayer systems

Pretransition and main transition of aqueous dipalmitoyl phosphatidylcholine (DPPC) dispersions were investigated by pulse NMR. The second moment M2 inter of the proton absorption line shows significant changes at 42 degrees C and about 35 degree C. Over the whole investigated temperature range between 25 and 50 degree C a superposition of at least two distinct second moments assigned to different molecular regions was observed.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.     

Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.