Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

The Kinetics of Hydrolysis of Animal Fat by Pancreatic Lipase

Hydrolysis of lipids from beef fat by pancreatic lipase was studied. The maximum release of free fatty acids was shown to occur at 40°C for the first 3 h of the experiment. After this, transetherification was predominant. The main kinetic parameters were the following: maximum hydrolysis rate, V = 1.25 ± 0.1 mg fat/ml min; Michaelis constant, K _M ^H = 100 ± 12 mg fat/ml; constant of substrate inhibition, K _S = 10.0 ± 0.8 mg fat/ml; equilibrium constant, K _P = 277 ± 170 mg fat/ml; and activation energy of beef fat hydrolysis by pancreatic lipase, E _a = 19.1 ± 1.1 kJ/mole. The kinetic method used could be applied to development of the method for biotransformation of poorly assimilated fats into more valuable products.     

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.     

Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus α-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H⁺, Zn²⁺ and Ca²⁺ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with α-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[¹²⁵I]iodophenyl)diazirine(¹²⁵I-TID) labels monomeric and—predominantly—hexameric forms of liposome-associated α-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H⁺ and Zn²⁺ but not of Ca²⁺—all of which inhibit calcein leakage—the distribution of label between hexamer and monomer is perturbed in favor of the latter. In α-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; they differ only in being more sensitive to ``closure' by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increases with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the α toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of α-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells. Received: 2 October 1995/Revised: 3 November 1995     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Are natural lipids UV-screening agents?

Summary Isolated lipids from Deinococcus radiodurans were reconstituted at final concentrations of 1 mg/ml into dioleoyl phosphatidyl choline (DOPC) vesicles and assayed for the ability to protect cells of Escherichia coli against killing by UV light (254 nm). Values of D₃₇ (UV dose required to reduce the number of surviving cells to 37% of the original number) were calculated from killing curves. E. coli was afforded the greatest protection with an individual lipid, identified as vitamin MK8 (D₃₇=310 J//m², compared to D₃₇=67 J/m² for E. coli irradiated in the presence of DOPC alone). Liposome-mediated protection was dependent on UV₂₅₄ absorbance and not on turbidity-related light-scattering. BOth vitamin MK8 from D. radiodurans and vitamin K₁, which is available commercially, showed a similar degree of UV₂₅₄-protection for E. coli. The UV-protective properties of vitamin K₁ were also investigated on mammalian cells in comparison with other natural lipids and known sunscreens. Survival curves were obtained for mouse fibroblast (L) cells irradiated at UV₂₅₄ in the absence or presence of DOPC liposomes into which were incorporated various natural lipids or standard sunscreen ingredients, all at final concentrations of 1 mg/ml. Experimentally determined values of D₃₇ were as follows: Vitamin K₁, 73 J/m²; \-carotene, 44 J/m₂; -tocopherol, 20 J/m₂; sulisobenzone, 156 J/m²; p-aminobenzoic acid (PABA), 113 J/m²; benzophenone, 80 J/m²; oxybenzone, 61 J/m² and DOPC alone. 23 J/m². Vitamin K₁, the most protective lipid tested, was also compared with PABA and oxybenzone (all at concentrations of 20 mg/ml; applied topicall) for its ability to protec Skh-hairless mice from UV₂₅₄-induced erythema, yielding a UV₂₅₄ protection factor of 3.5. In addition, vitamin K₁ (at 100 mg/ml) was able to provide hairless mice with a small degree of UVB protection, as indicated by an experimentally determined Solar Protection Factor of 1.5–2.0. Although it is concluded that vitamin K is not likely to account for the extraordinarily high degree of UV-resistance of D. radiodurans, vitamin K does show characteristics worthy of its consideration as a UV-screening agent. Offprint requests to: R. Anderson     

Toxicity and Biodistribution of Liposomes of the Main Phospholipid from the Archaebacterium Thermoplasma Acidophilum in Mice

Abstract

Toxicity and biodistribution of negatively charged liposomes of the main phospholipid (MPL) from the archaebacterium Thermoplasma acidophilum were tested in mice. MPL liposomes with a diameter of 160–220 nm were prepared by extrusion through polycarbonate filters, or by means of a French pressure cell and screened for central nervous system effects after intraperitoneal (i.p.) injection of 4–324 mg of liposomes per kg body weight in NMRI-mice. Besides increased behavioural activity no pharmacological or toxic effects were detected. No alterations were seen in the morphology of the tissues analyzed. Longterm toxicity after life-long oral application of 30 mg MPL per kg body weight per day starting at the age of 10 weeks was tested in immunosuppressed NMRI-mice. Again, there were no toxic effects on survival. Biodistribution of MPL liposomes labeled with ¹¹¹In-diethylenetriaminepentaacetic acid stearylamide was examined 15 min and 2.5 h after intravenous injection into ICR-mice. The liposomes were rapidly cleared from the circulation and the majority accumulated in the liver, followed by the spleen.     

PEGylation enhances the therapeutic potential of peptide antagonists of the neonatal Fc receptor, FcRn

Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease.     

Development and characterization of magnetic cationic liposomes for targeting tumor microvasculature

Cationic liposomes preferentially target tumor vasculature compared to vessels in normal tissues. The distribution of cationic liposomes along vascular networks is, however, patchy and heterogeneous. To target vessels more uniformly we combined the electrostatic properties of cationic liposomes with the strength of an external magnet. We report part I of development. We evaluated bilayer physical properties of our preparations. We investigated interaction of liposomes with target cells including the role of PEG (polyethylene-glycol), and determined whether magnetic cationic liposomes can respond to an external magnetic field. The inclusion of relatively high concentration of MAG-C (magnetite) at 2.5 mg/ml significantly increased the size of cationic liposomes from 105 ± 26.64 to 267 ± 27.43 nm and reduced the zeta potential from 64.55 ± 16.68 to 39.82 ± 5.26 mv. The phase transition temperature of cationic liposomes (49.97 ± 1.34 °C) reduced with inclusion of MAG-C (46.05 ± 0.21 °C). MAG-C cationic liposomes were internalized by melanoma (B16-F10 and HTB-72) and dermal endothelial (HMVEC-d) cells. PEG partially shielded cationic charge potential of MAG-C cationic liposomes, reduced their ability to interact with target cells in vitro, and uptake by major RES organs. Finally, application of external magnet enhanced tumor retention of magnetic cationic liposomes.     

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl₂. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl₂ the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K⁺ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl₂ and probably played a key role in determining growth capacity. (b) Internal concentrations of Na⁺, Ca²⁺, or Cl⁻ could not be directly correlated with growth. (C) Internal Mg²⁺ concentration could be significant only in the presence of high external Ca²⁺ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca²⁺)/(Na⁺)² concentration is crucial for growth. Ratios above 0.5 × 10⁻⁴ per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca²⁺)/(Na⁺)² ratio and the absolute external concentration of NaCl. However, a correlation between internal K⁺ concentration and growth capacity seemed independent of external NaCl concentration.     

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K _m and k _cat values are 0.4 mM and 15 sec^?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K _m and k _cat values are 1.3 mg/ml and 67 sec^?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K _m and k _cat values are 0.08 mM and 35 sec^?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.     

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol)

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.     

Immunologic response to protein immobilized on the surface of liposomes via covalent azo-bonding

A new method for immobilizing protein on the surface of liposomes is described. Inclusion of $\text{N-(p-}\text{aminophenyl)}$stearylamide in the lipid composition of vesicles resulted In liposomes that could be ‘activated’ by diazotization with NaNO₂/HCl, and subsequently coupled with protein. Using this method $\text{39.7 ? 7.5 μ}\text{g}$ egg albumin / μmol phospholipid has been coupled to multilamellar vesicles composed of phosphatidylcholine, cholesterol, and $\text{N-(p-}\text{aminophenyl}\text{)}$stearylamide in a molar ratio of 15:7.5:1.1. Furthermore, when the immunologic response of mice to egg albumin that was encapsulated in, nonspecifically adsorbed, or covalently linked to liposomes was investigated, only the covalent protein-liposome conjugates elicited pronounced and sustained elevations in antibody titers. These results suggest that the immunoadjuvant effects of liposomes can be maximized by covalently linking protein antigens to their surface.     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

Temporal variability and production of Euterpina acutifrons (Copepoda: Harpacticoida) in the Cananéia Lagoon estuarine system,São Paulo,Brazil

Diel and seasonal variations in abundance, population structure, biomass and production rate of the harpacticoid copepod Euterpina acutifrons were studied in the Cananéia Lagoon estuarine system, São Paulo, Brazil. Zooplankton samples were collected at 4-h intervals during multiple 24-h periods, from February 1995 to January 1996. Copepodites and adults of E. acutifrons were present in the plankton throughout the year (temperature, 18.6–29.4 °C; salinity, 4.5–33.0 psu; chlorophyll-a concentration, 1.32–20.42 g l^–1). Abundance of E. acutifrons showed considerable diel variations. On most sampling dates, higher abundances were recorded at times when salinity was higher. Biomass varied from 0.044 ± 0.046 (daily mean ± SD) to 5.264±3.425 mg C m^–3. The estimated production rates (minimum ± SD–maximum ± SD) were 0.034±0.035–4.95±3.25 (Ikeda-Motoda model), 0.035±0.036–5.123±3.347 (Huntley-Lopez model), and 0.016±0.017–2.101±1.372 mg C m^–3 d^–1 (Hirst-Sheader model).     

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C _t (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C _t decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.     

Conserved residues in the aromatic acid/H symporter family are important for benzoate uptake by NCgl2325 in Corynebacterium glutamicum

Corynebacterium glutamicum is a model organism for genetic and physiological studies in Gram-positive bacteria. NCgl2325 in C. glutamicum, a transporter belonging to the aromatic acid/H⁺ symporter family, has previously been reported to be involved in benzoate assimilation. Here, we showed that this transporter, fused with GFP, was associated with the cell membrane in Escherichia coli and C. glutamicum. Uptake assays with [¹⁴C]-labeled benzoate demonstrated that NCgl2325 transported benzoate into the cells at a V_max of 0.19 ± 0.01 nmol/min/mg of dry weight, and the K_m value was determined to be 1.11 ± 0.24 ??M. Among the competing substrates tested, hydroxyl-substituted benzoates resulted in significant inhibition (>50%) of benzoate uptake. Site-directed mutagenesis of conserved residues in the hydrophilic cytoplasmic loops (Gly-80, Asp-84 and Asp-312) and the hydrophobic transmembrane regions (Asp-35, Arg-119, Glu-139 and Arg-386) resulted in loss of benzoate transport activity. This is the first study to investigate the molecular basis of benzoate transport.