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PubMed2001

Biochemical and molecular characterization of Staphylococcus simulans lipase.

Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric protein (160 kDa) corresponding to the association of four lipase molecules. The 30 N-terminal amino acid residues were sequenced. This sequence is identical to the one of Staphylococcus aureus PS54 lipase (SAL PS54) and exhibits a high degree of homology with Staphylococcus aureus NCTC8530 lipase (SAL NCTC8530), Staphylococcus hyicus lipase (SHL) and Staphylococcus epidermis RP62A lipase (SEL RP62A) sequences. But the cloning and sequencing of the part of the gene encoding the mature lipase show some differences from SAL PS54 sequence, which suggest that it is a new sequence. The lipase activity was maximal at pH 8.5 and 37 degrees C. SSL is able to hydrolyze triacylglycerols without chain length specificity. A specific activity of about 1000 U/mg was measured on tributyrin or triolein as substrate at 37 degrees C and at pH 8.5 in the presence of 3 mM CaCl(2). In contrast to other staphylococcal lipases previously characterized, Ca(2+) is not required to express the activity of SSL. SSL was found to be stable between pH 4 and pH 9. The enzyme is inactivated after a few minutes when incubated at 60 degrees C. Using tripropionin as substrate, SSL does not present the interfacial activation phenomenon. In contrast to many lipases, SSL is able to hydrolyze its substrate in the presence of bile salts or amphiphilic proteins.

Amino Acid Sequence↗
PubMed2005

Preparation of alkanethiol monolayers on mild steel surfaces studied with sum frequency generation and electrochemistry.

An n-alkanethiol, octadecanethiol (ODT), monolayer was successfully prepared onto an oxide-free mild steel (MS) surface under cathodic polarization in a 0.1 M LiCl/CH(3)OH solution containing 1 mM ODT. Cyclic voltammetry (CV) and electrochemical impedance (EIS) and sum frequency generation (SFG) spectroscopy were applied to study and characterize the adsorption of ODT at a MS surface. In 0.1 M LiCl/CH(3)OH solution containing 1 mM ODT, CV of the MS electrode shows a dramatic decrease in charging current and a positive shift in oxidation potential when compared to a solution without ODT. The interfacial capacitance was obtained as 2.52 microF/cm(2) from the impedance data. An average chain tilt angle of 48 degrees for the ODT molecules was deduced from the comparison of the interfacial capacitances of the ODT/MS and ODT/Au monolayers. X-ray photoelectron spectroscopy confirmed the formation of the ODT monolayer on mild steel. The ppp SFG spectrum of the ODT-modified MS features three strong methyl vibrational modes at 2877, 2943, and 2967 cm(-1), indicating the formation of the oriented and densely packed ODT monolayer. However, the appearance of the two weak CH(2) groups' vibrational modes at 2850 and 2914 cm(-1) implies the presence of defects in the ODT monolayer. ODT/Au films were prepared to compare with the ODT/MS films. Orientation analysis of the air/solid interface suggests that the methyl group of ODT/Au films has a tilt angle of 30 degrees , while the methyl group of ODT/MS films has a tilt angle of 23 degrees . Water was found to have an impact on the shape of the SFG spectra of ODT/MS. This suggests that the solution penetrated through the defects to reach the MS surface.

Journal Article↗
PubMed2000

Horizontal transmission of Amblyospora albifasciati García and Becnel, 1994 (Microsporidia: amblyosporidae), to a copepod intermediate host and the neotropical mosquito, Aedes albifasciatus (Macquart, 1837).

The life cycle of Amblyospora albifasciati is characterized by three sporulation sequences involving the definitive mosquito host and a copepod intermediate host. Meiospores of A. albifasciati were infectious per os to adult females of the copepod Mesocyclops annulatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sporontogenic interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of M. annulatus, were large, pyriform, and measured 10.4 x 4.8 microm. They infected Aedes albifasciatus larvae when ingested to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oval binucleate spores (9.3 x 3.1 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation.

Aedes↗
PubMed2001

Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images.

The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation. An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed. The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method. OTS is based on the statistical properties of local gray-values and gradients in the image. By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition. The automated OTS method was shown to reproduce values obtained manually by an experienced operator.

Automation↗
PubMed2004

The affinity of GXXXG motifs in transmembrane helix-helix interactions is modulated by long-range communication.

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and approximately 18 A in structural space), work in concert to control TM-TM affinity synergistically.

Amino Acid Motifs↗
PubMed2002

Dynamic light scattering from colloidal fractal monolayers.

We address experimentally the problem of how the structure of a surface monolayer determines the viscoelasticity of the interface. Optical microscopy and surface quasielastic light scattering have been used to characterize aggregation of CaCO3 particles at the air-water interface. The structures formed by cluster-cluster aggregation are two-dimensional fractals that grow to eventually form a percolating network. This process is measured through image analysis. On the same system we measure the dynamics of interfacial thermal fluctuations (surface ripplons), and we discuss how the relaxation process is affected by the growing clusters. We show that the structures start damping the ripplons strongly when the two length scales are comparable. No macroscopic surface pressure is measured and this is in contrast to lipid, surfactant, or polymer monolayers at concentrations corresponding to surface coverage. This observation and the difficulty in fitting the ripplon spectrum with traditional models suggest that a different physical mechanism might be responsible for the observed damping of ripplons in this system.

Journal Article↗
PubMed2000

Characterization of two Acacia gums and their fractions using a langmuir film balance.

The mechanical properties of monolayers from two Acacia gums [Acacia senegal (L.) Willd. and Acacia seyal Del.] and their three fractions isolated by hydrophobic interaction chromatography were studied with a Langmuir film balance to obtain a more complete understanding of their action mode. The analysis of compression isotherms revealed that A. senegal gums globally exhibit better interfacial properties than A. seyal ones. The behavior of the whole gums appeared to be strongly influenced by their arabinogalactan-protein complex.

Chromatography↗
PubMed1987

Surface characterization of heparin-complexing poly(amido amine) chains grafted on polyurethane and glass surfaces.

Poly(amido-amine) chains grafted onto polyurethanes and glass form stable complexes with heparin yielding potential nonthrombogenic surfaces. The characterization of the surfaces, and the product of each chemical reaction including final heparinized surfaces, has been studied by contact angle data and scanning electron microscopy (SEM). Air in water, octane in water, and drop-on-plate contact angle data were used to estimate surface (gamma sv) and interfacial (gamma sw) free energies. Solid-water work of adhesion (Wa) and its dispersive (Wda) and polar (Ipsw) components were calculated for all studied surfaces. It has been found that the viscosity of polyurethane solution used for film casting influences wetting properties of these films. It has also been found that a direct correlation exists between the Ipsw/Wda values and the degree of coverage of the surfaces by cellular deposits after their exposure to platelet-rich plasma. Final heparinized polyurethane and glass materials are hydrophilic, their Ipsw/Wda ratio is high, and little or no cellular deposit is observed on their surfaces.

Biocompatible Materials↗
PubMed1995

Effect of surfactants on the physical stability of recombinant human growth hormone.

The physical stability of a human growth hormone (hGH) formulation upon exposure to air/water interfaces (with vortex mixing) and to nonisothermal stress [determined by differential scanning calorimetry (DSC)] was investigated. The effect of these stresses on the formation of soluble and insoluble aggregates was studied. The aggregates were characterized and quantified by size exclusion-HPLC and UV spectrophotometry. Vortex mixing of hGH solutions (0.5 mg/mL) in phosphate buffer, pH 7.4, for just 1 min caused 67% of the drug to precipitate as insoluble aggregates. These aggregates were noncovalent in nature. Non-ionic surfactants prevented the interfacially induced aggregation at their critical micelle concentration (cmc) for Pluronic F-68 (polyoxyethylene polyoxypropylene block polymer) and Brij 35 (polyoxyethylene alkyl ether) and above the cmc for Tween 80 (polyoxyethylene sorbitan monooleate). However, the same surfactants failed to stabilize hGH against thermal stress in DSC studies. Higher concentrations of surfactants actually destabilized hGH as evidenced by the decrease in the onset temperature for the denaturation endotherm.

Chromatography, High Pressure Liquid↗
PubMed1997

Interactions between Talc Particles and Water and Organic Solvents

Three talc samples have been studied by adsorption and immersion methods after a classical characterization of their properties. The combination of adsorption isotherms and of immersion measurements allows the calculation of enthalpies and entropies of adhesion. The studied talcs are characterized as "middle energy" solids. The differences between the particle shapes of the different samples are shown to be of great importance, indicating a linkage between cristallinity and surface properties. The whole results are explained by the influence of intermolecular forces such as acid-base interactions in the interfacial layer. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed2004

Interaction of antiinflammatory drugs with EPC liposomes: calorimetric study in a broad concentration range.

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (K(p)), and/or the equilibrium constant (K(b)). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DeltaH, and indirectly, DeltaG and DeltaS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction-possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity.

The main objective of this study was to develop a polymeric drug delivery system for paclitaxel, intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of Cremophor EL. To achieve this goal paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-Nps) were prepared by the interfacial deposition method. The influence of different experimental parameters on the incorporation efficiency of paclitaxel in the nanoparticles was evaluated. Our results demonstrate that the incorporation efficiency of paclitaxel in nanoparticles was mostly affected by the method of preparation of the organic phase and also by the organic phase/aqueous phase ratio. Our data indicate that the methodology of preparation allowed the formation of spherical nanometric (<200 nm), homogeneous and negatively charged particles which are suitable for intravenous administration. The release behaviour of paclitaxel from the developed Nps exhibited a biphasic pattern characterised by an initial fast release during the first 24 h, followed by a slower and continuous release. The in vitro anti-tumoral activity of Ptx-PLGA-Nps developed in this work was assessed using a human small cell lung cancer cell line (NCI-H69 SCLC) and compared to the in vitro anti-tumoral activity of the commercial formulation Taxol. The influence of Cremophor EL on cell viability was also investigated. Exposure of NCI-H69 cells to 25 microg/ml Taxol resulted in a steep decrease in cell viability. Our results demonstrate that incorporation of Ptx in nanoparticles strongly enhances the cytotoxic effect of the drug as compared to Taxol, this effect being more relevant for prolonged incubation times.

Chemical Phenomena↗
PubMed2006

Gate dielectric chemical structure-organic field-effect transistor performance correlations for electron, hole, and ambipolar organic semiconductors.

This study describes a general approach for probing semiconductor-dielectric interfacial chemistry effects on organic field-effect transistor performance parameters using bilayer gate dielectrics. Organic semiconductors exhibiting p-/n-type or ambipolar majority charge transport are grown on six different bilayer dielectric structures consisting of various spin-coated polymers/HMDS on 300 nm SiO(2)/p(+)-Si, and are characterized by AFM, SEM, and WAXRD, followed by transistor electrical characterization. In the case of air-sensitive (generally high LUMO energy) n-type semiconductors, dielectric surface modifications induce large variations in the corresponding OTFT performance parameters although the film morphologies and microstructures remain similar. In marked contrast, the device performance of air-stable n-type and p-type semiconductors is not significantly affected by the same dielectric surface modifications. Among the bilayer dielectric structures examined, nonpolar polystyrene coatings on SiO(2) having minimal gate leakage and surface roughness significantly enhance the mobilities of overlying air-sensitive n-type semiconductors to as high as approximately 2 cm(2)/(V s) for alpha,omega-diperfluorohexylcarbonylquaterthiophene polystyrene/SiO(2). Electron trapping due to silanol and carbonyl functionalities at the semiconductor-dielectric interface is identified as the principal origin of the mobility sensitivity to the various surface chemistries in the case of n-type semiconductors having high LUMO energies. Thiophene-based n-type semiconductors exhibiting similar film morphologies and microstructures on various bilayer gate dielectrics therefore provide an incisive means to probe TFT performance parameters versus semiconductor-dielectric interface relationships.

Journal Article↗
PubMed2006

Surface properties of dioleoyl-sn-glycerol-3-ethylphosphocholine, a cationic phosphatidylcholine transfection agent, alone and in combination with lipids or DNA.

Long-chain cationic amphipaths are routinely used for transfecting DNA into cells, although the mechanism of DNA delivery by these agents is poorly understood. Since their interfacial properties are undoubtedly involved at some stage in the process, a comprehensive study of the surface behavior of at least one of these compounds is highly desirable. Hence, the behavior of the cationic transfection agent EDOPC (dioleoyl-sn-glycerol-3-ethylphosphocholine or O-ethyldioleoylphosphatidylcholine), has been characterized at the air-water interface, by itself and in mixtures with other phospholipids. Surface pressure-molecular area isotherms obtained at the argon-buffer interface revealed that EDOPC is considerably (5-10 A(2)) more expanded than the parent phosphatidylcholine (DOPC) and even more expanded than the corresponding phosphatidylglycerol (DOPG), which has a similar charge density (of opposite polarity) as EDOPC. A 1:1 mixture of EDOPC and DOPG is very slightly condensed relative to DOPG and considerably condensed relative to EDOPC. The surface/dipole potential of this mixture is the mean of those of EDOPC and DOPG and is almost the same as that of DOPC. When the composition of EDOPC mixtures was varied, several surface parameters, including surface dipole moment, collapse pressure, and compressibility, exhibited discontinuities at a 1:1 mole ratio. EDOPC is unusually surface-active; the equilibrium surface tension of its dispersion was lower and the rate of fall of the surface tension (dynamic surface activity) of a dispersion with an initially clean surface was more than an order of magnitude greater than that for dispersions of DOPG. A 1:1 mixture of the cationic lipoid and phosphatidylglycerol had lower surface activity than DOPC in water but similar surface activity in 0.1 NaCl. Analysis, in terms of surface concentration, of the formation of EDOPC monolayers at the air interface of vesicle dispersions revealed a simple exponential rise to a maximum, at least for higher concentrations. Addition of a small proportion of DNA to EDOPC increased its dynamic surface activity even though DNA alone has no detectable surface activity at the concentrations used. This enhancement by DNA is presumably due to the disruption of the continuity of the bilayer and creation of defects from which lipoid spreads readily. The surface properties of this cationic compound, both alone and in combination with anionic lipids, provide insight into the previously described nonbilayer phase preferences of cationic-anionic lipid mixtures. In addition, they provide critical data (area condensation of mixed cationic-anionic monolayers) supporting a previously proposed mechanism of fusion of cationic bilayers with anionic bilayers. Such a process, involving anionic cellular membranes, is believed to be required for release of DNA from lipoplexes and is therefore a key stage of transfection.

Argon↗
PubMed1991

Purification and biochemical characterization of dog gastric lipase.

A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.

Amino Acid Sequence↗
PubMed1994

Interactions of synthetic peptide analogs of the class A amphipathic helix with lipids. Evidence for the snorkel hypothesis.

Class A amphipathic helixes present in exchangeable plasma apolipoproteins are characterized by the location of positively charged amino acid residues at the non-polar-polar interface and negatively charged amino acid residues at the center of the polar face. The objectives of the present study were: (i) to investigate the role of hydrocarbon side chain length of the interfacial positively charged amino acid residues in the lipid affinity of class A amphipathic helixes, and (ii) to investigate the importance of the nature of interfacial charge in the lipid affinity of class A amphipathic helixes. Toward this end, lipid interactions of the following two analogs of the class A amphipathic helix, Ac-18A-NH2 (acetyl-Asp-Trp-Leu-Lys-Ala-Phe-Tyr- Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe-NH2), and Ac-18A(Lys > Haa)-NH2 (acetyl-Asp-Trp-Leu-Haa-Ala-Phe-Tyr-Asp-Haa-Val-Ala-Glu-Haa-Leu-Haa-Glu- Ala-Phe-NH2) (Haa = homoaminoalanine), were studied. The side chain of Haa has two CH2 groups less than that of lysine. The lipid affinities of these two peptide analogs were compared with that of Ac-18R-NH2, an analog of Ac-18A-NH2 with positions of the charged amino acid residues reversed. The techniques used in these studies were circular dichroism, fluorescence spectroscopy, right-angle light scattering measurements, and differential scanning calorimetry. The results of these studies indicated the following rank order of lipid affinity: Ac-18A-NH2 > Ac-18A(Lys > Haa)-NH2 > Ac-18R-NH2. These results are in agreement with the "snorkel" model proposed earlier to explain the higher lipid affinity of class A amphipathic helixes (Segrest, J. P., Loof, H. D., Dohlman, J. G., Brouillette, C. G., and Anantharamaiah, G. M. (1990) Proteins Struct. Funct. Genetics 8, 103-117). In addition, it was observed from the differential scanning calorimetry studies that Ac-18A-NH2 and Ac-18A(Lys > Haa)-NH2 interact more strongly than Ac-18R-NH2 with negatively charged dimyristoyl phosphatidylglycerol. The weaker interaction of Ac-18R-NH2 with dimyristoyl phosphatidylglycerol is suggested to be due to electrostatic repulsion between the negatively charged lipid and the interfacial negative charges of the peptide.

Amino Acid Sequence↗
PubMed2001

Electrochemical evaluation of the interfacial capacitance upon phosphorylation of amino acid analogue molecular films.

An approach based on electrochemistry to differentiate between phosphorylated and nonphosphorylated amino acid analogues adsorbed on gold is presented. Analogues of serine, threonine, and tyrosine, containing thiohexadecyl headgroups, were synthesized and assembled on gold, and the surface capacitance was evaluated using electrochemical impedance spectroscopy. A procedure for deprotection of tert-butyl phosphate protecting groups, on the monolayer, is also described. Characterizations of the assembled analogues by cyclic voltammetry, infrared spectroscopy, and ellipsometry are used to confirm the insulating properties of the monolayers and the outcome of surface modifications. The results from cyclic voltammetry show good insulating properties for the monolayers even after phosphate deprotection. The infrared measurements reveal well-ordered monolayers, and the thickness from ellipsometry is in good agreement with expectations from molecular modeling. The impedance experiments show a capacitance increase up to 0.6 microF/cm2 as phosphate groups are introduced. The results in this study indicate the possibility of using a surface chemical and impedance spectroscopy approach to detect the kinase/phosphatase activity and kinetics involved in phosphorylation reactions.

Amino Acids↗
PubMed1989

Xanthene-dye-labelled phosphatidylethanolamines as probes of interfacial pH. Studies in phospholipid vesicles.

We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.

Cell Membrane↗