Partial Charge Neutralization Research Articles

Synthesis of Crosslinked Poly(Vinylphosphonic Acid) Particles by Water-in-Oil (W/O) Suspension Polymerization Method and Their Adsorption Properties of Metal Ions

Crosslinked poly(vinylphosphonic acid) (PVPA) particles were successfully synthesized from vinylphosphonic acid and a crosslinker by water-in-oil (W/O) suspension polymerization using three silicone oils. The structure, morphology, and size of the PVPA particles were confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, and optical microscopy. The surface of the spherical particles was wrinkled, and their size ranged from 5 – 260 µm. The adsorption of divalent and trivalent metal ions (Cu2+, Zn2+, La3+, Tb3+, Lu3+) from aqueous solutions was studied using the synthesized PVPA particles under batch conditions at various metal ion concentrations and pHs for the first time. For all metal ions, pH-dependent adsorptions were observed. Partial charge neutralization of metal ions occurred because the PVPA particles are highly hydrophilic. The extraction efficiencies of La3+, Tb3+, and Lu3+ are higher than those of Cu2+ and Zn2+. The separation ability for La3+, Tb3+, and Lu3+ is not so high, but they can be used for the group extraction of these metal ions.

Simvastatin Coadministration Modulates the Electrostatically Driven Incorporation of Doxorubicin into Model Lipid and Cell Membranes.

Understanding the interactions between drugs and lipid membranes is a prerequisite for finding the optimal way to deliver drugs into cells. Coadministration of statins and anticancer agents has been reported to have a positive effect on anticancer therapy. In this study, we elucidate the mechanism by which simvastatin (SIM) improves the efficiency of biological membrane penetration by the chemotherapeutic agent doxorubicin (DOX) in neutral and slightly acidic solutions. The incorporation of DOX, SIM, or a combination of them (DOX:SIM) into selected single-component lipid membranes, zwitterionic unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), neutral cholesterol, and negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) was assessed using the Langmuir method. The penetration of neutral lipid monolayers by the codelivery of SIM and DOX was clearly facilitated at pH 5.5, which resembles the pH conditions of the environment of cancer cells. This effect was ascribed to partial neutralization of the DOX positive charge as the result of intermolecular interactions between DOX and SIM. On the other hand, the penetration of the negatively charged DMPS monolayer was most efficient in the case of the positively charged DOX. The efficiency of the drug delivery to the cell membranes was evaluated under in vitro conditions using a panel of cancer-derived cell lines (A172, T98G, and HeLa). MTS and trypan blue exclusion assays were performed, followed by confocal microscopy and spheroid culture tests. Cells were exposed to either free drugs or drugs encapsulated in lipid carriers termed cubosomes. We demonstrated that the viability of cancer cells exposed to DOX was significantly impaired in the presence of SIM, and this phenomenon was greatly magnified when DOX and SIM were coencapsulated in cubosomes. Overall, our results confirmed the utility of the DOX:SIM combination delivery, which enhances the interactions between neutral components of cell membranes and positively charged chemotherapeutic agents.

Controllable enzymatic superactivity of α-chymotrypsin activated by the electrostatic interaction with cationic gemini surfactants.

Surfactant plays a critical role in enzymatic multi-functionalization processes. However, a deep understanding of surfactant-enzyme interactions has been lacking up until now due to the extreme complexity of the mixed system. This work reported the effect of cationic gemini surfactants, alkanediyl-α,ω-bis(dimethyldodecylammonium bromide) (C12CSC12Br2, S = 2, 6, and 10) on the enzymatic activity and conformation of α-chymotrypsin (α-CT) in phosphate buffer solution (PBS, pH 7.3). The enzymatic activity was assessed by the rate of 2-naphthyl acetate (2-NA) hydrolysis measured by UV-vis absorption. The superactivity of α-CT in the presence of C12CSC12Br2 appears in the concentration region below the critical micelle concentration (cmc) of the surfactant, and its maximum superactivity is correlated to the spacer length of C12CSC12Br2. Subtle regulation of the charge density of headgroups of the cationic surfactant can be achieved through partial charge neutralization of cationic headgroups by introducing inorganic counterions or oppositely charged surfactant, demonstrating that the electrostatic interaction plays the crucial role for emergence of the superactivity. The interaction between C12CSC12Br2 (S = 2,6, and 10) and α-CT was characterized by isothermal titration calorimetry (ITC), and the obtained endothermic enthalpy change indicates that the interaction induces the change in conformation and enzymatic superactivity. The methodologies of fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC) show that the gemini surfactants with different spacer lengths induct and regulate the secondary, tertiary and even fourth structures of the protein. The present work is significant to get deeper insight into the mechanism of the activation and denaturation of enzymes.

Protein adsorption to poly(2-aminoethyl methacrylate)-grafted Sepharose gel: Effects of chain length and charge density

Grafting functional polymer chains onto porous resins has been found to drastically increase both adsorption capacity and uptake rate in protein chromatography. In this work, 2-aminoethyl methacrylate (AEM) was used for grafting onto Sepharose FF gel, and six anion-exchangers of different polyAEM (pAEM) chain lengths (ionic capacities, ICs), FF-pAEM, were obtained for protein adsorption and chromatography. It was found that protein adsorption capacity (qm) increased with increasing pAEM chain length, but the uptake rate, represented by the ratio of effective pore diffusivity to the free solution diffusivity (De/D0), showed an up-down trend, reaching a peak value (De/D0=0.55) at an IC of 313 mmol/L. Partial charge neutralization of the AEM-grafted resin of the highest IC (513 mmol/L) by reaction with sodium acetate produced three charge-reduced resins, FF-pAEM513-R. With reducing the charge density, the adsorption capacity kept unchanged and then decreased, but the uptake rate monotonically increased, reaching a maximum (about 2-fold increase) at a residual IC of 263 mmol/L. It is notable that, at the same IC, the charge-reduced resin (FF-pAEM513-R) presented similar or even higher values of qm and De/D0 than its FF-pAEM counterpart. Particularly, at the same IC of 263 mmol/L, a ~50% enhancement of De/D0 was observed. Both adsorption capacity and uptake rate in the charge-reduced resin with a residual IC of 339 mmo/L (FF-pAEM513-R339) decreased more sharply with increasing NaCl concentration by comparison with FF-pAEM513, indicating its increased salt-sensitivity than FF-pAEM513. That is, charge reduction on the AEM-grafted resin could accelerate protein uptake at 0 mmol/L NaCl but decrease salt tolerance. Column breakthrough experiments showed that FF-pAEM513-R339 was favorable for high flow rate protein chromatography at low NaCl concentration (0 mmol/L), whereas FF-pAEM513 was a good choice in a wide range of salt concentrations at low flow rate. This research proved the excellent protein chromatography performance of the AEM-based anion-exchangers.

Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization and Auto-phosphorylation.

Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1-6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930-34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1-6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR.

Protein adsorption to poly(allylamine)-modified Sepharose FF: Influences of polymer size and partial charge neutralization

Recently, poly(allylamine) (PAA) of 17.5 and 900 kDa were grafted onto Sepharose FF gel to study protein adsorption and chromatography. In order to further investigate the influences of polymer size and partial charge neutralization, PAAs of 160 and 450 kDa were chosen to synthesize two more types of ion-exchangers (denoted as FF-PAA-M and FF-PAA-ML, respectively). Protein adsorption capacity (qm) of these resins showed a monotonic increasing trend till the maximum ionic capacity (IC) available (970–990 mmol/L), while the uptake rates (De/D0) showed a decreasing-then-increasing trend. The maximum qm and De/D0 reached 328 mg/mL and 0.715, respectively, with FF-PAA-M990 (IC = 990 mmol/L). Partial charge neutralization by modification of FF-PAA-M990 with acetate produced four charge-reduced resins (FF-PAA-Rs), of which the lowest IC reached 210 mmol/L. Both the qm and De/D0 of FF-PAA-Rs presented an increasing-then-decreasing trend with reducing the charge density. The maximum qm obtained with FF-PAA-R860 (IC = 860 mmol/L) was 40% higher than FF-PAA-M990, and all the charge-reduced resins presented 2–3 times higher uptake rates than FF-PAA-M990. Both the qm and De/D0 of FF-PAA-Rs decreased sharply with increasing salt concentration, indicating their increased salt-sensitivities. The FF-PAA-R860 and FF-PAA-R710 columns presented much higher dynamic binding capacities than the FF-PAA-M990 column. FF-PAA-M990 and the charge-reduced resins were suitable for protein chromatography handling feedstocks containing different salt concentrations due to their different sensitivities to ionic strength.

Characterization of new polymer-grafted protein cation exchangers developed by partial neutralization of carboxyl groups derivatized by modification of poly(ethylenimine)-Sepharose with succinic anhydride

Previously, we have studied protein adsorption and chromatographic behaviors on poly(ethylenimine) (PEI)-grafted Sepharose FF anion-exchange resins, and found that protein uptake rates increased greatly when PEI grafting density reached over a critical ionic capacity (cIC) due to the occurrence of the “chain delivery” effect. Moreover, by partial charge neutralization of starting resin FF-PEI-L740 (IC = 740 mmol/L, larger than the cIC) with sodium acetate to FF-PEI-R440, it exhibited a three-fold increase in uptake rate over FF-PEI-L740. In this work, to take the advantages of PEI and extend the applications of the PEI-grafted resins in cation-exchange chromatography, a series of cation exchangers of five different ICs were developed. First, the charged of FF-PEI-L740 was reversed from positive to negative by reaction with excess succinic anhydride, which created a cation-exchanger with an IC of 970 mmol/L (FF-FEI-C970). FF-PEI-C970 was further modified with ethanolamine for partial charge neutralizations, leading to the preparation of four charge-reduced cation exchangers with IC values (in mmol/L) of 780, 630, 560 and 430, which were denoted as FF-PEI-CR780, −CR630 −CR560 and −CR430, respectively. Protein adsorption and chromatographic behaviors were investigated using lysozyme (Lys) as the model protein. It was found that, the resins of high and moderate IC values (IC ≥ 560 mmol/L) afforded adsorption capacities up to over 230 mg/mL. Besides, the uptake rate, represented by the effective pore diffusivity (De/D0), exhibited significant increase from 0.067 (FF-PEI-C970 and FF-PEI-CR780) to 0.343 (FF-PEI-CR630 and FF-PEI-CR560) and then to 1.035 (FF-PEI-CR430) with decreasing IC. It was considered that decreasing IC led to the decreased protein binding sites (binding strength), which encouraged the occurrence of the “chain delivery” effect. Moreover, the resins of high and moderate IC values, particularly, the resins of moderate IC values (FF-PEI-CR630 and FF-PEI-CR560), presented both high adsorption capacities and uptake kinetics at 0–100 mmol/L NaCl. Besides, dynamic binding capacity achieved 150 mg/mL for the resins of moderate IC values at 0 mmol/L NaCl concentration, and afforded >110 mg/mL for the resin of high IC values at 0–100 mmol/L NaCl concentration. The results proved the excellent IEC performance of the PEI-derived cation exchangers.

Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction.

The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1μg/ml (0.41 μM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC100) is 400 times higher. Also, in relation to in vitro activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.

Membrane-Spanning DNA Ion Channels: Conductance Mechanism, Electro-Osmotic Transport and Mechanical Gating

DNA self-assembly has emerged as a new paradigm for design of biomimetic ion channels and transporters. Several experimental groups have already reported spontaneous insertion of DNA constructs into lipid bilayer membranes and measurements of ionic flux through the channels. However, the structure such DNA ion channels in the lipid bilayer membrane environment and their conductance mechanisms have remained undetermined. Two years ago, we reported the first atomistic molecular dynamics (MD) simulations of DNA origami objects, characterizing their chemical and mechanical properties in unprecedented detail [PNAS, 110(50), 20099 (2013)]. Here, we extend our MD method to characterize the biophysical properties of DNA ion channels embedded in lipid bilayer membranes. First, we investigate the effect of two popular methods of DNA construct insertion—partial neutralization of DNA charge and hydrophobic anchoring—on the structure of the surrounding lipid bilayer. Although our simulations confirm that both methods yield stable assembly, profound differences are found in the structure of the DNA—lipid interface. Second, we find that the transmembrane ionic current flows not only through the central pore of the channel, but also around the walls of the channel and through the gaps in the DNA structure. The ratio of the current through each path depends on the design of the channel and the lipid—DNA interface. Surprisingly, we find the conductance of a DNA channel to depend on the membrane tension, making them potentially suitable for force-sensing applications. Finally, we show that electro-osmosis governs the transport of drug-like molecules through the DNA ion channels, suggesting possible applications of such systems in biosensing and drug delivery.

Origin of the Instability of Octadecylamine Langmuir Monolayer at Low pH

It has been reported that an octadecylamine (ODA) Langmuir monolayer becomes unstable at low pH values with no measurable surface pressure at around pH 3.5, suggesting significant dissolution of the ODA molecule into the subphase solution (Albrecht, Colloids Surf. A 2006, 284-285, 166-174). However, by lowering the pH further, ODA molecules reoccupy the surface, and a full monolayer is recovered at pH 2.5. Using surface sum-frequency spectroscopy and pressure-area isotherms, it is found that the recovered monolayer at very low pH has a larger area per molecule with many gauche defects in the ODA molecules as compared to that at high pH values. This structural change suggests that the reappearance of the monolayer is due to the adsorbed Cl(-) counterions to the protonated amine groups, leading to partial charge neutralization. This proposition is confirmed by intentionally adding monovalent salts (i.e., NaCl, NaBr, or NaI) to the subphase to recover the monolayer at pH 3.5, in which the detailed structure of the monolayer is confirmed by sum frequency spectra and the adsorbed anions by X-ray reflectivity.

Internal structure and thermo-viscoelastic properties of agar ionogels

Ionic liquids (IL) can alter the physical properties of agar hydrogels. Rheology studies show that gels with wide range of storage moduli (gel strength) G0 values ranging from 1 to 20KPa could be made in imidazolium based IL solutions where the IL concentration may not exceed 5% (w/v). Gelation and gel melting temperatures (tgel and Tm) could be altered by as much as≈10°C. Small angle neutron scattering studies revealed the presence of fibre bundles of agar double helices having typical length of 120nm that increased to ≈180nm under favorable conditions. These structures gain flexibility from the cladding of the agar bundles by IL molecules which in turn caused partial charge neutralization of its surface. Raman spectroscopy revealed differential hydration of these bundles. It was found that IL molecules with longer alkyl chain (more hydrophobic) altered the gel homogeneity, and changed its thermal and mechanical properties significantly. Therefore, customization of agar hydrogels in green solvent medium (IL solutions) widens the scope of its application potential that may include sensing.

Elucidation of photophysical changes and orientation of acridine orange dye on the surface of borate capped gold nanoparticles using multi-spectroscopic techniques.

In the present work, we have carried out a detailed investigation on the binding interaction of acridine orange (AO) with borate capped gold nanoparticles (Au NPs) by absorption spectroscopy, steady state emission spectroscopy, time resolved fluorescence spectroscopy, high resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), zeta potential measurements, Fourier transform infrared (FT-IR) and Raman spectroscopy. The phenomena of hypochromism and the appearance of coupled localized surface plasmon resonance (LSPR) bands in the absorption spectral studies of the AO-Au NP system suggested the vital role of electrostatic interactions between AO dye and Au NPs. The results from HR-TEM and DLS measurements confirmed the formation of aggregated clusters of nanoparticles. The zeta potential studies of Au NPs and AO coated Au NPs suggested the occurrence of partial surface charge neutralization of negatively charged Au NPs by cationic AO dye. The interparticle edge to edge separation distance of adjacent Au NPs revealed the presence of AO molecules between the nano-gaps of Au NPs. Emission spectral studies of AO in the presence of increasing concentrations of Au NPs indicated the existence of a static quenching mechanism. Time resolved fluorescence spectroscopic study further validated the findings of steady state emission measurements. The comparison of experimental emission quenching data with the theoretically calculated value suggested the probable multi-layered assembly of AO on the surface of Au NPs. FT-IR and Raman spectral studies further revealed the existence of electrostatic interactions and possible horizontal orientation of AO dye molecules on Au NP surfaces.

DNA–Gelatin Complex Coacervation, UCST and First-Order Phase Transition of Coacervate to Anisotropic ion gel in 1-Methyl-3-octylimidazolium Chloride Ionic Liquid Solutions

Study of kinetics of complex coacervation occurring in aqueous 1-octyl-3-methylimidazolium chloride ionic liquid solution of low charge density polypeptide (gelatin A) and 200 base pair DNA, and thermally activated coacervate into anisotropic gel transition, is reported here. Associative interaction between DNA and gelatin A (GA) having charge ratio (DNA:GA = 16:1) and persistence length ratio (5:1) was studied at fixed DNA (0.005% (w/v)) and varying GA concentration (C(GA) = 0-0.25% (w/v)). The interaction profile was found to be strongly hierarchical and revealed three distinct binding regions: (i) Region I showed DNA-condensation (primary binding) for C(GA) < 0.10% (w/v), the DNA ζ potential decrease from -80 to -5 mV (95%) (partial charge neutralization), and a size decrease by ≈60%. (ii) Region II (0.10 < C(GA) < 0.15% (w/v)) indicated secondary binding, a 4-fold turbidity increase, a ζ potential decrease from -5 to 0 mV (complete charge neutralization), which resulted in the appearance of soluble complexes and initiation of coacervation. (iii) Region III (0.15 < C(GA) < 0.25% (w/v)) revealed growth of insoluble complexes followed by precipitation. The hydration of coacervate was found to be protein concentration specific in Raman studies. The binding profile of DNA-GA complex with IL concentration revealed optimum IL concentration (=0.05% (w/v)) was required to maximize the interactions. Small angle neutron scattering (SANS) data of coacervates gave static structure factor profiles, I(q) versus wave vector q, that were remarkably similar and invariant of protein concentration. This data could be split into two distinct regions: (i) for 0.0173 < q < 0.0353 Å(-1), I(q) ~ q(-α) with α = 1.35-1.67, and (ii) for 0.0353 < q < 0.35 Å(-1), I(q) = I(0)/(1 + q(2)ξ(2)). The correlation length found was ξ = 2 ± 0.1 nm independent of protein concentration. The viscoelastic length (≈8 nm) was found to have value close to the persistence length of the protein (≈10 nm). Rheology data indicated that the coacervate phase resided close to the gelation state of the protein. Thus, on a heating-cooling cycle (heating to 50 °C followed by cooling to 20 °C), the heterogeneous coacervate exhibited an irreversible first-order phase transition to an anisotropic ion gel. This established a coacervate-ion gel phase diagram having a well-defined UCST.

Contribution of hydrophobic interactions to the folding and fibrillation of histone H1 and its carboxy-terminal domain

Histone H1 is involved in chromatin structure and gene regulation. H1 also performs functions outside cell nuclei, which may depend on its properties as a lipid-binding protein. The H1 CTD behaves as an intrinsically disordered protein (IDP) with coupled binding and folding. Here, we used neutral detergents and anionic SDS to study the contribution of hydrophobic interactions to the folding of the CTD. In the presence of neutral detergents, the CTD folded with proportions of secondary structure motifs similar to those observed in the DNA complexes. These results identify a folding pathway for the CTD based on hydrophobic interactions, and independent of charge compensation. The CTD is phosphorylated to different extents by cyclin-dependent kinases. The general effect of phosphorylation in the presence of detergents was a decrease in the α-helix content and an increase in that of the β-structure. The greatest effect was observed in the fully phosphorylated CTD (three phosphate groups) in the presence of anionic SDS (7:1, detergent/CTD molar ratio); in these conditions, the CTD became an all-β protein, with 83% β-structure and no α-helix. The CTD in all-β conformation readily formed ribbon-like fibers. The entire H1 also formed fibers when fully phosphorylated in the CTD. Fibers were of the amyloid type, as judged by strong birefringence in the presence of Congo red and thioflavin fluorescence enhancement. Amyloid fiber formation was only observed in SDS, suggesting that it requires the joint effects of partial charge neutralization and hydrophobic interactions, together with the all-β potential provided by full phosphorylation.

Mechanism for translocation of fluoroquinolones across lipid membranes

Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins. Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach the membrane in stacks, but they diffuse through the membrane as neutral CPFX monomers due to intermolecular transfer of protons favored by partial solvation loss. The mechanism is expected to be of importance in the permeation and translocation of a variety of ampholitic drugs with stacking tendencies.

Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50°C) and below (25°C) the main phase transition temperature of the LUVs (∼41°C). The binding constant values, Kb, estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the Kb value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation.

Influence of γ-irradiation (60Со) on the concentration and mobility of carriers in Ge and Si single crystals of n-type

The influence of γ-irradiation ( n , the mobility is shown to grow anomalously with the irradiation dose in the region of combined scattering of carriers. Proposed in this paper is the model based on accounting partial neutralization of charge of scattering centers by charge of radiation defects produced mainly around the scattering centers. This model qualitatively explains the experimental data.

Inhibition of Candida albicans CC biofilms formation in polystyrene plate surfaces by biosurfactant produced by Trichosporon montevideense CLOA72

This study evaluated the effects of glycolipid-type biosurfactant produced by Trichosporon montevideense CLOA72 in the formation of biofilms in polystyrene plate surfaces by Candida albicans CC isolated from the apical tooth canal. Biofilm formation was reduced up to 87.4% with use of biosurfactant at 16 mg/ml concentration. It has been suggested that the interaction with the cell or polystyrene plate surface could ultimately be responsible for these actions. Therefore, the interaction of C. albicans CC cells with the biosurfactant, as well as the corresponding thermodynamic parameters, have been determined by isothermal titration calorimetry and zeta potential measurements. This process is endothermic (((int)H°=+1284±5 cal/mg OD(600)) occurring with a high increase of entropy (T((int)S°=+10635 cal/mg OD(600)). The caloric energy rate data released during the titulation indicates saturation of the cell-biosurfactant at 1.28 mg/ml OD(600). Also, the zeta potential of the cell surface was monitored as a function of the biosurfactant concentration added to cell suspension showing partial neutralization of net surface charge, since the value of zeta potential ranged from -16 mV to -6 mV during the titration. The changes of cell surface characteristics can contribute to the inhibition of initial adherence of cells of C. albicans in surface. The CMC of the purified biosurfactant produced from T. montevideense CLOA72 is 2.2 mg/ml, as determined both by ITC dilution experiments and by surface tension measurements. This biomolecule did not presented any cytotoxic effect in HEK 293A cell line at concentrations of 0.25-1 mg/ml. This study suggests a possible application of the referred biosurfactant in inhibiting the formation of biofilms on plastic surfaces by C. albicans.

Interactions between whey protein isolate and gum Arabic

In this study we have attempted to understand the nature of “charge interactions” between two negatively charged biopolymers (whey protein isolate, WPI and gum Arabic, GA) and, consequently, why their mixture exhibits better interfacial activity. Surface tension ( γ 0) measurements indicated that at ca. 1 wt.% of the biopolymer mixture (3:1 wt. ratio) the air/water surface is saturated. At 5 wt.% the γ 0 of the mixture is lower than the calculated co-operative value. The ζ-potential measurements revealed that the isoelectric point of the WPI:GA 3:1 wt. ratio mixture is 3.8. The ζ-potential values up to pH 6 are below those calculated. Similarly, the electrical conductivities of the mixture are lower than those calculated. All these measurements indicate: (1) partial charge neutralization in spite of the fact that both biopolymers are negative or (2) partial charge–charge interactions between the two biopolymers. The thermal heating behavior of the frozen water in the aqueous mixture studied by DSC (heating cycle of the frozen sample) clearly indicates that the two biopolymers are interacting. We calculated the enthalpy, the free energy and the chemical potential of the interactions. We found that the interactions of the biopolymers are rather weak. They are likely derived from some local positively charged domains (pH 7) on the protein that neutralize some of the negatively charged GA. These interactions form weak charge adducts. These charge adducts are sufficient to improve its adsorption into the oil–water interface and enhance the emulsion stability.

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.