Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum.

Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.     

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the Zeta potential

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values.At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.     

The effect of electrostatic charge of food particles on capture efficiency by Oxyrrhis marina Dujardin (dinoflagellate)

Laboratory experiments were carried out to investigate the effect of food quality, measured as surface charge of the particles, on capture efficiency and ingestion rate by the heterotrophic dinoflagellate Oxyrrhis marina. Fluorescent particles in two size classes of around 1 and 4 microm and of 7 different qualities were offered to the flagellate: carbohydrate and albumin particles, the algae Synechocystis spec. and Chlorella spec., carboxylated microspheres, silicate particles and bacteria. Rates of particle uptake showed significant differences depending on particle size and quality, and ranged from 0 to 4 particles cell(-1) h(-1). Ingestion rates were up to 4 times higher for 4 pm particles than for 1 microm particles, which indicates strong size-selective feeding. Our main result is that the surface charge or zeta potential, of artificial particles, i.e. carboxylated microspheres (> or = -107 mV) and silicate particles, strongly differ from more natural and natural food (< or = -17 mV). For both size classes Oxyrrhis had ingestion rates up to 4 times higher for particles with less negative charge, such as albumin particles or algae. Thus, the zeta potential of the model food should be considered in experimental design. Particles with a zeta potential similar to that of natural food, e.g. albumin, seem to be the preferred model food.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

Effects of the surface wettability and zeta potential of bioceramics on the adhesiveness of anchorage-dependent animal cells

Using calcium phosphate ceramics that have high biocompatibility with the living body, the effects of the surface characteristics of the bioceramics on cell adhesiveness were investigated. In the case of carriers with contact angles from 35° to 60°, the cell adhesiveness increased according to the increase in the wettability. Measurement of the zeta potentials of HAP-TCP sinters showed that these bioceramics had negative potentials from −2 mV to −6 mV. Electrochemical analysis suggested that the initial cell anchoring ratio (R_ia) and adhesive strength (F_a,enz) were affected by the surface ionic condition of the ceramic material. To clarify the effects of the surface potential of the ceramics on cell adhesiveness, the ceramic surface was modified chemically by means of various silane coupling reagents. The surface potential was regulated from −20 mV to +24 mV. Using these ceramics, the affinity and adhesiveness of the cells to the ceramics were found to be dominantly regulated by the surface potential. A negative potential was effective in increasing the adhesiveness, even though living cells have negative charges.     

Optimization of PEGylation Conditions for BSA Nanoparticles Using Response Surface Methodology

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.     

Characterization of silver nanoparticles synthesized by using marine isolate Streptomyces albidoflavus

Silver nanoparticles production by the green chemistry approach was investigated using an isolated marine actinomycetes strain. The isolated strain was identified as Streptomyces albidoflavus based on chemotaxonomic and ribotyping properties. The strain revealed production of silver nanoparticles both extracellular and intracellularly. Surface Plasmon Resonance analysis with the function of time revealed that particle synthesis by this strain is reaction time dependent. The produced particles were spherical shaped and monodispersive in nature and showed a single surface plasmon resonance peak at 410 nm. Size distribution histograms indicated production of 10-40- nm-size nanoparticles with a mean size of 14.5 nm. FT-IR spectra of nanopartilces showed N-H, C-H, and C-N stretching vibrations, denoting the presence of amino acid/ peptide compounds on the surface of silver nanoparticles produced by S. albidoflavus. Synthesized nanoparticles revealed a mean negative zeta potential and electrophoretic mobility of -8.5 mV and -0.000066 cm2/Vs, respectively. The nanoparticles produced were proteinaceous compounds as capping agents with -8.5 mV zeta potential and revealed antimicrobial activity against both Gram-negative and -positive bacterial strains. Owing to their small size, these particles have greater impact on industrial application spectra.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress

The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media. With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV. With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability. The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy. Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth. L. acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol.     

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles.

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Deltap) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Deltap. The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.     

Zeta potential of synthetic and biological skin substitutes: effects on initial adherence

Early adherence of a skin substitute to the wound surface is paramount if it is to function as a skin equivalent. A surface electrical property (the zeta potential) was evaluated, and a positive correlation was found in which 5-hour adherence properties increased as the zeta potential became more positive. The following materials were tested: nylon-silicone composite (Z = -24.8 mV), Biobrane (Z = -15.2 mV), fresh-frozen porcine skin (Z = +12.5 mV), Opsite (Z = +14.9 mV), human amnion (Z = +18.2 mV), and human skin (Z = +23.0 mV). This order was also followed for increasing adherence values at 5 hours, which ranged from a low of 48.9 gm/cm2 for the nylon-silicone composite to a high of 88 gm/cm2 for human skin. Also determined was that both adherence and zeta potential decreased as increasing concentrations of glutaraldehyde were used to cross-link fresh-frozen porcine skin. Values ranged from a maximum of 85.5 gm/cm2 (0% glutaraldehyde; Z = +12.5 mV) to a minimum of 42.5 gm/cm2 (10% glutaraldehyde; Z = -26.4 mV). Additionally, deliberate biochemical modifications of porcine skin were undertaken in an attempt to increase zeta potential and adherence.     

三种不同表面活性剂对羟基磷灰石纳米颗粒的表面修饰的比较

目的:本研究旨在通过不同方法修饰羟基磷灰石纳米颗粒并检测其稳定性及分散性。方法:首先采用水合热合成法制备羟基磷灰石纳米颗粒,然后用透射电镜(TEM)和场发射扫描电镜(SEM)对其表面形态结构进行表征。我们首次用溴化十六烷三甲基铵(CTAB),PEG2000和人血清对羟基磷灰石纳米颗粒通过共价结合或表面吸附的方式进行表面嫁接,并利用透射电镜,傅里叶红外光谱(FT-IR)和X射线衍射(XRD)对新合成的这三种纳米羟基磷灰石复合物的形貌,结构和晶粒粒径进行表征。对这三种羟基磷灰石纳米颗粒悬浮液的时间沉降曲线进行分析。在分散性上通过检测这三种羟基磷灰石复合物悬浮液在不同pH值下的Zeta电位并绘制Zeta-pH曲线。结果:我们发现CTAB修饰的羟基磷灰石纳米颗悬浮液的悬浮稳定性最佳,其次是PEG2000,最后是人血清。在pH=7.0时,CTAB修饰的羟基磷灰石纳米颗粒的zeta电位值是25.68 m V,而PEG2000修饰的Zeta电位是4.32m V,人血清修饰的Zeta电位是-13.23m V。结论:CTAB表面修饰的羟基磷灰石纳米颗粒相对于其它两种表面活性剂复合物具有更好的分散性和悬浮稳定性,与DNA/RNA结合能力更强。本课题的结果给羟基磷灰石纳米颗粒载体的应用提供了一种新的选择,有望利用亲和力更高的基因载体实现基因治疗,具有广阔的应用前景。     

pH modification of human T-type calcium channel gating

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.     

<Emphasis Type="Italic">In Vitro</Emphasis> Anti-inflammatory and Antimicrobial Activities of Azithromycin After Loaded in Chitosan- and Tween 20-Based Oil-in-Water Macroemulsion for Acne Management

The objectives of the current investigation are (1) to prepare and characterize (particle size, surface charge (potential zeta), surface morphology by transmission electron microscopy, drug content, and drug release) the azithromycin (AZM, 100 mg)-loaded oil-in-water (o/w) macroemulsion, (2) to assess the toxicity of macroemulsion with or without AZM using RBC lysis test in comparison with AZM in phosphate buffer solution of pH 7.4, (3) to compare the in vitro antimicrobial activity (in Escherichia coli using zone inhibition assay) of AZM-loaded macroemulsion with its aqueous solution, and (4) to assess the in vitro anti-inflammatory effect (using egg albumin denaturation bioassay) of the AZM-loaded macroemulsion in comparison with diclofenac sodium in phosphate buffer solution of pH 7.4. The AZM-loaded macroemulsion possessed the dispersed oil droplets with a mean diameter value of 52.40?±?1.55 μm. A reversal in the zeta potential value from negative (?2.16?±?0.75 mV) to positive (+6.52?±?0.96 mV) was noticed when AZM was added into the macroemulsion. At a 1:5 dilution ratio, 2.06?±?0.03 mg of drug was released from macroemulsion followed by 1.01?±?0.01 and 0.25?±?0.08 mg, respectively, for 1:10 and 1:40 dilution ratios. Antimicrobial activity maintenance and significant reduction of RBC lysis property were noticed for AZM after loaded in the macroemulsion. However, an increment in the absorbance values for emulsion-treated samples in comparison to the control samples was noticed in the anti-inflammatory test. This speculates the potential of the AZM-loaded emulsion to manage inflammatory conditions produced at Acne vulgaris.     

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.     

Variability in Bioreactivity Linked to Changes in Size and Zeta Potential of Diesel Exhaust Particles in Human Immune Cells

Acting as fuel combustion catalysts to increase fuel economy, cerium dioxide (ceria, CeO₂) nanoparticles have been used in Europe as diesel fuel additives (Envirox™). We attempted to examine the effects of particles emitted from a diesel engine burning either diesel (diesel exhaust particles, DEP) or diesel doped with various concentrations of CeO₂ (DEP-Env) on innate immune responses in THP-1 and primary human peripheral blood mononuclear cells (PBMC). Batches of DEP and DEP-Env were obtained on three separate occasions using identical collection and extraction protocols with the aim of determining the reproducibility of particles generated at different times. However, we observed significant differences in size and surface charge (zeta potential) of the DEP and DEP-Env across the three batches. We also observed that exposure of THP-1 cells and PBMC to identical concentrations of DEP and DEP-Env from the three batches resulted in statistically significant differences in bioreactivity as determined by IL-1β, TNF-α, IL-6, IFN-γ, and IL-12p40 mRNA (by qRT-PCR) and protein expression (by ELISPOT assays). Importantly, bioreactivity was noted in very tight ranges of DEP size (60 to 120 nm) and zeta potential (−37 to −41 mV). Thus, these physical properties of DEP and DEP-Env were found to be the primary determinants of the bioreactivity measured in this study. Our findings also point to the potential risk of over- or under- estimation of expected bioreactivity effects (and by inference of public health risks) from bulk DEP use without taking into account potential batch-to-batch variations in physical (and possibly chemical) properties.     

Effects of bacteriophage on the surface properties of chalcopyrite (CuFeS₂), and phage-induced flocculation of chalcopyrite, glacial till, and oil sands tailings

The binding of mineral-specific phage to the surface of chalcopyrite (CuFeS(2)) was investigated by using X-ray photoelectron spectroscopy and scanning Auger microscopy. These studies confirmed the elemental composition of the minerals and confirmed that bacteriophage were bound to the mineral surface. These techniques also revealed that the phage were not forming a continuous film over the entire surface of the CuFeS(2) particles, but selectively bound to the slimes coating the particles. In addition, the effect of mineral-specific phage binding to the surface of CuFeS(2) was investigated using induction time and zeta potential measurements. Bacteriophage (10(12) /mL) increased the induction time (contact time resulting in 50% particle attachment to a bubble) from ～7.5 to ～17 ms and reversed the zeta potential from negative to positive. In the course of performing the zeta potential measurements on particles <45 μm in diameter, phage-induced aggregation was observed. The mechanism of aggregation was explored using a range of pH (3-11) and cation concentrations. Aggregation was observed across the tested pH range and with all cations. Phage also mediated aggregation of glacial till and oil sands tailings in a dose-dependent and particle size-dependent manner. We conclude that binding of bacteriophage to the surface of CuFeS(2) does alter its surface properties.     

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.