The stability of prostaglandin E1 in dilute physiological solutions at 37 degrees C

The stability of prostaglandin E₁ (PGE₁) in three physiologic solutions was studied at body temperature (37°C) over 32 days. The solutions were 100 mcg/ml PGE₁ in isotonic saline (pH 4.5), 0.1 M phoshate buffered water (pH 7.4) or 0.01 M phosphate buffered isotonic saline (pH 4.7). PGE₁ was found to be more stable in the saline and buffered saline solutions at the pH values of 4.5 and 4.7 respectively. Twenty-five per cent of the PGE₁ remained at 32 days in these solutions while 95% of the PGE₁ in the solution at pH 7.4 was degraded by day 14. The degradation of PGE₁ in the acidic solutions appeared to be nearly linear when plotted on a semi-log graph. This data allows one to use PGE₁ in an aqueous, slightly acidic solution in a system that requires it to be kept at 37°C for up to 30 days such as a biologically implantable pump. Investigators can use such a system to study the effect of known concentrations of PGE₁ given over a period of time to a specific area of interest.     

Pulmonary effects of the cocaine pyrolysis product,methylecgonidine, in guinea pigs

The pulmonary effects of the cocaine pyrolysis product, methylecgonidine (MEG; anhydroecgonine methyl ester), were assessed in guinea pigs. Specific airway conductance (SGaw), which decreases during bronchoconstriction, was measured in guinea pigs exposed to atmospheres containing a condensation aerosol of MEG free base (13 ± 1 mg/liter of air), nebulized MEG fumarate (3 and 12% in phosphate buffered saline) or nebulized acetylcholine chloride (0.2 and 0.4% in phosphate buffered saline). A decrease in SGaw to 24.0 ± 4.2% (mean ± 2 S.E.M.) of baseline levels was observed in guinea pigs breathing MEG free base. A decrease to 28.4 + 4.5% of baseline was observed following administration of 0.4% acetylcholine. No change in SGaw was measured in guinea pigs exposed to 3% MEG fumarate but SGaw was reduced to 69.3 ± 5.3% of baseline after exposure to 12% MEG fumarate. MEG free base poses an alkaline challenge to the lung, 3% MEG fumarate is neutral (pH ≈ 7.4) and 12% MEG fumarate is acidic (pH ≈ 4.3); thus, MEG free-base and 12% MEG fumarate might provoke a reflex bronchoconstriction due to direct pulmonary irritant effects. These results suggest that MEG free base produced during crack pyrolysis may play a role in bronchoconstriction observed in crack smokers.     

Synthesis and characterisation of a new pH-sensitive amphotericin B--poly(ethylene glycol)-b-poly(L-lysine) conjugate

This paper reports on the synthesis, characterisation, and efficiency of a new intravenous conjugate of amphotericin B (AMB). Twelve molecules of AMB were attached to block copolymer poly(ethylene glycol)-b-poly(L-lysine) via pH-sensitive imine linkages. In vitro drug release studies demonstrated the conjugate (M(w)=26,700) to be relatively stable in human plasma and in phosphate buffer (pH 7.4, 37 degrees C). Controlled release of AMB was observed in acidic phosphate buffer (pH 5.5, 37 degrees C) with the half-life of 2 min. The LD(50) value determined in vivo (mouse) is 45 mg/kg.     

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactideco-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37°C and at elevated temperatures (50°C–60°C) in 0.1 M phosphate buffered saline (PBS) pH 7.4 and 0.1 M acetate buffer pH 4.0. The data were analyzed using a modification, of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37°C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37°C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, α, a scaling factor for the apparent rate constant. Complete release at 37°C was shortened from ∼30 days to 5 days at 50°C, 3.5 days at 55°C, 2.25 days at 60°C in PBS pH 7.4, and 3 days at 50°C in acetate buffer pH 4.0. Values for the model parameter β indicated that the shape of the release profiles at 55°C in PBS pH 7.4 (2.740) and 50°C in 0.1 M acetate buffer pH 4.0 (2.711) were similar to that at 37°C (2.577). The E_a for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E_a of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with longterm studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches. Selected for the 2005 AAPS Outstanding Graduate Student Research Award in Pharmaceutical Technologies Sponsored by Solvay Pharmaceuticals.     

Some molecular properties of rat-liver lysosomal beta-glucuronidase isoenzymes.

The isoenzymes of rat-liver lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase (EC 3.2.1.31)) were inactivated at different rates at 0 degrees C in 3M guanidinium chloride solutions adjusted to pH 5.0 In 4 M urea buffered by 0.01 M glycylglycine, pH 7.0 isoenzymes I, III, and V were reversibly inhibited 80%. Sodium dodecyl sulfate (SDS), 0.1% in 0.01 M phosphate buffer, pH 7.0 irreversibly inhibited at 37 degrees C all five isoenzymes. Sedimentation analysis showed that loss of catalytic activity in these denaturing media is accompanied by dissociation into slower sedimenting subunits. SDS gel electrophoresis revealed that the isoenzymes are apparently tetramers made up of different proportions of subunits alpha, beta, and gamma having apparent molecular weights of 62,900, 60,200, and 58,700, respectively. The three subunits appear to be glycoproteins.     

Glutaraldehyde mediated echinocyte/discocyte transformation is Ca2+ dependent.

Echinocytes, which were produced from freshly banked blood by repeated washes in phosphate buffered saline, undergo a transformation to the discoid shape within less than 30 seconds of incubation in isotonic 0.05% glutaraldehyde pH 7.4. This echinocyte/discocyte transformation is not associated with a change of cell volume or critical hemolysis volume although a slight decrease of cellular deformability and a 4-8 fold increase of K+ efflux within 1 hour after glutaraldehyde incubation provide evidence of the fixative's attack on the cell membrane. Trypsination prior to the incubation in isotonic glutaraldehyde could not inhibit the shape change. Hypertonic glutaraldehyde solutions partially prevent the E/D transformation with regard to both the osmolarity of the medium and the permeability of the cell membrane. The glutaraldehyde stimulated transformation is entirely inhibited in the presence of a chelating agent the efficiency of which is overcome by addition of a more-than-equivalent amount of Ca2+. The mutual action of either agent is discussed, however, the mechanism of the phenomenon remains unclear.     

Self-association studies on the EphB2 receptor SAM domain using analytical ultracentrifugation

The self-association behavior of the Eph-kinases SAM domain has been studied in phosphate buffer, pH 7.4, containing 0.14 M NaCl using concentration-dependent sedimentation equilibrium experiments. Only weak interactions typical for a monomer-dimer equilibrium up to at least 12 mg/mL were observed. Such concentrated solutions require a consideration of the non-ideality expressed by virial coefficients. A special centrifuge equation was used for the global analysis to estimate equilibrium constants based on the thermodynamic activities of the reactants. When neglecting this, the parameters deviate by about 20%. Association constants for dimerization of the EphB2-SAM domain vary between 163 M(-1) at 10 degrees C and 395 M(-1) at 32 degrees C, indicating hydrophobic forces are involved in the dimerization process. In solutions of about 12 mg/mL, less than 50% dimers are in solution and higher oligomers can be excluded.     

Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca2+, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50-100 times faster at 22 degrees C than at 4 degrees C and was 5-10 times faster at 37 degrees C than at 22 degrees C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20-30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10-15 times faster in the absence of 1 mM Ca2+ in the buffer solution and was approximately 100 times faster in the presence of 0.1 M D-mannose as compared to 0.1 M D-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A2 and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation, Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2-4 h incubation at 4 degrees C was observed.     

Preparation and in vitro evaluation of primaquine-conjugated gum arabic microspheres

Gum arabic, a branched polysaccharide, was oxidized using periodate to generate reactive aldehyde groups on the biopolymer. Primaquine, an 8-aminoquinoline, was covalently coupled onto oxidized gum arabic via an imine bond and simultaneously fabricated into microspheres of less than 2 microm in size by heat denaturation in a reverse emulsion of 1:1 light paraffin oil and toluene stabilized by sorbitan sesquioleate as the surfactant. The covalent binding of primaquine to the polysaccharide using the clinically used water-soluble form of the drug primaquine phosphate was achieved in the presence of borate buffer of pH 11. Up to 35% of the drug could be bound to the polymer backbone depending on the concentration of the drug employed initially and the degree of oxidation of the polysaccharide. Interestingly, both the aliphatic and the hindered aromatic amino groups of primaquine were found to react with the aldehyde functions through Schiff base formation leading to cross-linking of the polysaccharide with the drug itself. In vitro release of the drug from microspheres into phosphate buffered saline (PBS, pH 7.4, 0.1 M) at 37 degrees C showed that the release of primaquine from the matrix was slow, although gradually increased with time. The maximum released was below 50% of the drug payload even after 10 days. Release into simulated gastric and intestinal fluids was faster compared to the release in PBS due to rapid hydrolysis of the Schiff's linkage in the gastric fluid. A possible reason for the poor hydrolytic susceptibility of the Schiff's linkage is suggested based on the unequal reactivity of the amino groups on primaquine and its relevance in possible therapeutic application of this polymer-drug conjugate discussed.     

Water balance and its relation to fermentation acid production in the intestinal parasites Hymenolepis diminuta (Cestoda) and Moniliformis moniliformis (Acanthocephala).

Water balance and its relation to carbohydrate metabolism was examined in Hymenolepis diminuta in parallel with the putative osmoconformer Moniliformis moniliformis. Worms were removed from rat intestines, weighed, and incubated (37 C) 1 hr in rat serum and various salines, some with mannitol to vary osmotic concentration from 150 to 400 mOsm/L. Worms were removed at 15-min intervals, weighed, and returned to the test solution. Rat serum and a Ringer's saline (pH 7.4 and 300 mOsm/L) with or without 5 mM glucose were isotonic to M. moniliformis, which behaved like an osmometer, shrinking, or swelling in proportion to external osmotic changes. Hymenolepis diminuta rapidly lost 20-25% wet weight in these solutions and regained lost water when 5 mM glucose was added to the saline. Tapeworms maintained constant body weight between 210 and 335 mOsm/L, but they rapidly gained or lost water outside of this range. Glucose metabolism and uptake of [3H]glucose from the medium increased progressively between 210 and 310 mOsm/L, whereas uptake rates of [3H]leucine, 22Na+, and 36Cl- were not affected. Unbuffered saline (initial pH 6.5 and 300 mOsm/L) had a lower pH (5.0) and higher osmolality (307 mOsm/L) after a 1-hr incubation with tapeworms. Such saline was less hypertonic than unconditioned saline to freshly obtained worms. A Ringer's saline (300 mOsm/L) containing 50 mM acetate- was also hypertonic (greater than 20% weight loss) to tapeworms at pH 7.4, but it was hypotonic (greater than 20% weight gain) at pH 5.0. Isotonicity at 300 mOsm/L was achieved with pH 5.0 and 20 mM acetate-, the approximate pH and fermentation acid concentration in an infected rat intestine. Rats infected with tapeworms (12 days old) were fasted for 2 days. Starved worms were smaller but had the same percentage of body water and internal osmolality as controls. These results show that H. diminuta can regulate its body water content and that water balance is closely related to the fermentation acid concentration and pH of the bathing medium.     

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P < 0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P < 0.05).     

Bacterial counts in canine duodenal fluid after exposure to saline, sodium bicarbonate and hypertonic dextrose solutions used to maintain patency of chronically implanted catheters

Flushing of intestinal vascular access ports (VAPs) is commonly performed to prevent the problems of blockage and infection, and in this study four different flushing solutions were compared. The growth of bacteria from canine duodenal contents was compared in: 0.9% saline, 50% dextrose, 8.4% sodium bicarbonate (NaHCO3) and 0.01 M phosphate buffered saline (PBS). Duodenal contents from three laboratory beagles were serially diluted in these four solutions, spread plated onto agar at 24 h periods for 7 days and bacterial counts were performed. Immediately after the duodenal juices were added, no significant differences could be seen in bacterial counts with any of the solutions. Over the 7 day period, bacterial numbers greatly increased in saline and phosphate buffered saline, but greatly decreased in dextrose and sodium bicarbonate solutions. Dextrose and sodium bicarbonate appeared to be the most promising flushing solutions tested to minimize infections of associated intestinal VAPs.     

Quick freezing of rat embryos

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Complex viscosity of bovine red blood cells in suspensions

The complex viscosity eta* has been measured of bovine red blood cells suspended in a medium of isotonic NaCl solutions including dextran and buffered with potassium phosphate at pH 7.0. A multiple lumped resonator apparatus was used at the frequencies of 144, 572, 1491, 3742, and 8026 Hz at 20.0 degrees C. Due to the high molecular weight of dextran the medium also exhibited some visco-elasticity eta s*. So we adopted the complex specific viscosity eta sp* = (eta*-eta s*)/[eta s*]. At 20.0 degrees C eta sp* decreased with the frequency where the hematocrit was 0.233 and eta s 0.34 poise. The measurements were made for the medium with different viscosity at 5.0 degrees C and 25.0 degrees C. The results are compared with the theory of elastic shells.     

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.

BACKGROUND: Two calibration methods have been proposed for determining the relation between the fluorescence ratio of a pH-sensitive fluorescent indicator and intracellular pH (pHi). The first method uses nigericin to clamp pHi to external pH (pHe) and the second is the null point method. We compared these different calibration methods, solution conditions, and temperatures by using flow cytometry and the fluorescent dye 1,5- (and-6)-carboxy seminaphtorhodafluor-1-acetoxymethyl ester with an NS0 cell line. METHODS: The nigericin method was performed in glucose solutions supplemented with KCl and 2-(N-morpholino)ethane sulphonic acid plus tris(hydroxymethyl)aminomethane (solution 1A), a mixture of K2HPO4/KH2PO4 in glucose-solution supplemented solutions (solution 2A), or bicarbonate buffered growth medium supplemented with K2HPO4/KH2PO4 (solution 2B); this allowed a range of pHe values to be used. The effect of temperature (22 degrees C or 37 degrees C) on the nigericin calibration curve was also investigated. The null point method was performed by using a series of solutions with a mixture of weak acid and base with a known pHi response. RESULTS: Using solution 1A as the calibration solution resulted in acidic values of pHi for cells cultured in medium as compared with the values achieved with solution 2A. Using solution 2B did not affect the calibration curve. For the temperatures considered in this study, there was no affect on the calibration curve, but temperature did affect the pHi value of cells in phosphate buffered saline. The pseudo-null point method used with flow cytometry resulted in a calibration curve that was significantly different (P<0.05) from that achieved using the nigericin method. CONCLUSIONS: Our data indicates that the choice of calibration solution can affect the reported pHi value; therefore, careful choice of solution is important.     

Direct force measurements between siRNA and chitosan molecules using force spectroscopy

Information on the interaction strength between small interfering RNA (siRNA) and chitosan can contribute to the understanding of the formation and stability of chitosan/siRNA nanoparticles used as siRNA delivery systems for gene silencing. In this study, we utilize atomic force microscopy to obtain force spectroscopy results of the interaction strengths between siRNA and chitosan measured in physiological phosphate buffered saline buffer at different pH. The force measurements revealed that the adhesive interactions decreased in force strength and force frequency as the pH was increased from 4.1 to 6.1, 7.4, and 9.5, exhibiting distinct multimodal distributions of the interaction forces between siRNA and chitosan molecules at acidic pH and only negligible adhesive forces were observed at neutral or high pH. The strong pH dependence of siRNA-chitosan interactions can provide a convincing rationale for siRNA/chitosan complex formation and nanoparticle stability under low acidic conditions. These findings demonstrate that the use of force spectroscopy for the adhesive force measurements allows an evaluation of the complexing ability between siRNA and chitosan that can be utilized to predict nanoparticle stability.     

The influence of pH on the inhibition of DNA cleavages induced by pyrogallol

Induction of DNA damage by pyrogallol has been shown at physiological pH, but mutagenesis data also suggest there is inhibition in acidic media. In the present work, the plasmid pBSK was incubated with pyrogallol, under aerobic conditions at 37 degrees C, at pH 7.4, 4.5 or 3.5, for 1, 3 or 5 h, in the absence or presence of Cu(2+). Cleavage of the supercoiled DNA form was analyzed through topology modifications by agarose gel electrophoresis and quantified by densitometry. Independently of the presence of Cu(2+), DNA cleavage at pH 7.4 was significantly (P < 0.001) induced and occurred extensively after 1-h incubation. At pH 4.5, the cleavage was significantly (P < 0.05) induced only after 5 h incubation in the absence of Cu(2+), but was extensive (P < 0.001) after 1-h incubation when the metal ion was present. At pH 3.5, DNA cleavage was inhibited (P > 0.05), after 5-h incubation, even in the presence of Cu(2+). Our results provide evidence that DNA cleavage by pyrogallol is pH-dependent, catalyzed by Cu(2+) , and extensively decreased in acidic pH. Due to the abundant presence of the pyrogallate ion in physiological media, we suggest that this conjugate base form is responsible for DNA cleavage.