Study of beta endorphin metabolism in inflamed tissue,serum and trypsin solution by liquid chromatography–tandem mass spectrometric analysis

Beta endorphin (β-END) is recognised as one of the most significant endogenous neuropeptides, responsible for a wide range of biological activities in the body. However, within the body β-END is exposed to hydrolysis by a variety of enzymes. In this study, we investigated the metabolism and fragmentation pattern of β-END in rat inflamed tissue, in rat serum and in trypsin solution. β-END (1-31)-rat was incubated at 37 °C in each matrix for different incubation times. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Structural information for the fragments was elucidated using tandem mass spectrometry. Incubation of β-END (1-31)-rat in trypsin solution and in rat serum resulted in 8 and 13 fragments, respectively. Incubation in inflamed rat paw tissue resulted in 22 fragments at pH 7.4 and 26 fragments at pH 5.5. Some of these fragments were common to both pH values. The degradation of β-END (1-31)-rat in inflamed tissue at pH 5.5 was faster than that at pH 7.4. Secondary fragmentation of some larger primary fragments was also observed in this study.     

Microgels formed by electrostatic and hydrophobic interaction of naphthaleneacetic acid with β-cyclodextrin-grafted polyethyleneimine

Microgels were prepared by physically cross-linking β-cyclodextrin-grafted polyethyleneimine (βCD-PEI) using a hydrophobic acidic compound, naphthaleneacetic acid (NAA). Under a strong acidic condition (e.g., pH 3.0), fibrous microgels were observed on a scanning electron microscope (SEM) possibly due to the intermolecular electrostatic interaction of NAA with PEI. In the range of pH 4.0 to pH 8.0, globular microgels were found possibly because an intramolecular electrostatic interaction prevails over an intermolecular interaction. At pH 9.0 and pH 10.0, neither fibrous nor globular microgels were observed due to lack of the electrostatic interaction and the hydrophobic interaction of NAA with βCD-PEI. The release of fluorescein isothiocyanate-dextran (FITC-dextran; M.W., 10,000) from the microgels increased with increasing pH. At pHs higher than pH 3.0, not only the diffusion of the solute, but also the dissolution of the microgels could contribute to the release.     

The behaviour of cationic NanoFibrillar Cellulose in aqueous media

This paper deals, with cationically modified NanoFibrillar Cellulose (cat NFC), obtained by reacting a dissolving pulp with 2,3-epoxypropyl trimethylammonium chloride (EPTMAC). The cat NFC was thoroughly characterized in terms of morphology and physical properties. The dimensions of individual cellulose nanofibrils were determined by atomic force microscopy (AFM) imaging in water and in air. Fibrils as thin as 0.8–1.2 nm were observed in water. The fibril diameter changed upon drying and the average size was further quantified by image analysis. The experiments showed the importance of characterizing nanocellulosic materials in situ before drying. The fibril size in air was confirmed by cryogenic transmission electron microscopy (cryo-TEM), and it was found to be 2.6–3.0 nm. Smooth ultrathin films of cationic NFC were prepared by spincoating on silica substrates. The effect of electrolyte concentration and pH on swelling of the cationic NFC film was studied using a quartz crystal microbalance with dissipation. The results showed that at pH = 8 the cat NFC film was insensitive to electrolyte changes while at pH = 4.5, the water content of the film decreased with increasing ionic strength. The electrophoretic mobility measurements showed a cationic zeta potential for the cat NFC that decreased at increasing pH, verifying the swelling behaviour.     

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.     

Impact of environmental conditions on the adsorption behavior of radionuclide Ni(II) onto hematite

In this work, adsorption of Ni(II) from aqueous solution onto hematite under various solution chemistry and temperature was investigated. The results indicated that the pseudo-second-order rate equation fitted the kinetic adsorption well. The adsorption of Ni(II) onto hematite was strongly dependent on pH and ionic strength. At low pH, the adsorption was dominated by outer-sphere surface complexation or ion exchange, whereas inner-sphere surface complexation was the main adsorption mechanism at high pH. A positive effect of FA on Ni(II) adsorption was found at pH < 8.0, whereas a negative effect was observed at pH > 8.0. The Langmuir, Freundlich, and D–R models were applied to simulate the adsorption isotherms at three different temperatures of 293.15, 313.15 and 333.15 K. The thermodynamic parameters were calculated from the temperature dependent adsorption, and the results indicated that the adsorption was endothermic and spontaneous.     

A CIDEP study of photolysis of duroquinone/hydrogen-donor homogeneous and triton micelle solutions

The CIDEP spectra of transient radicals during photolysis of the duroquinone (DQ)/ethylene glycol (EG) system in acid, basic, and micellar environments were measured with a home-made highly time-resolved ESR spectrometer. In the DQ/EG homogeneous solution, the enhanced emissive CIDEP signal of the neutral durosemiquinone radical DQH^• was observed. When the DQ/EG solution at pH 9 or the DQ/EG/TX-100/H₂O micelle system was photolyzed, the CIDEP signal of the duroquinone anion radical (DQ^•−) was obtained. When the DQ/EG solution at pH 2.5 was irradiated, the CIDEP signal of DQH^• appeared. These experimental results indicate that the neutral radical DQH^• was formed by proton transfer from EG to ³DQ*, and that DQ^•− was formed by dissociation of DQH^• accompanying polarization transfer.     

Single‐Molecule Visualization of the Activity of a Zn2+‐Dependent DNAzyme

We demonstrate the single‐molecule imaging of the catalytic reaction of a Zn²⁺‐dependent DNAzyme in a DNA origami nanostructure. The single‐molecule catalytic activity of the DNAzyme was examined in the designed nanostructure, a DNA frame. The DNAzyme and a substrate strand attached to two supported dsDNA molecules were assembled in the DNA frame in two different configurations. The reaction was monitored by observing the configurational changes of the incorporated DNA strands in the DNA frame. This configurational changes were clearly observed in accordance with the progress of the reaction. The separation processes of the dsDNA molecules, as induced by the cleavage by the DNAzyme, were directly visualized by high‐speed atomic force microscopy (AFM). This nanostructure‐based AFM imaging technique is suitable for the monitoring of various chemical and biochemical catalytic reactions at the single‐molecule level.     

AFM measurements of forces between silica surfaces

Interaction forces and adhesion between a silica sphere and a flat silica surface in aqueous electrolyte solutions were investigated by atomic force microscopy. The forces were measured as a function of surface separation, pH and NaCl concentration as the surfaces were approaching each other. The adhesion force was determined upon retraction with respect to pH, NaCl concentration and contact time. The magnitude of the long range repulsive force was decreasing with decreasing pH. A short range repulsive force was observed at pH = 2, but no long range repulsive forces were observed at this pH. Force measurements showed that adhesion of silica surfaces in water was obstructed by short and long range repulsive forces. Adhesion was enhanced when both the long and the short range repulsive force was mitigated. A maximum adhesion force of 7.8 mN/m was measured at pH = 12.5 when the short range force vanished and the long range repulsive force was reduced by increasing the NaCl concentration. At pH = 12.5, the work of adhesion was calculated to be 1.2 mJ/m² according to the Derjaguin–Muller–Toporov (DMT) model. Adhesion energy was much less at pH = 2 (0.3 mJ/m²) due to persistive short range repulsion.     

Synthesis and pH-sensitive micellization of doubly hydrophilic poly(acrylic acid)-b-poly(ethylene oxide)-b-poly(acrylic acid) triblock copolymer in aqueous solutions

A doubly hydrophilic triblock copolymer poly(acrylic acid)-b-poly(ethylene glycol)-b-poly(acrylic acid) (PAA-b-PEO-b-PAA) with M _w/M _n = 1.15 was synthesized by atom transfer radical polymerization of t-butyl acrylate (tBA), followed by acidolysis of the PtBA blocks. The pH-sensitive micellization of PAA-b-PEO-b-PAA in acidic solution was investigated by potentiometric titration, fluorescence spectrum, dynamic light scattering and zeta potential. The pK _a was 6.6 and 6.0 in deionized water and in 0.1 mol/L NaCl solution, respectively. The copolymer formed micelles composed of a weakly hydrophobic core of complexed PAA and PEO and a hydrophilic PEO shell in 1 mg/mL solution at pH < 5.5 due to hydrogen bonding. The critical micelle concentration was 0.168 mg/mL at pH 2.0. At pH < 4.5, steady and narrow distributed micelles were formed. Increasing pH to 5.0, unsteady and broad distributed micelles were observed. At pH > 5.5, the micelle was destroyed owing to the ionization of the PAA blocks.     

Self-aggregates of deoxycholic acid-modified chitosan as a novel carrier of adriamycin

A novel and simple method for delivery of adriamycin (ADR) was developed using self-aggregates of deoxycholic acid-modified chitosan. Deoxycholic acid was covalently conjugated to chitosan via EDC-mediated reaction to generate self-aggregated chitosan nanoparticles. ADR was physically entrapped inside the self-aggregates and the characteristics of ADR-loaded chitosan self-aggregates were analyzed by dynamic light scattering, fluorescence spectroscopy, and atomic force microscopy (AFM). The maximum amount of entrapped ADR reached 16.5 wt% of chitosan self-aggregates, suggesting a loading efficiency of 49.6 wt%. The size of ADR-loaded self-aggregates increased with increasing the loading content of ADR. AFM images showed spherical shape of ADR-loaded self-aggregates, and ADR was slowly released from chitosan self-aggregates in PBS solution (pH 7.2). Received: 24 April 2000/Accepted: 11 July 2000     

Electrocatalytic reduction of hydrogen peroxide at Prussian blue modified electrodes: a RDE study

Electrocatalytic reduction of hydrogen peroxide at Prussian blue modified electrode has been studied with rotating disk electrode in pH 5.5 and 7.3 solutions. It has been shown that the electrocatalytic cathodic reduction obeys Koutecky–Levich relationship at electrode potentials ranging from 0.1 to −0.4 V vs. Ag/AgCl for low concentrations of peroxide not exceeding 0.3 mM. Within this potential window, the calculated kinetic cathodic current ranges within the limits of 2.15–6.09 and 1.00–3.60 mA cm⁻² mM⁻¹ for pH 5.5 and 7.3, respectively. For pH 5.5 and 7.3 solutions, a linear slope of the dependence of kinetic current on electrode potential of −10.8 and −2.89 mA cm⁻² mM⁻¹ V⁻¹, respectively, has been obtained. At a higher concentration of peroxide, exceeding 0.6 mM, deviations from Koutecky–Levich relationship have been observed. These deviations appear more expressed at higher potentials and higher solution pH. The results obtained have been interpreted within the frame of two-step reaction mechanism, including (1) dissociative adsorption of hydrogen peroxide with the formation of OH radicals and (2) one-electron reduction of these radicals to OH⁻ anions. At a higher concentration of peroxide, and especially at a higher pH, the second process becomes rate limiting.     

Preparation of Co–Al and Ni–Al layered double hydroxide thin films by a sol–gel process with hot water treatment

Using hot water treatment of sol–gel derived precursor gel films, Co–Al and Ni–Al layered double hydroxide (LDH) thin films were prepared. The precursor gel films of Al₂O₃–CoO or Al₂O₃–NiO were prepared from cobalt or nickel nitrates and aluminum tri-sec-butoxide using the sol–gel method. Then, the precursor gel films were immersed in a NaOH aqueous solution of 100 °C. Nanocrystallites of Co–Al and Ni–Al LDH were precipitated with the hot water treatment with NaOH solution. The largest amounts of nanocrystals were obtained with a solution of pH = 10 for Co–Al LDH, and with that of pH = 9 for Ni–Al LDH. X-ray diffraction measurements confirmed that this process formed CO₃ ²⁻ intercalated LDHs. Both Co–Al and Ni–Al LDH thin films were confirmed to work as electrodes for electrochemical devices by cyclic voltammogram measurements.     

Biodegradable star-shaped poly(ethylene glycol)-poly(β-amino ester) cationic pH/temperature-sensitive copolymer hydrogels

Biodegradable star-shaped copolymers comprised of four-arm poly(ethylene glycol) (4-arm PEG) and poly(β-amino ester) (PAE) were synthesized by conjugating PAE to 4-arm PEG. The synthesized copolymers were characterized by ¹H and ¹³C NMR and gel permeation chromatography. The PAE showed pH/temperature-sensitive properties in an aqueous solution. The copolymer solutions (30 wt.%) showed a gel-to-sol phase transition as a function of temperature in the pH range 7.2–7.8. The gel window covers the physiological conditions (37 °C and pH 7.4) and can be controlled by varying the PAE block length, copolymer solution concentration and PEG molecular weight. After a subcutaneous injection of the copolymer solution into a SD rat, a gel formed rapidly in situ which remained for more than 2 weeks in the body. This copolymer is expected to be a potential candidate for biomedical applications.     

Synthesis and self-assembly behavior of novel polyaspartamide derivatives for anti-tumor drug delivery

A series of amphiphilic graft copolymers based on polyaspartamide were synthesized by a successive aminolysis reaction of polysuccinimide using 2-diisopropylaminoethyl (DIP) and laurylamine as a pH sensitive and hydrophobic group, respectively. The pH-dependent self-assembly behavior of the aqueous copolymer solution was investigated. Nano-aggregation occurred at a pH in the vicinity of the pKa of the DIP group, which was induced by a hydrophilic–hydrophobic shift of the DIP group in solution. The mean diameter of the nano-aggregate could be modulated by changing the composition of both pendants. The mean diameter of the nanoparticles increased with increasing solution pH from 6.5 to 8. At pH 8, the mean diameter of the nanoparticles increased rapidly at the temperature above 45 °C, probably due to the change in hydrophilic–hydrophobic balance of the pH-sensitive DIP moiety. The dissolution of paclitaxel (PTX) into this amphiphilic nanoparticle was attempted and the pH-dependent release behavior was examined with the stability study of the particle. The results showed significantly faster release of PTX at pH 6.5, which is a tumoral acidic pH, than in a neutral physiological pH. These thermo- and pH-sensitive polyaspartamide derivatives have potential use as a tumor-targeting delivery.     

Rapid quantification of tilidine,nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r ² > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.     

Improved rapid assay of plasma uric acid by short-end injection capillary zone electrophoresis

A rapid and simple short-end injection capillary zone electrophoresis method was developed for the quantification of plasma uric acid. The separation was performed in an uncoated fused-silica capillary (50 μm ID, 60 cm total length, 10.2 cm effective length) by using as a background electrolyte a 75 mmol/L glycylglycine solution titrated with NaOH 5 mol/L to pH 9.0, a voltage of 28 kV, a cartridge temperature of 15 °C, and direct UV detection at 292 nm. Under optimized conditions, uric acid was determinate in little more than 1 min (1.076 minutes). In order to verify the accuracy of the analysis, urate levels were measured in 543 apparently healthy volunteers by the new assay and our previous method, and the obtained data were compared by Passing–Bablock regression, Bland–Altman test, and a new regression-based approach, which showed a good agreement between two methods.     

Multi-wall carbon nanotubes and TiO2 as a sensor for electrocatalytic determination of epinephrinein the presence of p-chloranil as a mediator

In this work, we describe an electrochemical method using p-chloranil as a mediator and multi-wall carbon nanotube and TiO₂ as sensors for sensitive determination of epinephrine (EP) in aqueous solution at pH = 10.0. It has been found that under optimum condition (pH 10.0) in cyclic voltammetry, the oxidation of EP occurred at a potential about 171 mV less positive than that unmodified carbon nanotube paste electrode. The diffusion coefficient (D) and the kinetic parameters, such as electron transfer coefficient, (α) and heterogeneous rate constant (k _h) for EP were also determined using electrochemical approaches. The electrocatalytic currents increase linearly with the EP concentration over the range 0.6–135 μM. The detection limits for EP will be equal to 0.25 μM. The relative standard deviation percentage values for 10.0 and 15.0 μM EP were 1.7% and 1.9%, respectively. Finally, this modified electrode was also examined as a selective, simple, and precise new electrochemical sensor for the determination of EP in real sample such as urine and epinephrine injection solution.     

NMR elucidation of monomer–dimer transition and conformational heterogeneity in histone‐like DNA binding protein of Helicobacter pylori

Helicobacter pylori (H. pylori) colonizes under harsh acidic/oxidative stress conditions of human gastrointestinal tract and can survive there for infinitely longer durations of host life. The bacterium expresses several harbinger proteins to facilitate its persistent colonization under such conditions. One such protein in H. pylori is histone‐like DNA binding protein (Hup), which in its homo‐dimeric form binds to DNA to perform various DNA dependent cellular activities. Further, it also plays an important role in protecting the genomic DNA from oxidative stress and acidic denaturation. Legitimately, if the binding of Hup to DNA is suppressed, it will directly impact on the survival of the bacterium, thus making Hup a potential therapeutic target for developing new anti‐H. pylori agents. However, to inhibit the binding of Hup to DNA, it is necessary to gain detailed insights into the molecular and structural basis of Hup‐dimerization and its binding mechanism to DNA. As a first step in this direction, we report here the nuclear magnetic resonance (NMR) assignments and structural features of Hup at pH 6.0. The study revealed the occurrence of dynamic equilibrium between its monomer and dimer conformations. The dynamic equilibrium was found to shifting towards dimer both at low temperature and low pH; whereas DNA binding studies evidenced that the protein binds to DNA in its dimeric form. These preliminary investigations correlate very well with the diverse functionality of protein and will form the basis for future studies aiming to develop novel anti‐H. pylori agents employing structure‐based‐rational drug discovery approach.     

Selectivity in substrate–enzyme complexation studied by surface forces measurement

The selectivity of substrate in substrate–enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM). This enzyme is composed of two dissociable subunits, which exhibits a catalytic activity only when they are associated together in the presence of a cofactor, Mg²⁺, and a substrate, farnesyl diphosphate (FPP). We have recently succeeded to directly demonstrate a specific interaction involved in this enzyme reaction and obtain new insights into the molecular mechanism of the reaction using the approach based on the colloidal probe AFM. The AFM measurement showed the adhesive force between the subunits only in the presence of both Mg²⁺ and FPP. In this study, we studied the substrate selectivity in the complexation by monitoring the adhesive force. The substrates studied are pyrophosphate (PPi), isopentenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl monophosphate (FP), and farnesyl geranyl diphosphate (FGPP). No adhesion was observed in the case of PPi, IPP, and GPP. On the other hand, the significant adhesion was observed for phosphate derivatives, which bear prenyl units longer than three. This is in good agreement with the selectivity of the substrates by this enzyme, which catalyzes the condensation reaction of four IPP molecules with FPP to give heptaprenyl (C₃₅) diphosphates. Our study showed a useful methodology for examining the elemental processes of biological reactions.     

Magnetic resonance studies of copper (II) sorbitol complex,in solution,reveal a supramolecular structure compatible to the crystal structure

Although the Cu²⁺-sorbitol complex [Cu²⁺-Sorb] structure in crystalline state has been determined by X rays, it is not known in solution, where most studies of this complex are performed. Therefore, the goal of this work was to obtain information about the structure of this complex in aqueous solution using nuclear magnetic resonance and electron paramagnetic resonance spectroscopies. The magnetic resonance results indicate that the complex is formed at approximately pH 12. In this pH the sorbitol ¹H relaxation times were so short (broad line) that was not possible to use standard nuclear magnetic resonance parameters (nuclear Overhauser effect and spin–spin coupling constants values) to solve the three-dimensional structure. However, valuable structural information about the complex in solution was obtained. The relaxation results indicate that the Cu²⁺ ions are buried in the structure and not accessible to solvent; the ¹H and ¹³C spectra shows strong paramagnetic shift effect indicating short distance between these nuclei and Cu²⁺ in the structure. No electron paramagnetic resonance signal was observed in pH 12 indicating strong Cu²⁺- Cu²⁺ dipolar interaction, compatible to Cu²⁺-Cu²⁺ distances measured in crystal, from 1.148 to 1.393 Angstroms. The complex self-diffusion coefficient (D) of 1.58 × 10⁻¹⁰ m²/s value, determined by Diffusion-Ordered Spectroscopy, is compatible to a molecular weight of 3–6 KDa. Therefore, these results corroborate that the [Cu²⁺-Sorb] complex is assembled in solution, at pH 12, with several structural parameters compatible to the toroidal hexadecacuprate supramolecular structure determined in solid state.