Sodium Phosphate Buffer Solution Research Articles

Expression and characterization of α‐glucosidase III in the dwarf honeybee, Apis florea (Hymenoptera: Apoidea: Apidae)

AbstractAlpha‐glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apis florea. To analyze the developmental stage‐specific expression of the α‐glucosidase gene in A. florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction (RT‐PCR), it was shown that the highest expression levels of the α‐glucosidase III gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete α‐glucosidase III cDNA was obtained by RT‐PCR and sequenced. The 1 701 bp cDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. melliferaα‐glucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract (0.30 U/mg) sequentially precipitated with 95% saturated ammonium sulfate (0.18 U/mg), and purified by DEAE cellulose ion exchange chromatography (0.17 U/mg), and gel filtration on Superdex 200 (0.52 U/mg). After resolution through sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, a single enzymically active band (73 kDa) was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) revealed six matching masses to the A. mellifera (Q17958) and predicted A. floreaα‐glucosidase III protein with 12% coverage, supporting the probable purification of the same α‐glucosidase III protein as that encoded by the cloned cDNA.

Simultaneous multiresponse optimization applied to epinastine determination in human serum by using capillary electrophoresis

Experimental design and optimization techniques were implemented for the development of a rapid and simple capillary zone electrophoresis method (CZE) for the determination of epinastine hydrochloride in human serum. The effects of five factors were studied on the resolution between the peaks for the target analyte (epinastine hydrochloride) and lidocaine hydrochloride, used as internal standard, as well as on the analysis time. The factors were the concentration and pH of the buffer, the injection time, the injection voltage and the separation voltage. The separation was carried out by using an uncoated silica capillary with 50 μm i.d. and total length 64.5 cm (150 μm of path length) and UV detection (200 nm). Multiple response simultaneous optimization by using the desirability function was used to find experimental conditions where the system generates desirable results. The optimum conditions were: sodium phosphate buffer solution, 16.0 mmol L −1; pH 8.50; injection voltage, 20.0 kV; injection time, 30 s; separation voltage, 26.7 kV. The method was confirmed to be linear in the range of 2.0–12 ng mL −1. The injection repeatability of the method was evaluated by six injections at three concentration levels, while intra-assay precision was assessed by analysing a single concentration level, yielding a CV's of ca. 1% for standard and 2% for serum samples. Accuracy was evaluated by recovery assays and by comparing with an HPLC method, the results being acceptable according to regulatory agencies. The rudgeness was evaluated by means of an experimental Plackett–Burman design, in which the accuracy was assessed when small changes were set in the studied parameters. Clean-up of human serum samples was carried out by means of a liquid–liquid extraction procedure, which gave a high extraction yield for epinastine hydrochloride (93.00%).

２段階液／液抽出法による水中のダラポンのＬＣ／ＭＳ分析

A simple pre-concentration method of dalapon (2,2-dichloropropionic acid) in water for LC/MS analysis by two-step liquid-liquid extraction is proposed. Dalapon was extracted from 200 ml of water sample containing 2 ml of sulfuric acid and 80 g of sodium chloride into 10 ml of methyl-t-butyl ether (MTBE). Then, dalapon was back-extracted from 5 ml portion of the MTBE extract into 1 ml of aqueous solution (water, several concentrations of sodium hydroxide solution or phosphate buffer solution). An aliquot of the aqueous phase was directly injected to LC/MS apparatus. Recovery of dalapon depended on equilibrium pH after the second step extraction. The best recovery was observed in the pH range from 6 to 8. When the phosphate buffer solution (pH 6.87) was used as the aqueous phase in the second step extraction, no change in the pH value of the aqueous phase was observed between before and after extraction. Therefore, phosphate buffer solution was suitable for the second step extraction.The recovery of dalapon (0.0008 mg/l) by the proposed method was about 120 % and the relative standard deviation of five replicate runs was below 3 %. This method is satisfactorily applicable to LC/MS analysis of detection limit of dalapon (0.0008 mg/l) in tap water provided by the Ministry of Health Labour and Welfare of Japan.

Water activity of bacterial suspension media unable to account for the baroprotective effect of solute concentration on the inactivation of Listeria monocytogenes by high hydrostatic pressure

Inactivation of Listeria monocytogenes (10 8 CFU/ml) by high hydrostatic pressure (HHP) from 400 to 600 MPa at 25 °C for 10 min was investigated with various concentrations of sodium chloride, sucrose, and sodium phosphate buffer solutions. Sodium chloride significantly inhibited HHP-induced inactivation of L. monocytogenes at concentrations higher than 2.6 M. A low concentration of sodium chloride within 1.7 M had no effect on HHP-induced inactivation. Almost complete inactivation at relatively low sodium chloride concentration solution was observed with treatments above 500 MPa. Sucrose also significantly inhibited HHP-induced inactivation of L. monocytogenes when greater than 1.2 M sucrose solutions were used. HHP-treatment at 400 MPa reduced the number of L. monocytogenes in 1.2 M, 1.5 M, and 1.8 M sucrose solutions by 4.8, 2.0, and 0.7 log cycles, respectively. Higher pressure did not yield significant reductions. Sodium phosphate buffer significantly inhibited HHP-induced inactivation of L. monocytogenes. In particular, 1 M phosphate buffer completely inhibited HHP-induced inactivation even at 600 MPa. HHP-treatment at 400 MPa reduced the number of L. monocytogenes in 0.1 M, 0.25 M, and 0.5 M phosphate buffer solutions by 5.6, 4.1, and 3.2 log cycles, respectively. The effect of HHP-induced inactivation of L. monocytogenes in the three kinds of solution was evaluated by adjusting water activity ( a w). However, the baroprotective effect differed depending on the kind of solute even at the same a w. This result showed no consistent correlation between a w and solute concentration in terms of the baroprotective effect. As an alternative approach, saturation of suspension solution was used for evaluating the effect of HHP-induced inactivation of L. monocytogenes. As the saturation of suspension media increased, the effect of HHP-induced inactivation of L. monocytogenes decreased regardless of the kinds of solute. The saturation of solution would be an alternative parameter of inhibition in terms of HHP-induced inactivation of bacteria.

Solute Crystallization in Frozen Systems–Use of Synchrotron Radiation to Improve Sensitivity

To demonstrate the sensitivity of low temperature synchrotron X-ray diffractometry (SXRD) for detecting solute crystallization in frozen sodium phosphate buffer solutions. To determine the effect of annealing on solute crystallization in frozen solutions. Sodium phosphate buffer solutions, at initial buffer concentrations ranging from 1 to 100 mM (pH 7.4) were cooled to -50 degrees C. The crystallization of disodium hydrogen phosphate dodecahydrate (Na(2)HPO(4) *12H(2)O) was monitored using a laboratory as well as a synchrotron source. At selected concentrations, the effect of annealing (at -20 degrees C) was investigated. With the laboratory source, solute crystallization, based on the appearance of one diagnostic peak with a d-spacing of 5.4 A, was evident only when the initial buffer concentration was at least 50 mM. In contrast, using SXRD, crystallization was detected at initial buffer concentrations down to 1 mM. In addition, the use of a high-resolution 2D detector enabled the visualization of numerous diffraction rings of the crystalline solute. At both 10 and 100 mM buffer concentration, there was no increase in solute crystallization due to annealing. By using synchrotron radiation, solute crystallization was detected with substantially increased sensitivity, making the technique useful for freeze-drying cycles of practical and commercial importance. Since numerous peaks of the crystalline solute appeared, the technique has potential utility in complex, multi-component systems.

Conformational Changes in Single-Strand DNA as a Function of Temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5′-ATGCTGATGC-3′, in sodium phosphate buffer solution at 10°C temperature increments from 25°C to 80°C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25°C to 43°C, an expansion in the pitch direction with a slight decrease in the diameter from 43°C to 53°C, and finally a dramatic increase in the pitch and diameter from 53°C to 80°C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 ± 0.5°C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 ± 3 kJ mol −1 that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 ± 1°C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.

Corrosion Behavior of Ti-Based Metallic Glasses

The corrosion behavior of the Ti 47.5 Cu 42.5 Ni 7.5 Zr 2.5 metallic glass as well as its modified glasses by the addition of 5 at% Co, Nb or Ta was investigated by electrochemical measurements. Potentiodynamic polarization was carried out in 0.14 kmol/m 3 NaCl solution and 0.2 kmol/m 3 phosphate buffer solution with 0.14 kmol/m 3 Cl - ion. All the metallic glasses examined were spontaneously passivated with significantly low passive current density in sodium chloride solution and phosphate buffer solution. The additional elements improve the corrosion resistance and the effect of Nb or Ta is particular. The results of XPS revealed that the passive films were rich in titanium and deficient in copper and nickel. The higher corrosion resistance for the modified glasses is attributed to stable and protective passive films enriched with titanium cation contained certain amount of additional elements. In phosphate buffer solution, the pitting potentials for all glasses are higher than those in NaCl solution possibly due to inhibiting ability of phosphate ion absorbing on the glassy surface.

A Nonequilibrium Stage Model To Simulate the Chemical Absorption of SO2

A nonequilibrium stage model was developed to simulate chemical absorption of SO2 in flue gas by sodium phosphate solution and ethylenediamine/phosphoric acid solution. The predicted values fit well with the experimental results. Using this model and the double-film theory, the accurate phase equilibrium constant in the nonequilibrium stage model was obtained. The absorption process of SO2 in the simulating of flue gas was predicted for different ratios of liquid flux to vapor flux (L/V) and absorber concentrations in a laboratory packed tower (0.03 m in diameter, and 0.6 m in packed section height,) using sodium phosphate buffer solution and ethylenediamine/phosphoric buffer solution. The relative error of SO2 concentration in exhaust gas between predicted and experimental results is <30%. When the SO2 concentration axial profile in liquid phase was predicted using the above nonequilibrium model, the relative error was <15%. The model can be used for the process design, optimization, and popularization.

Multiple response optimization applied to the development of a capillary electrophoretic method for pharmaceutical analysis

Multiple response simultaneous optimization by using the desirability function was used for the development of a capillary electrophoresis method for the simultaneous determination of four active ingredients in pharmaceutical preparations: vitamins B 6 and B 12, dexamethasone and lidocaine hydrochloride. Five responses were simultaneously optimized: the three resolutions, the analysis time and the capillary current. This latter response was taken into account in order to improve the quality of the separations. The separation was carried out by using capillary zone electrophoresis (CZE) with a silica capillary and UV detection (240 nm). The optimum conditions were: 57.0 mmol l −1 sodium phosphate buffer solution, pH 7.0 and voltage = 17.2 kV. Good results concerning precision (CV lower than 2%), accuracy (recoveries ranged between 98.5 and 102.6%) and selectivity were obtained in the concentration range studied for the four compounds. These results are comparable to those provided by the reference high performance liquid chromatography (HPLC) technique.

Kinetics of Thiamin Degradation in Solutions under Ambient Storage Conditions

ABSTRACT:Thiamin is being incorporated into a variety of nutritionally enhanced beverages. Although thiamin degradation during thermal processing has been extensively studied, minimal data exist regarding its stability in solution during ambient storage. This study collected kinetic data for thiamin degradation in solutions as a function of pH, buffer type, and buffer concentration at 25 °C. Thiamin was incorporated into sodium phosphate and citrate buffer solutions, each prepared at 0.02 and 0.1 M and pH 4, 5, 6, and 7. Experimental solutions were stored in an incubator at 25 °C. Thiamin concentrations were analyzed at 9 time points over a 42‐wk period. Thiamin degradation data were modeled using pseudo‐1st‐order kinetics. Thiamin stability generally decreased as pH and buffer concentration increased in both phosphate and citrate buffers. In 0.1 M phosphate buffer, the time for a 10% loss of thiamin decreased from 79 wk at pH 4 to 3 wk at pH 7. At pH 6 and 7, thiamin stability was greater in citrate buffer than in phosphate buffer. However, at pH 4 and 5, thiamin stability was greater in phosphate buffer than citrate buffer. To optimize thiamin stability in low pH beverages, phosphate buffer would be more appropriate to use than citrate buffer, whereas citrate buffer should be used for better thiamin stability in high pH beverages.

Enzymic asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester in aqueous ionic liquid co-solvent mixtures.

An aqueous solution of ionic liquid BMIM.BF(4) (1-butyl-3-methylimidazolium tetrafluoroborate) was used as the reaction medium for the asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester to enantiopure L-HPG (L-p-hydroxyphenylglycine) for the first time. Papain was screened out from five proteases and two lipases tested because of its high activity and enantioselectivity for this reaction. A comparative study demonstrated that papain is more active, enantioselective and stable in the sodium phosphate buffer solution of BMIM.BF(4) than in aqueous buffer and in the buffer solutions of several typical organic solvents examined. L-HPG with an e.e. (enantiomeric excess) of 95.6% and the yield of 47.4% (on a mole basis) could be obtained when the reaction was performed in a buffer solution of 12.5% (v/v) BMIM.BF(4) (pH 7.0). The aqueous ionic liquid co-solvent increased papain's half-life by 3.1-, 3.8-, 4.7- and 16.3-fold when compared with aqueous buffer, aqueous propan-2-ol, aqueous acetonitrile and aqueous 1,4-dioxan co-solvent respectively at 40 degrees C. When hydrolysis was conducted under reduced pressure, the yield, the e.e. of the L-HPG and the e.e. of remaining D-hydroxyphenylglycine methyl ester achieved were as high as 49.8, 98.1 and >99.0% respectively.

QSAR Analysis for ADA upon Interaction with a Series of Adenine Derivatives as Inhibitors

The kinetic parameters of adenosine deaminase such as Km and Ki were determined in the absence and presence of adenine derivatives (R1–R24) in sodium phosphate buffer (50 mM; pH 7.5) solution at 27°C. These kinetic parameters were used for QSAR analysis. As such, we found some theoretical descriptors to which the binding affinity of adenosine deaminase (ADA) towards several adenine nucleosides as inhibitors is correlated. QSAR analysis has revealed that binding affinity of the adenine nucleosides upon interaction with ADA depends on the molecular volume, dipole moment of the molecule, electric charge around the N1 atom, and the highest of positive charge for the related molecules.

Structure and antitumor activities of the water-soluble polysaccharides from Ganoderma tsugae mycelium

Six water-soluble polysaccharides coded as GTM1 to GTM6 were extracted sequentially from the mycelium of Ganoderma tsugae with 0.2 M sodium phosphate buffer solution at 25, 40, and 80 °C, water at 120 °C, 0.5 M sodium hydroxide at 25 and 65 °C. The chemical structures were determined by using IR, EA, GC and 13C NMR. The weight-average molecular mass ( M w) was characterized by size exclusion chromatography combined with laser light scattering (SEC-LLS). The results indicated that the samples GTM1 and GTM2 were heteropolysaccharide–protein complexes with the protein content of 13.5 and 20.1%, and apparent-mean M w of 62.8×10 4, 81.8×10 4, respectively. GTM3 and GTM4 contained (1→3)-β- d-glucans and (1→4)-α- d-glucans, while GTM5 and GTM6 were mainly a (1→6)-branched (1→3)-β- d-glucan. Two peaks were found in the SEC patterns of the four samples GTM1 to GTM4, representing fractions 1 composed of branched (1→4)-α- d-glucan with high M w and fraction 2 composed of galactose, glucose and mannose with relatively low M w. With the progress of isolation, the content of fraction 2 decreased from 90.2 to 57.1%, accompanying with enhancing antitumor activity. The polysaccharides GTM1, GTM2 and GTM3 had significantly higher antitumor activity against solid tumor Sarcoma 180 with the inhibition ratio beyond 50%. The results suggested that the effects of the moderate content of galactose and bound protein, as well as relatively lower M w, on the improvement of antitumor activities of polysaccharides could not be negligible.

Quality characteristics of spent layer surimi during frozen storage

The effects of different washing solutions and cryoprotectants on quality characteristics of spent layer surimi were studied during frozen storage at −18 °C. The highest (P<0.05) process yield was obtained with 0.5% sodium bicarbonate and 0.04 M sodium phosphate buffer solutions. While washing resulted in decreases (P<0.05) in protein, fat, cholesterol and ash contents, collagen and myofibrillar protein showed increases compared to the unwashed mince. A cryoprotectant mixture of 2% sucrose+2% sorbitol+0.3% sodium pyrophosphate resulted in higher water-holding capacity, except for surimi washed with 0.04 M sodium phosphate buffer solution, and lower cooking loss (P<0.05) than a cryoprotectant mixture of 4% sucrose+2.8% sorbitol. Increased lightness and decreased redness was observed as a result of washing with all solutions (P<0.05).

Gelatin–alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 °C in aqueous sodium phosphate buffer (pH=6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc=8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin–AOS micelle complexes. The micellar radii ( R m), the effective gelatin–surfactant complex radii ( R c), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin–surfactant complex ( k D 1 ) and inter-micellar interactions ( k D 2 ), were determined by fitting the concentration dependence of R m and R c to a virial expansion in reduced concentration ( c−cmc), which are compared. While k D 1 showed strong ionic strength dependence, k D 2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer–surfactant interactions.

Heat-Treated Transition Metal Hexacyanometallates as Electrocatalysts for Oxygen Reduction Insensitive to Methanol

Highly active catalysts for electrochemical reduction of oxygen insensitive to the presence of methanol were prepared from transition metal hexacyanometallate precursors by heat-treatment with carbon black under an inert atmosphere. The catalytic activity for oxygen reduction was examined with the floating electrode technique under an air atmosphere at room temperature. The electrolyte used in most of the measurements was 1 M sodium phosphate buffer solution (pH 7.5), whereas acid and alkaline solutions were also used in addition to the neutral buffer solution to examine the catalytic activity of the prepared catalyst over a wide range of pH. Remarkable enhancements in the catalytic activity were observed for samples heat-treated at temperatures higher than 500°C. Among several 3d-transition metals incorporated in the inorganic precursor, the combination of cobalt and iron incorporated at neighboring sites gave the highest activity, comparable to that of platinum black catalyst (Pt/C). The catalytic activity for oxygen reduction was not affected by the presence of 2.5 M methanol in the electrolyte, while that of Pt/C was severely impaired by the presence of methanol. The catalysts prepared from the inorganic precursors were characterized by X-ray diffraction (XRD), infrared (IR), and X-ray photoelectron spectroscopy (XPS) measurements. The XRD and IR data indicated that the cyanide structure of the inorganic precursor was decomposed when heating beyond 500°C. The XPS data indicated that the oxidation states of cobalt and iron are close to metallic ones and two types of nitrogen forming new bonding are present in the heat-treated samples. The same structural and spectral changes were observed for samples heat-treated without carbon black. From these results, the evolution of the high catalytic activity by heat-treating the inorganic precursors is discussed. © 2004 The Electrochemical Society. All rights reserved.

Adsorption/desorption of acidic and basic proteins on needle-like hydroxyapatite filter prepared by slip casting

The adsorption/desorption of proteins, such as the bovine serum albumin (BSA) as an acidic protein and the lysozyme chloride (LYZ) as a basic protein, was investigated with the needle-like hydroxyapatite(N-HAp) filters after fired at several fixed temperatures. The filters were prepared by a slip casting with the aqueous N-HAp(commercial powder:Ca/P=1.54) slips supplemented with calcium hydroxide [Ca(OH) 2] for the lack of calcium. The cast filters were fired in H 2O vapor at the range of 600–1200 °C. The adsorbed amounts were measured with the N-HAp fired filters. For both the BSA and LYZ, the adsorbed amounts increased with increasing the fired temperatures. Furthermore, the proteins desorption was also investigated by a column chromatograph using the linear gradient method with the sodium phosphate buffer solution (PBS). The PBS concentration at which the BSA was desorbed from the filters was independent to the firing temperature of the filters. However, for the LYZ, the PBS concentration increased with increasing the fired temperatures. From the results, it was found on the filters fired more than 1000 °C that the LYZ adsorbed stronger to the HAp filters than the BSA.

Nanoscale Intermolecular Interactions between Human Serum Albumin and Alkanethiol Self-Assembled Monolayers

To study the molecular origins of hemocompatibility, the blood plasma protein human serum albumin (HSA) was covalently grafted to a nanosized probe tip at the end of a soft, microfabricated cantilever force transducer. The net force versus separation distance between the HSA-modified probe tip and three different model surfaces, including (1) gold; (2) a hydrophobic, CH3-terminated alkanethiol self-assembling monolayer (SAM); and (3) a hydrophilic, COO--terminated alkanethiol SAM in aqueous sodium phosphate buffer solution (PBS, ionic strength (IS) = 0.01 M, pH = 7.4), was recorded and compared to the values of various theoretical models. The approach interaction of the HSA probe tip on the COO--terminated SAM and Au substrates was found to be purely repulsive for D < 15 nm, nonlinear with decreasing separation distance, and consistent with electrostatic double layer repulsion. The approach interaction of the HSA probe tip on the CH3-terminated SAM substrate was found to be purely attractive, long range (D < 80 nm), nonlinear with decreasing separation distance, and much greater in magnitude and range than that known for van der Waals interactions between hydrocarbon SAMs terminated with hydrophilic chemical groups on Au. Large adhesive energies were observed for the HSA probe tip on both the CH3-terminated SAM and Au surfaces (≤−29 mN/m, −22 to −73kBT/protein), while smaller adhesive energies were observed on the COO--terminated SAM surface (≤ −4.9 mN/m, −5.3 to −12kBT/protein). It was shown that short-range adhesive contacts between the HSA chain segments and these surfaces give rise to energy dissipating mechanisms, such as HSA entropic molecular elasticity and enthalpic unfolding forces (deformation and rupture of noncovalent intramolecular bonds) and noncovalent bond rupture of the HSA chain segments adsorbed to the surface.

Deactivation of norepinephrine by peroxynitrite as a new pathogenesis in the hypotension of septic shock.

Vascular hyporeactivity to catecholamines limits successful treatment of hypotension in septic shock. Large amounts of nitric oxide (NO) and superoxide anion (O(2)(-1).) are produced in response to bacterial endotoxins and/or inflammatory cytokines. NO reacts with O(2)(-1). to form the potentially toxic NO metabolite, peroxynitrite (ONOO(-1)). The purpose of this study was to investigate whether ONOO(-1) decreases the vasocontractile activity of norepinephrine. Norepinephrine was treated with ONOO(-1) or 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1; an ONOO(-1) producer) in a 5 x 10(-2) m sodium phosphate buffer solution at pH 7.4, and absorbance of the product was measured spectrophotometrically at 295 and 370 nm. Norepinephrine pretreated with ONOO(-1) was administered to isolated rat thoracic aortas to observe contractions in functional experiments. The rate constant between norepinephrine and ONOO(-1) was determined via a competition assay with cysteine in functional experiments. Norepinephrine pretreated with ONOO(-1) was injected intravenously into anesthetized rats to measure blood pressure. Norepinephrine pretreated with ONOO(-1) was confirmed spectrally as oxidized norepinephrine. Norepinephrine pretreated with ONOO(-1) decreased its vasocontractile force in an ONOO(-1) (10(-6), up to 3 x 10(-4) m) concentration-dependent manner (EC(50) = 5.1 x 10(-5) m). The decrease in its force was lower at pretreatment with ONOO(-1) in a lower pH buffer. A rate constant for the ONOO(-1)-norepinephrine reaction was 6 x 10(2) m/s. Norepinephrine (10(-7) m) incubated with SIN-1 (10(-3) m) decreased its vasocontractile force in an incubation time-dependent manner. Administration of norepinephrine pretreated with ONOO(-1) to anesthetized rats caused no significant change in arterial blood pressure. These results indicate that norepinephrine was oxidized and deactivated by ONOO(-1). This deactivation may, at least in part, account for the hyporeactivities of vasocontraction to norepinephrine in septic shock.

Controlled gene delivery system based on thermosensitive biodegradable hydrogel.

Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The release of 32P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37 degrees C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37 degrees C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.