Tris Buffer pH Research Articles

Simultaneous Identification of Different Domains of Calpain from Blood and Turkey Meat Samples Using Casein Zymography

The aim of this study was to develop a simple method for simultaneous identification of two different domains of calpains (μ- and m-calpains) in blood and turkey meat samples. The method is based on Tris-based extraction techniques followed by casein zymography detection. The extracts were purified by dialysis, and target compounds were separated on Diethylaminoethyl (DEAE)-Sephacel anion-exchange chromatography. Results indicated that a buffer pH of 6.7 produced optimum extraction efficiency. The separation of calpastatin, μ- and m-calpains on column chromatography was carried out using stepwise elution profiles of 100, 200, and 400 mM NaCl solutions, respectively. Casein zymography study clearly demonstrated the presence of μ- and m-calpains in the crude extracts of breast and blood samples; however, μ-calpain was absent in the thigh sample. Similarly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis identified both the calpains from muscle and blood samples, but presence of calpastatin was observed in both the breast and thigh muscle samples only. The activity analysis of purified fractions indicated that both the μ- and m-calpain concentrations were differed significantly (P < 0.05) amongst breast, thigh and blood samples. Collectively, it was concluded that Tris buffer (pH 6.7) and DEAE-Sephacel column for extraction and separation of μ- and m-calpains and casein zymography and SDS-PAGE detection methods were most suitable for accurate identification of calpains and calpastatin from blood and turkey meat samples.

Light Levels Affect Carbon Utilisation in Tropical Seagrass under Ocean Acidification.

Under future ocean acidification (OA), increased availability of dissolved inorganic carbon (DIC) in seawater may enhance seagrass productivity. However, the ability to utilise additional DIC could be regulated by light availability, often reduced through land runoff. To test this, two tropical seagrass species, Cymodocea serrulata and Halodule uninervis were exposed to two DIC concentrations (447 μatm and 1077 μatm pCO2), and three light treatments (35, 100, 380 μmol m-2 s-1) for two weeks. DIC uptake mechanisms were separately examined by measuring net photosynthetic rates while subjecting C. serrulata and H. uninervis to changes in light and addition of bicarbonate (HCO3-) use inhibitors (carbonic anhydrase inhibitor, acetazolamide) and TRIS buffer (pH 8.0). We observed a strong dependence on energy driven H+-HCO3- co-transport (TRIS, which disrupts H+ extrusion) in C. serrulata under all light levels, indicating greater CO2 dependence in low light. This was confirmed when, after two weeks exposure, DIC enrichment stimulated maximum photosynthetic rates (Pmax) and efficiency (α) more in C. serrulata grown under lower light levels (36–60% increase) than for those in high light (4% increase). However, C. serrulata growth increased with both DIC enrichment and light levels. Growth, NPP and photosynthetic responses in H. uninervis increased with higher light treatments and were independent of DIC availability. Furthermore, H. uninervis was found to be more flexible in HCO3- uptake pathways. Here, light availability influenced productivity responses to DIC enrichment, via both carbon fixation and acquisition processes, highlighting the role of water quality in future responses to OA.

Effect of incubation time, buffer type and concentration on gamma-aminobutyric acid (GABA) production using Khao Dawk Mali 105 rice bran

Rice bran of Khao Dawk Mali 105 (KDML105) variety was selected for production of gamma-aminobutyric acid (GABA). The effect of incubation time on GABA production was studied and the maximum GABA was produced after 6 h of incubation. Different types of 50 mM buffers (containing 0.2% glutamic acid) consisting of Tris, citric acid, boric acid and phosphate buffer (pH 5.6) were used to stabilize the pH of the reaction system. The highest GABA content (5.05 mg/g of bran) was found in the phosphate buffer system. Therefore, the effect of phosphate buffer concentrations (0–200 mM) on GABA production was investigated. The results showed that rice bran with phosphate buffer at a concentration of 80 mM at pH 5.6 with a rice bran to phosphate buffer at a ratio of 1–8 (weight per volume) produced the highest GABA content (p ≤ 0.05). GABA production was increased about 2.7 times in the phosphate buffer system compared with the control and about 11 times compared to the initial GABA content (0.58 mg/g of bran) in the rice bran. The results indicated that incubation time, buffer type and concentration significantly affect GABA production using rice bran.

Structural characteristics and surface properties of fluoride-containing calcium phosphates derived from octacalcium phosphate

Event Abstract Back to Event Structural characteristics and surface properties of fluoride-containing calcium phosphates derived from octacalcium phosphate Yukari Shiwaku1, 2, Takahisa Anada1 and Osamu Suzuki1 1 Tohoku University Graduate School of Dentistry, Division of Craniofacial Function Engineering, Japan 2 Tohoku University Graduate School of Dentistry, Liaison Center for Innovative Dentistry, Japan Introduction: Fluoride ion (F-) is well known to be incorporated in hydroxyapatite (HA) and form fluoridated hydroxyapatite (FHA). Fluoride ion stimulates osteoblastic cells[1],[2] and modifies protein adsorption on the calcium phosphate. We previously reported that synthetic octacalcium phosphate (OCP), a precursor of HA, promotes osteoblastic cell differentiation and facilitates bone regeneration[3],[4]. Because of the structure similarity to HA, OCP prepared with F- possibly improves osteogenic capability of original OCP. The aim of this study is to investigate the structural characteristics, protein adsorption, and cellular response of fluoride-containing calcium phosphate (F-CaP) derived from OCP. F-CaPs were prepared two different methods, such as hydrolysis of OCP in F- solution and co-precipitation of OCP with F- . Materials and Methods: F-CaP prepared by hydrolysis (HF-CaP) was synthesized by incubating OCP in 150 mM Tris buffer (pH = 7.1~9.9) containing 50 ppm F- at 37oC. F-CaP prepared by co-precipitation (CF-CaP) was synthesized by adding calcium solution into phosphate solution including 12 and 96 ppm F- at 70oC[5]. The chemical composition and crystal structure of the F-CaPs were examined using inductively coupled plasma (ICP), fluoride electrode and x-ray diffraction (XRD), and field emission scanning electron microscope (FE-SEM). The solubility of the products was evaluated by Ca2+ concentration released after immersing them at pH = 7.4 and 4.0. Protein adsorption on F-CaPs was analyzed using bovine serum albumin (BSA) and cytochrome c. For cell attachment and proliferation test, mouse bone marrow stromal ST-2 cells were seeded onto coatings of F-CaPs and OCP. The number of cells on the coatings or polystyrene culture plate (no coating) was determined using the WST-8 assay. Results: Chemical composition and XRD analysis showed that HF-CaP tended to convert to FHA depending on the F- content of the solids. On the other hand, CF-CaP possessed not only the reflection of OCP, but also that of fluoridated hydroxyapatite (FHA). The solubility of HF-CaP was decreased depending on the fluoride content, whereas that of CF-CaP was comparable to OCP regardless of fluoride contens. HF-CaP adsorbed more cytochrome c than CF-CaP and OCP, whereas BSA adsorption was inhibited on HF-CaP. The difference of protein adsorption behaviors between two types of F-CaPs affected cell attachment and proliferation of ST-2 cells. CF-CaP with high F- content promoted cell attachment and proliferation more than OCP. In contrast, the cell numbers on the HF-CaP with middle F- content were lowest among all the groups. Discussion and Conclusion: The present study confirmed that one of the CF-CaP with high F- content enhances the attachment and proliferation of mouse bone marrow stromal cells. The synthesis processes of incorporating F- to OCP had crucial effects on the crystal structure and morphology as well as the protein adsorption on F-CaP. CF-CaP preserved a small amount of OCP in FHA and maintained and Ca2+ concentration in the medium at a level similar to the original OCP. It seems likely that CF-CaP with high F- content may work as a better substrate for coating an implant surface or bone substitute material.

Optical and relaxometric properties of monometallic (EuIII, TbIII, GdIII) and heterobimetallic (ReI/GdIII) systems based on a functionalized bipyridine-containing acyclic ligand

A series of lanthanide complexes of [LnL(H2O)](2-) composition where Ln = Eu(III), Tb(III) or Gd(III) has been studied for determining their photophysical and relaxometric properties in aqueous solution. The bifunctional ligand L (H5BPMNTA) is an acyclic chelator based on a central functionalized 2,2'-bipyridine core and two iminodiacetate coordinating arms. The mono-aqua Eu(III) and Tb(III) complexes display attractive spectroscopic properties with an excitation wavelength at 316 nm, similar excited state lifetimes and overall quantum yields (in the ranges 0.5-0.6 ms and 10-13%, respectively) in Tris buffer (pH 7.4). The proton longitudinal relaxivity, r1, of the Gd(III) complex is 4.4 mM(-1) s(-1) at 20 MHz and 310 K, which is comparable to that of the clinically used Gd-DTPA (Magnevist®). Interestingly, the water exchange rate between the coordination site and the bulk solvent is very fast (Kex = 2.6 × 10(8) s(-1) at 310 K). The ability of the complex to bind non-covalently to human serum albumin (HSA) was also examined by relaxometric measurements. We also report the synthesis and properties of a bimetallic complex based on Gd-BPMNTA and Re(I)(bpy)(CO)3 components. In this system, the Re core exhibits interesting photophysical properties (λem = 588 nm, Φ = 1.4%) and the Gd-BPMNTA core displays improved relaxivity (r1 = 6.6 mM(-1) s(-1) at 20 MHz and 310 K), due to an increase of the rotational correlation time. Besides these appealing optical and relaxometric properties, the presence of a reactive function on the structure proposes this potential dual imaging probe for conjugation to biomolecules or nanomaterials.

Strontium substituted bioactive glasses for tissue engineered scaffolds: the importance of octacalcium phosphate.

Porous bioactive glasses are attractive for use as bone scaffolds. There is increasing interest in strontium containing bone grafts, since strontium ions are known to up-regulate osteoblasts and down regulate osteoclasts. This paper investigates the influence of partial to full substitution of strontium for calcium on the dissolution and phase formation of a multicomponent high phosphate content bioactive glass. The glasses were synthesised by a high temperature melt quench route and ground to a powder of <38 microns. The dissolution of this powder and its ability to form apatite like phases after immersion in Tris buffer (pH 7.4) and simulated body fluid (SBF) was followed by inductively coupled plasma optical emission spectroscopy (ICP), Fourier transform infra red spectroscopy (FTIR), X-ray powder diffraction (XRD) and 31P solid state nuclear magnetic resonance spectroscopy up to 42 days of immersion. ICP indicated that all three glasses dissolved at approximately the same rate. The all calcium (SP-0Sr-35Ca) glass showed evidence of apatite like phase formation in both Tris buffer and SBF, as demonstrated after 3 days by FTIR and XRD analysis of the precipitate that formed during the acellular dissolution bioactivity studies. The strontium substituted SP-17Sr-17Ca glass showed no clear evidence of apatite like phase formation in Tris, but evidence of an apatite like phase was observed after 7 days incubation in SBF. The SP-35Sr-0Ca glass formed a new crystalline phase termed “X Phase” in Tris buffer which FTIR indicated was a form of crystalline orthophosphate. The SP-35Sr-0Ca glass appeared to support apatite like phase formation in SBF by 28 days incubation. The results indicate that strontium substitution for calcium in high phosphate content bioactive glasses can retard apatite like phase formation. It is proposed that apatite formation with high phosphate bioactive glasses occurs via an octacalcium phosphate (OCP) precursor phase that subsequently transforms to apatite. The equivalent octa-strontium phosphate does not exist and consequently in the absence of calcium, apatite formation does not occur. The amount of strontium that can be substituted for calcium in OCP probably determines the amount of strontium in the final apatite phase and the speed with which it forms.

A simple, sensitive and compact electrochemical ELISA for estradiol based on chitosan deposited platinum wire microelectrodes

Abstract A simple and inexpensive method for fabricating a sensitive and compact indirect sandwich type electrochemical ELISA for the detection of estradiol using a chitosan electrodeposited platinum (Pt) wire microelectrode was proposed. In this assay, anti-17β estradiol antibody produced in goat (goat anti-estradiol Ab) was used as the capture antibody which was immobilized on the chitosan coated Pt wire microelectrode, anti-17β estradiol antibody produced in mouse (mouse anti-estradiol Ab) was used as the detection antibody, and goat anti-mouse IgG (immunoglobulin G) conjugated with alkaline phosphatase (AP) was used as the secondary antibody. The effect of pre-coated layers (chitosan, the capture antibody, and the blocking reagent BSA) on the electron transfer resistance (R et ) at the surface of ELISA electrodes has been investigated and analyzed by the electrochemical impedance spectrum (EIS). 4-Aminophenyl phosphate (4-APP) was chosen as the AP substrate and the oxidation potential of the electroactive AP product, 4-aminophenol (4-AP), on the Pt electrode was determined to be + 0.14 V ( vs . Ag/AgCl). The electrochemical ELISA was detected by constant potential amperometry at + 0.14 V in the Tris buffer (pH 9.0). The limit of detection of this assay was 2.7 × 10 − 1 pg/mL with a wide detection range from 2.7 × 10 − 1 pg/mL up to 1.0 × 10 5 pg/mL. The assay specificity evaluated by testing the cross-reactivity of the assay for progesterone and 17α-ethynylestradiol was found to be 0.033% and 3.4%, respectively. This assay has been tested with estradiol in spiked serum samples; however, further pretreatment of serum samples may be required to enhance precision and recovery.

Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography

Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide

Cytochrome bc1 is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Qo site in cytochrome b with cytochrome c1. The Qo site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Qo site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Qo site. This renders cytochrome bc1 non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Qo site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc1 to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which "semireverse" electron transfer back to the Qo site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique "analyte focusing by micelle collapse-MEKC (AFMC-MEKC)" were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single-variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10mM Tris buffer (pH 9.5) containing 60mM SDS, 7mM γ-CD, and 20% v/v ethanol. The sample was prepared in 100mM Tris buffer (pH 9.0) containing 7.5mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000-fold dilution were successfully determined by AFMC-MEKC.

An optimized and validated SPE-LC–MS/MS method for the determination of caffeine and paraxanthine in hair

Caffeine is the probe drug of choice to assess the phenotype of the drug metabolizing enzyme CYP1A2. Typically, molar concentration ratios of paraxanthine, caffeine’s major metabolite, to its precursor are determined in plasma following administration of a caffeine test dose. The aim of this study was to develop and validate an LC–MS/MS method for the determination of caffeine and paraxanthine in hair. The different steps of a hair extraction procedure were thoroughly optimized. Following a three-step decontamination procedure, caffeine and paraxanthine were extracted from 20mg of ground hair using a solution of protease type VIII in Tris buffer (pH 7.5). Resulting hair extracts were cleaned up on Strata-X™ SPE cartridges. All samples were analyzed on a Waters Acquity UPLC® system coupled to an AB SCIEX API 4000™ triple quadrupole mass spectrometer. The final method was fully validated based on international guidelines. Linear calibration lines for caffeine and paraxanthine ranged from 20 to 500pg/mg. Precision (%RSD) and accuracy (%bias) were below 12% and 7%, respectively. The isotopically labeled internal standards compensated for the ion suppression observed for both compounds. Relative matrix effects were below 15%RSD. The recovery of the sample preparation procedure was high (>85%) and reproducible. Caffeine and paraxanthine were stable in hair for at least 644 days. The effect of the hair decontamination procedure was evaluated as well. Finally, the applicability of the developed procedure was demonstrated by determining caffeine and paraxanthine concentrations in hair samples of ten healthy volunteers. The optimized and validated method for determination of caffeine and paraxanthine in hair proved to be reliable and may serve to evaluate the potential of hair analysis for CYP1A2 phenotyping.

Cryopreservation of Siberian sturgeon (Acipenser baerii , Brandt, 1869) and sterlet (Acipenser ruthenus , Linnaeus, 1758) semen and its influence on sperm motility parameters assessed using a computer-assisted sperm analysis (CASA) system

Using a computer assisted sperm analysis (CASA) system, sperm motility parameters were determined in semen of the Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus) prior to and after cryopreservation. Semen was collected from 15 Siberian sturgeon and 15 sterlet males, diluted 1:1 (sperm:freezing medium) in 23.4 mm sucrose, 0.25 mm KCl, 30 mm Tris (pH of 8.0) and 10% methanol, loaded in 0.25 ml straws and frozen over liquid nitrogen vapor. Post-thaw sperm motility was activated in 10 mm Tris buffer (pH 8.5), containing 20 mm NaCl and 2 mm CaCl2. A significant decrease in the percentage of motile sperm (MOT: from 41.3% to 25.3%), curvilinear velocity (VCL: from 121.9 to 112.2 μm s−1), amplitude of lateral head displacement (ALH: from 10.4 to 5.4 μm) and momentum (MOM: from 4591 to 1038 m s−1) was observed as effect of the cryopreservation process in the Siberian sturgeon semen. A significant decrease in the values of most CASA parameters, including MOT, VAP, VCL, VSL, ALH and MOM, were also noted following the cryopreservation of sterlet semen. In the sterlet, an additional decrease in VAP (from 102.4 to 80.3 μm s−1) and VSL (from 86.8 to 69.8 μm s−1) values was noted after cryopreservation when compared to fresh semen. Unfortunately, the cryopreservation of semen from both species led to a drop in CASA parameters, which are important to the successful fertilization of eggs. MOT and VCL parameters were the most affected in these species. They can be used as sensitive criteria to check changes in motility.

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10μM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.

Determining ultraviolet absorbents in sunscreen products by combining direct injection with micelle collapse on-line preconcentration capillary electrophoresis

The on-line preconcentration technique of analyte focusing by micelle collapse-micellar electrokinetic chromatography (AFMC-MEKC) was combined with direct injection without extraction to determine ultraviolet absorbents in sunscreen products. The stacking mechanism is based on the transport, release, and accumulation of analytes bound to micelle carriers that are collapsed into the micelle dilution zone. The following optimized conditions were determined: the running buffer was 10mM Tris buffer (pH 9.5) containing 60mM SDS, 7mM γ-CD and 20% ethanol; the SDS concentration was required to be slightly above the critical micelle concentration (cmc) value (7.4mM) in the sample matrix, which allowed the micelle dilution zone to form when voltage was applied; and finally, the sample was prepared in 100mM Tris buffer (pH 9.0) containing 7.5mM SDS and 20% (v/v) ethanol to provide sufficient resolution and to improve the sensitivity. Samples were injected at 0.5psi for 40s, and the separation voltage was set at 15kV for first 15min and then increased to 23kV to decrease the analysis time. The detection sensitivity for ultraviolet absorbents was enhanced by approximately 41-fold using AFMC-MEKC compared to conventional MEKC. The limit of detection (S/N=3) was 98nM for benzophenone-2 and benzophenone-4. The correlation of the regression curve was greater than 0.995. The relative error and relative standard deviation were lower than 9.94% with high precision and accuracy. The recoveries of nine ultraviolet absorbents in a homemade emulsion were between 95.08% and 104.57%. After optimization and validation, this AFMC-MEKC method combined with direct injection is considered to be established and successfully applicable to commercial sunscreen products.

Long-term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer-independent peroxidase purified from Jatropha curcas leaves.

Isoenzyme c of horseradish peroxidase (HRP-C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP-C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H2O2). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H2O2. Compared with HRP-C, the JcGP1-induced reaction was enhancer independent, which made the enzyme-linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long-term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H2 O2, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long-term stable CL signal combined with enhancer-independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection.

First Report of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica in Ivory Coast.

Xanthomonas citri pv. mangiferaeindicae causing bacterial canker (or black spot) is a major mango (Mangifera indica L.) pathogen in tropical and subtropical areas (3). The bacterium infects a wide range of mango cultivars, and induces raised, angular, black leaf lesions, sometimes with a yellow chlorotic halo. Fruit symptoms first appear as small water-soaked spots on the lenticels turning into star-shaped, erumpent lesions, which exude an infectious gum, yielding tear-stain patterns. Severe infections cause severe defoliation and/or premature fruit drop. Twig cankers are potential sources of inoculum and weaken branch resistance to winds. Drastic yield losses have been reported at grove scale for susceptible cultivars (3). Mango leaves showing typical angular, black, raised leaf lesions were first observed and collected in April 2014 from trees cv. Kent in five localities of the Korhogo province of Ivory Coast (i.e., the major commercial mango-growing area in this country). Non-pigmented Xanthomonas-like colonies were isolated on KC semi-selective medium (4). Five strains (LL60-1, LL61-1, LL62-1, LL63-1, and LL64-1), one from each locality, were compared by multilocus sequence analysis (MLSA) to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes, as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Leaves of mango cv. Maison Rouge from the youngest vegetative flush were infiltrated (10 inoculation sites/leaf for three replicate leaves on different plants/bacterial strain) as detailed previously (1) with the same five strains. Bacterial suspensions (~1 × 105 cfu/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old cultures on YPGA (7 g yeast, 7 g peptone, 7 g glucose, and 18 g agar/liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites/leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h day/night cycle) at 80 ± 5% RH. All leaves inoculated with the strains from Ivory Coast showed typical symptoms of bacterial canker a week after inoculation. No lesions were recorded from the negative controls. The pathogen was recovered at high population densities (>1 × 106 cfu/lesion) from leaf lesions, typical of a compatible interaction (1) and isolated colonies were identified as the target by atpD sequencing (2). Koch's postulates have therefore been fully verified. This is the first report of the disease in Ivory Coast, a country which has been an internationally significant mango exporter (up to 15,000 tons per year) over the last two decades. A high disease incidence and severity were observed, outlining the need for implementing integrated pest management in mango groves and the production of disease-free nursery stock. This report further expands the distribution of the pathogen in West Africa after its first description from Ghana in 2011 (5) and subsequently in other neighboring countries.

High-Performance Liquid Chromatographic Determination of Cefalexin Monohydrate and Kanamycin Monosulfate with Precolumn Derivatization

A liquid chromatographic method for the determination of cefalexin monohydrate and kanamycin monosulfate in their synthetic binary mixture was developed and validated according to ICH guidelines. Cefalexin monohydrate and kanamycin monosulfate both contain primary amine and were derivatized with phenylisocyanate in presence of triethylamine at 70°C for 10 min forming corresponding N-aryl-N′-phenyl urea derivative. The separation was achieved on Phenomenex column, C18, 250 × 4.6 mm, 5 µm under isocratic conditions, using acetonitrile: 1% tris buffer (40:60 v/v) pH adjusted to 6.5 with 1 N sulfuric acid, as a mobile phase and flow rate was 1.0 mL/min. The retention times were 3.7 and 8.5 min for cefalexin monohydrate and kanamycin monosulfate, respectively, at 242 nm. The linear concentration range was 7.5–22.5 µg/mL for cefalexin monohydrate and 5–15 µg/mL for kanamycin monosulfate. The mean percentage recoveries for binary mixture were found to be 93.33–99.11 for cefalexin monohydrate and 92–98 for kanamycin monosulfate. The pooled % RSD value for validation parameters for both the methods was less than 2. The results of method confirm that this high-performance liquid chromatographic method can be applicable, for quality control and assay of bulk and binary synthetic mixture.

Retraction Note to: Thermodynamic study on the interaction of cyanide ion and jack bean urease at different temperatures

A method based on Isothermal Titration Calorimety (ITC) is described for the thermodynamic assay of jack bean urease. Inhibitory activity of cyanide ion was examined against jack bean urease (JBU), at 27 and 37 oC in 30 mM Tris buffer of pH = 7. The binding parameters of the CN− + JBU complexation have been calculated. It was found that in the low and high concentrations of the cyanide ions, the JBU structure was destabilized, resulting in a decrease in its biological activity.

Processing and characterisation of a novel electropolymerized silk fibroin hydrogel membrane.

Silk fibroin can be made into various forms of biocompatible medical materials, including hydrogel due to its excellent properties. Here, we report a novel method for the preparation of electropolymerized silk fibroin hydrogel membrane (ESFHM), which is formed on a nanoporous film as a barrier using a homemade device at a higher DC voltage. Regenerated silk fibroin solution in Tris buffer (pH 6.55–7.55) was added into a reservoir with a negative charge, and the silk molecules migrated toward the positive charge at 80VDC, resulting in the formation of the ESFHM on the barrier film. Barrier film with a MWCO of 10 kDa is favourable to the formation of the ESFHM. Semi-transparent ESFHM with a swelling ratio of 1056.4% predominantly consisted of a mixture of β-sheets and α-helix crystalline structures. SEM studies revealed that the ESFHM consisted of a 3D mesh structure woven by a chain of silk fibroin nanoparticles with a size of approximately 30 nanometres, similar to a pearl necklace. In vitro studies indicated that the ESFHM was degradable and was sufficient for cell adhesion and growth. Thus, ESFHM is a promising candidate for loading bioactive protein and appropriate cells, as artificial skin or for use in transplantation.

Sensitive and selective trypsin detection using redox cycling in the presence of L-ascorbic acid.

We report a simple, sensitive, and selective electrochemical method for trypsin detection that can cover a wide range of concentrations. The method is based on the proteolytic generation of an electroactive species (P) by trypsin followed by a signal-amplified electrochemical measurement of P using electrochemical-chemical (EC) or electrochemical-chemical-chemical (ECC) redox cycling. The detection is performed using bare indium-tin oxide (ITO) electrodes without washing steps. P is generated by the cleavage of an amide bond between P and oligopeptide (Gly-Pro-Arg) at the C-terminal of Gly-Pro-Arg-P. Four trypsin products including 4-amino-1-naphthol (AN) and their trypsin substrates are investigated to obtain a high signal-to-background ratio in ECC redox cycling. AN and its trypsin substrate produce the highest signal-to-background ratio. The detection limits obtained with ECC redox cycling involving AN (approximately 1 ng mL(-1) and 100 ng mL(-1) with an incubation period of 2 h and 30 min, respectively) in Tris buffer (pH 8.0) are lower than those obtained with EC redox cycling involving AN (approximately 5 ng mL(-1) and 200 ng mL(-1) with an incubation period of 2 h and 30 min, respectively). In trypsin detection using ECC redox cycling, the interference effects of electroactive species such as l-ascorbic acid and uric acid are not significant.