Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation.

PubMed

Zhao, Renyong; Bean, Scott R; Crozier-Dodson, Beth Ann; Fung, Daniel Y C; Wang, Donghai

2009-01-01

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0-5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R (2) = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41-47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (mu(max)) were 0.42 and 0.32 h(-1) for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases. PMID:29487850

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices.

PubMed

Busto, M D; García-Tramontín, K E; Ortega, N; Perez-Mateos, M

2006-09-01

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.

Electrophoretic analysis of the major polypeptides of human erythrocyte membranes prepared by low and high osmolarity haemolysis.

PubMed

Zail, S S; Hoek, V D

1975-04-16

Human erythrocyte membranes were prepared in three ways: washing in hypotonic Tris buffer, pH 7.6, by lysis in isotonic Tris buffer pH 7.6 after incubation at 37 degrees C for 2 hours and by ultrasonication in an isotonic medium, pH 7.6. Analysis of the major polypeptides of the erythrocyte membranes by sodium dodecylsulphate polyacrylamide gel electrophoresis revealed a selective depletion of a major polypeptide representing glyceraldehyde-3-phosphate dehydrogenase in the membranes prepared by high osmolarity lysis. The pattern of seperation of the remaining polypeptides was identical in the 3 different membrane preparations.

Optimizing buffering chemistry to maintain near neutral pH of broiler feed during pre-enrichment for Salmonella.

PubMed

Berrang, M E; Cosby, D E; Cox, N A; Cason, J A; Richardson, K E

2015-12-01

Salmonella is a human pathogen that can accompany live broilers to the slaughter plant, contaminating fully processed carcasses. Feed is one potential source of Salmonella to growing broilers. Monitoring feed for the presence of Salmonella is part of good agricultural practice. The first step in culturing feed for Salmonella (which may be at low numbers and sub-lethally stressed) is to add it to a pre-enrichment broth which is incubated for 24 h. During the course of pre-enrichment, extraneous bacteria metabolize carbohydrates in some feed and excrete acidic byproducts, causing the pH to drop dramatically. An acidic pre-enrichment pH can injure or kill Salmonella resulting in a failure to detect, even if it is present and available to infect chickens. The objective of this study was to test an array of buffering chemistries to prevent formation of an injurious acidic environment during pre-enrichment of feed in peptone water. Five grams of feed were added to 45 mL of peptone water buffered with carbonate, Tris pH 8, and phosphate buffering ingredients individually and in combination. Feed was subjected to a pre-enrichment at 35°C for 24 h; pH was measured at 0, 18, and 24 h. Standard phosphate buffering ingredients at concentrations up to 4 times the normal formulation were unable to fully prevent acidic conditions. Likewise, carbonate and Tris pH 8 were not fully effective. The combination of phosphate, carbonate, and Tris pH 8 was the most effective buffer tested. It is recommended that a highly buffered pre-enrichment broth be used to examine feed for the presence of Salmonella. Published by Oxford University Press on behalf of Poultry Science Association 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

An improved glycerol biosensor with an Au-FeS-NAD-glycerol-dehydrogenase anode.

PubMed

Mahadevan, Aishwarya; Fernando, Sandun

2017-06-15

An improved glycerol biosensor was developed via direct attachment of NAD + -glycerol dehydrogenase coenzyme-apoenzyme complex onto supporting gold electrodes, using novel inorganic iron (II) sulfide (FeS)-based single molecular wires. Sensing performance factors, i.e., sensitivity, a detection limit and response time of the FeS and conventional pyrroloquinoline quinone (PQQ)-based biosensor were evaluated by dynamic constant potential amperometry at 1.3V under non-buffered conditions. For glycerol concentrations ranging from 1 to 25mM, a 77% increase in sensitivity and a 53% decrease in detection limit were observed for the FeS-based biosensor when compared to the conventional PQQ-based counterpart. The electrochemical behavior of the FeS-based glycerol biosensor was analyzed at different concentrations of glycerol, accompanied by an investigation into the effects of applied potential and scan rate on the current response. Effects of enzyme stimulants ((NH 4 ) 2 SO 4 and MnCl 2 ·4H 2 O) concentrations and buffers/pH (potassium phosphate buffer pH 6-8, Tris buffer pH 8-10) on the current responses generated by the FeS-based glycerol biosensor were also studied. The optimal detection conditions were 0.03M (NH 4 ) 2 SO 4 and 0.3µm MnCl 2 ·4H 2 O in non-buffered aqueous electrolyte under stirring whereas under non-stirring, Tris buffer at pH 10 with 0.03M (NH 4 ) 2 SO 4 and 30µm MnCl 2 ·4H 2 O were found to be optimal detection conditions. Interference by glucose, fructose, ethanol, and acetic acid in glycerol detection was studied. The observations indicated a promising enhancement in glycerol detection using the novel FeS-based glycerol sensing electrode compared to the conventional PQQ-based one. These findings support the premise that FeS-based bioanodes are capable of biosensing glycerol successfully and may be applicable for other enzymatic biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural and Biochemical Studies of the Ovarian Tumor Domain

DTIC Science & Technology

2007-05-01

solution containing Bis-Tris pH 5.5-6.5, 16-20% PEG 3350 , and 100-200 mM of a magnesium cation. These crystals belong to spacegroup P64 with unit...drop method using a reservoir solution containing Bis-Tris pH 5.5-6.5, 16-20% PEG 3350 , and 50-200 mM ammonium acetate . Orthorhombic crystals

Interaction of HEPES buffer with glass-ceramic scaffold: Can HEPES replace TRIS in SBF?

PubMed

Rohanová, Dana; Horkavcová, Diana; Paidere, Laine; Boccaccini, Aldo Roberto; Bozděchová, Pavlína; Bezdička, Petr

2018-01-01

An international standard (ISO: 23317:2014) exists for the in vitro testing of inorganic biomaterials in simulated body fluid (SBF). This standard uses TRIS buffer to maintain neutral pH in SBF, but in our previous paper, we showed that the interaction of a tested glass-ceramic material with TRIS can produce false-positive results. In this study, we evaluated whether the HEPES buffer, which also belongs to the group of Good´s buffers, would be more suitable for SBF. We compared its suitability in two media: SBF with HEPES and demineralized water with HEPES. The tested scaffold (45S5 bioactive glass-based) was exposed to the media under a static-dynamic arrangement (solutions were replaced on a daily basis) for 15 days. Leachate samples were collected daily for the analysis of Ca 2+ ions and Si (AAS), (PO 4 ) 3- ions (UV-VIS), and to measure pH. The glass-ceramic scaffold was analyzed by SEM/EDS, XRD, and WD-XRF before and after 0.3, 1, 3, 7, 11, and 15 days of exposure. Our results confirmed the rapid selective dissolution of the glass-ceramic crystalline phase (Combeite) containing Ca 2+ ions due to the presence of HEPES, hydroxyapatite supersaturation being reached within 24 h in both solutions. These new results suggest that, like TRIS, HEPES buffer is not suitable for the in vitro testing of highly reactive inorganic biomaterials (glass, glass-ceramics). The ISO standard for such tests requires revision, but HEPES is not a viable alternative to TRIS buffer. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 143-152, 2018. © 2016 Wiley Periodicals, Inc.

Stability and tribological performances of fluid phospholipid bilayers: effect of buffer and ions.

PubMed

Dekkiche, F; Corneci, M C; Trunfio-Sfarghiu, A-M; Munteanu, B; Berthier, Y; Kaabar, W; Rieu, J-P

2010-10-15

We have investigated the mechanical and tribological properties of supported Dioleoyl phosphatidylcholine (DOPC) bilayers in different solutions: ultrapure water (pH 5.5), saline solution (150 mM NaCl, pH 5.8), Tris buffer (pH 7.2) and Tris saline buffer (150 mM NaCl, pH 7.2). Friction forces are measured using a homemade biotribometer. Lipid bilayer degradation is controlled in situ during friction tests using fluorescence microscopy. Mechanical resistance to indentation is measured by force spectroscopy with an atomic force microscope. This study confirms that mechanical stability under shear or normal load is essential to obtain low and constant friction coefficients. In ultrapure water, bilayers are not resistant and have poor lubricant properties. On the other hand, in Tris saline buffer, they fully resist to indentation and exhibit low (micro=0.035) and stable friction coefficient with no visible wear during the 50 min of the friction test. The unbuffered saline solution improves the mechanical resistance to indentation but not the lubrication. These results suggest that the adsorption of ions to the zwiterrionic bilayers has different effects on the mechanical and tribological properties of bilayers: higher resistance to normal indentation due to an increase in bilayer cohesion, higher lubrication due to an increase in bilayer-bilayer repulsion. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers.

PubMed

Lu, Shih-Chin; Lin, Sung-Chyr

2012-01-05

Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

The effect of CO2 and non-CO2-generating buffers on cerebral acidosis after cardiac arrest: A 31P NMR study.

PubMed

Rosenberg, J M; Martin, G B; Paradis, N A; Nowak, R M; Walton, D; Appleton, T J; Welch, K M

1989-04-01

There is controversy regarding the use of alkalinizing agents during reperfusion after cardiac arrest. The potential deleterious effects of sodium bicarbonate (bicarb) administration, including paradoxic cerebral acidosis, have led to the search for alternative agents. Tromethamine (tris) is a non-CO2-generating buffer that has been proposed for use during cardiopulmonary resuscitation. The purpose of this experiment was to compare the ability of tris with bicarb to correct brain pH (pH B) during reperfusion after a 12-minute cardiac arrest. Adult mongrel dogs were instrumented and placed in the bore of a Bruker Biospec 1.89 tesla superconducting magnet system. Ventricular fibrillation was induced; after 12 minutes, cardiopulmonary bypass was initiated and maintained for two hours with minimum flows of 80 mL/kg/min. Bicarb (n = 5) or tris (n = 5) were administered to correct arterial pH as rapidly as possible. 31P NMR spectra were obtained at baseline and throughout ischemia and reperfusion. The pH B was determined with the inorganic phosphate relative to the phosphocreatine resonance signal shift. Profile analysis indicates a difference between groups (P less than .02) related to an initial delay in pH B correction in the tris group. By 48 minutes of reperfusion, pH B did not differ between the groups. Moreover, there was no evidence of paradoxic cerebral acidosis in the bicarb group. Although tris corrects blood pH as quickly as bicarb, it is less effective in correcting pH B. Absence of paradoxic acidosis may be caused by efficient elimination of CO2 by cardiopulmonary bypass.

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas).

PubMed

Fu, Xue-Yan; Xue, Chang-Hu; Miao, Ben-Chun; Liang, Jun-Ni; Li, Zhao-Jie; Cui, Feng-Xia

2006-02-08

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.

Purification of α-glucosidase from mouse intestine by countercurrent chromatography coupled with a reverse micelle solvent system.

PubMed

He, Kai; Zou, Zongyao; Hu, Yinran; Yang, Yong; Xiao, Yubo; Gao, Pincao; Li, Xuegang; Ye, Xiaoli

2016-02-01

Countercurrent chromatography coupled with a reverse micelle solvent was applied to separate α-glucosidase, which is stable at pH 6.0-8.8, 15-50°C. The separation conditions are as follows: stationary phase: pH 4.0 Tris-HCl buffer phase containing 50 mM Tris-HCl and 50 mM KCl; mobile phase A: isooctane containing 50 mM anionic surfactant sodium di(2-ethylhexyl)sulfosuccinate; mobile phase B: 50 mM Tris-HCl buffer containing 500 mM KCl (pH 8.0); In total, 25 mL (23.9 mg) crude enzyme was injected through the injection valve, the enzymatic reaction and sodium dodecylsulfate polyacrylamide gel electrophoresis results imply that the activity of purified α-glucosidase is 6.63-fold higher than that of the crude enzyme. Therefore, countercurrent chromatography coupled with a reverse micelle solvent is capable for protein separation and enrichment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

PubMed

Metrick, Michael A; Temple, Joshua E; MacDonald, Gina

2013-12-31

The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions. © 2013. Published by Elsevier B.V. All rights reserved.

Tris-base buffer: a promising new inhibitor for cancer progression and metastasis.

PubMed

Ibrahim-Hashim, Arig; Abrahams, Dominique; Enriquez-Navas, Pedro M; Luddy, Kim; Gatenby, Robert A; Gillies, Robert J

2017-07-01

Neutralizing tumor external acidity with oral buffers has proven effective for the prevention and inhibition of metastasis in several cancer mouse models. Solid tumors are highly acidic as a result of high glycolysis combined with an inadequate blood supply. Our prior work has shown that sodium bicarbonate, imidazole, and free-base (but not protonated) lysine are effective in reducing tumor progression and metastasis. However, a concern in translating these results to clinic has been the presence of counter ions and their potential undesirable side effects (e.g., hypernatremia). In this work, we investigate tris(hydroxymethyl)aminomethane, (THAM or Tris), a primary amine with no counter ion, for its effects on metastasis and progression in prostate and pancreatic cancer in vivo models using MRI and bioluminescence imaging. At an ad lib concentration of 200 mmol/L, Tris effectively inhibited metastasis in both models and furthermore led to a decrease in the expression of the major glucose transporter, GLUT-1. Our results also showed that Tris-base buffer (pH 8.4) had no overt toxicity to C3H mice even at higher doses (400 mmol/L). In conclusion, we have developed a novel therapeutic approach to manipulate tumor extracellular pH (pHe) that could be readily adapted to a clinical trial. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

The effects of biological buffers TRIS, TAPS, TES on the stability of lysozyme.

PubMed

Pannuru, Pavani; Rani, Anjeeta; Venkatesu, Pannuru; Lee, Ming-Jer

2018-06-01

To explore the mechanism of lysozyme stabilization in buffer system, we have investigated the interactions between lysozyme and the biological buffers (TRIS, TAPS, and TES) using spectroscopic techniques, including ultraviolet-visible (UV-Vis), fluorescence, thermal fluorescence, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. From the series of spectroscopic studies, it is found that the native structure of the protein remains intact in the different concentrations (0.05, 0.1, 0.25, 0.5, and 1.0M) of the biological buffer aqueous solutions at pH7.0. Moreover, all these three investigated buffers are able to protect lysozyme against thermal denaturation, particularly in high concentration (1.0M) of the buffer aqueous solutions. Copyright © 2018 Elsevier B.V. All rights reserved.

Isolation of Protective Antigen from Anthrax Toxin by Preparative Isotachophoresis

DTIC Science & Technology

1982-06-14

caosule of polymerized D-olutamic acid which inhibits phago- cytosis and allows the bacillus to establish an infectious focus and elaborate a tripartite...The terminating buffer Tris-c- aminocaproate (Tris-EACA), pH 8.4, was used in the upper electrode (cathode) chamber and in the column. fteen ml of toxin

DNA stretching on the wall surfaces in curved microchannels with different radii

NASA Astrophysics Data System (ADS)

Hsieh, Shou-Shing; Wu, Fong-He; Tsai, Ming-Ju

2014-08-01

DNA molecule conformation dynamics and stretching were made on semi-circular surfaces with different radii (500 to 5,000 μm) in microchannels measuring 200 μm × 200 μm in cross section. Five different buffer solutions - 1× Tris-acetate-EDTA (TAE), 1× Tris-borate-EDTA (TBE), 1× Tris-EDTA (TE), 1× Tris-phosphate-EDTA (TPE), and 1× Tris-buffered saline (TBS) solutions - were used with a variety of viscosity such as 40, 60, and 80 cP, with resultant 10-4 ≤ Re ≤ 10-3 and the corresponding 5 ≤ Wi ≤ 12. The test fluids were seeded with JOJO-1 tracer particles for flow visualization and driven through the test channels via a piezoelectric (PZT) micropump. Micro particle image velocimetry (μPIV) measuring technique was applied for the centered-plane velocity distribution measurements. It is found that the radius effect on the stretch ratio of DNA dependence is significant. The stretch ratio becomes larger as the radius becomes small due to the larger centrifugal force. Consequently, the maximum stretch was found at the center of the channel with a radius of 500 μm.

Photo-degradation behaviour of roseoflavin in some aqueous solutions

NASA Astrophysics Data System (ADS)

Tyagi, A.; Penzkofer, A.; Mathes, T.; Hegemann, P.

2010-03-01

An absorption and emission spectroscopic characterization of roseoflavin (8-dimethylamino-8-demethyl-riboflavin, RoF) in aqueous solutions was carried out. The studies were concentrated on roseoflavin in pH 8 phosphate buffer. Absorption cross-section spectra, fluorescence excitation spectra, fluorescence quantum distributions, fluorescence quantum yields and fluorescence lifetimes were determined. The fluorescence of RoF is quenched by photo-induced intra-molecular charge-transfer at room temperature. The photo-degradation of RoF in un-buffered water, in Tris-HCl buffer, and in phosphate buffer was studied. Phosphate buffer and to a smaller extent Tris buffer catalyse the RoF photo-degradation. Photo-excitation of the primary photoproduct, 8-methylamino-riboflavin (8-MNH-RF), enhanced the RoF degradation by triplet 8-MNH-RF - singlet RoF excitation transfer with subsequent triplet-state RoF degradation.

Identification of Bacillus anthracis by Using Monoclonal Antibody to Cell Wall Galactose-N-Acetylglucosamine Polysaccharide

DTIC Science & Technology

1990-02-01

which appear to be directed to an epitope associated with the galactose-N-acetyl-D- glucosamine polysaccharide. Both demonstrated specificity in their...liquid composed primarily of D-galactose and N-acetyl-D-glu - R medium (28) buffered with 50 mM Tris hydrochloride , pH cosamine (12, 13) (Gal-NAG...Ascites fluid (5 ml) was dialyzed (Cel-Line Associates, Inc., Newfield, N.J.). Suspensions against 20 mM Tris hydrochloride (pH 8.0) for 18 to 20 h, were

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

PubMed

Zeng, Z; Clark, S M; Mathies, R A; Glazer, A N

1997-10-01

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Dialysis buffer with different ionic strength affects the antigenicity of cultured nervous necrosis virus (NNV) suspensions.

PubMed

Gye, Hyun Jung; Nishizawa, Toyohiko

2016-09-02

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus (Nodaviridae). It is highly pathogenic to various marine fishes. Here, we investigated the antigenicity changes of cultured NNV suspensions during 14days of dialyses using a dialysis tube at 1.4×10(4) molecular weight cut off (MWCO) in three different buffers (Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), and deionized water (DIW)). Total NNV antigen titers of cultured NNV suspension varied depending on different dialysis buffers. For example, total NNV antigen titer during D-PBS dialysis was increased once but then decreased. During Tris-HCl dialysis, it was relatively stable. During dialysis in DIW, total NNV antigen titer was increased gradually. These antigenicity changes in NNV suspension might be due to changes in the aggregation state of NNV particles and/or coat proteins (CPs). ELISA values of NNV suspension changed due to changing aggregates state of NNV antigens. NNV particles in suspension were aggregated at a certain level. These aggregates were progressive after D-PBS dialysis, but regressive after Tris-HCl dialysis. The purified NNV particles self-aggregated after dialysis in D-PBS or in Tris-HCl containing 600mM NaCl, but not after dialysis in Tris-HCl or DIW. Quantitative analysis is merited to determine NNV antigens in the highly purified NNV particles suspended in buffer at low salt condition. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA stretching on the wall surfaces in curved microchannels with different radii.

PubMed

Hsieh, Shou-Shing; Wu, Fong-He; Tsai, Ming-Ju

2014-01-01

DNA molecule conformation dynamics and stretching were made on semi-circular surfaces with different radii (500 to 5,000 μm) in microchannels measuring 200 μm × 200 μm in cross section. Five different buffer solutions - 1× Tris-acetate-EDTA (TAE), 1× Tris-borate-EDTA (TBE), 1× Tris-EDTA (TE), 1× Tris-phosphate-EDTA (TPE), and 1× Tris-buffered saline (TBS) solutions - were used with a variety of viscosity such as 40, 60, and 80 cP, with resultant 10(-4) ≤ Re ≤ 10(-3) and the corresponding 5 ≤ Wi ≤ 12. The test fluids were seeded with JOJO-1 tracer particles for flow visualization and driven through the test channels via a piezoelectric (PZT) micropump. Micro particle image velocimetry (μPIV) measuring technique was applied for the centered-plane velocity distribution measurements. It is found that the radius effect on the stretch ratio of DNA dependence is significant. The stretch ratio becomes larger as the radius becomes small due to the larger centrifugal force. Consequently, the maximum stretch was found at the center of the channel with a radius of 500 μm.

DNA stretching on the wall surfaces in curved microchannels with different radii

PubMed Central

2014-01-01

DNA molecule conformation dynamics and stretching were made on semi-circular surfaces with different radii (500 to 5,000 μm) in microchannels measuring 200 μm × 200 μm in cross section. Five different buffer solutions - 1× Tris-acetate-EDTA (TAE), 1× Tris-borate-EDTA (TBE), 1× Tris-EDTA (TE), 1× Tris-phosphate-EDTA (TPE), and 1× Tris-buffered saline (TBS) solutions - were used with a variety of viscosity such as 40, 60, and 80 cP, with resultant 10−4 ≤ Re ≤ 10−3 and the corresponding 5 ≤ Wi ≤ 12. The test fluids were seeded with JOJO-1 tracer particles for flow visualization and driven through the test channels via a piezoelectric (PZT) micropump. Micro particle image velocimetry (μPIV) measuring technique was applied for the centered-plane velocity distribution measurements. It is found that the radius effect on the stretch ratio of DNA dependence is significant. The stretch ratio becomes larger as the radius becomes small due to the larger centrifugal force. Consequently, the maximum stretch was found at the center of the channel with a radius of 500 μm. PMID:25147488

Retention of ionisable compounds on high-performance liquid chromatography XVII. Estimation of the pH variation of aqueous buffers with the change of the methanol fraction of the mobile phase.

PubMed

Subirats, Xavier; Bosch, Elisabeth; Rosés, Martí

2007-01-05

The use of methanol-aqueous buffer mobile phases in HPLC is a common election when performing chromatographic separations of ionisable analytes. The addition of methanol to the aqueous buffer to prepare such a mobile phase changes the buffer capacity and the pH of the solution. In the present work, the variation of these buffer properties is studied for acetic acid-acetate, phosphoric acid-dihydrogenphosphate-hydrogenphosphate, citric acid-dihydrogencitrate-hydrogencitrate-citrate, and ammonium-ammonia buffers. It is well established that the pH change of the buffers depends on the initial concentration and aqueous pH of the buffer, on the percentage of methanol added, and on the particular buffer used. The proposed equations allow the pH estimation of methanol-water buffered mobile phases up to 80% in volume of organic modifier from initial aqueous buffer pH and buffer concentration (before adding methanol) between 0.001 and 0.01 mol L(-1). From both the estimated pH values of the mobile phase and the estimated pKa of the ionisable analytes, it is possible to predict the degree of ionisation of the analytes and therefore, the interpretation of acid-base analytes behaviour in a particular methanol-water buffered mobile phase.

Extraction of glycogen on mild condition lacks AIG fraction.

PubMed

Ghafouri, Z; Rasouli, M

2016-12-01

Extraction of animal tissues with cold water or perchloric acid yields less glycogen than is obtained with hot-alkaline. Extraction with acid and alkaline gives two fractions, acid soluble (ASG) and insoluble glycogen (AIG). The aim of this work is to examine the hypothesis that not all liver glycogen is extractable by Tris-buffer using current techniques. Rat liver was homogenized with Tris-buffer pH 8.3 and extracted for the glycogen fractions, ASG and AIG. The degree of homogenization was changed to remove all glycogen. The content of glycogen was 47.7 ± 1.2 and 11.6 ± 0.8 mg/g wet liver in the supernatant and pellet of the first extraction respectively. About 24% of total glycogen is lost through the first pellet. Increasing the extent of homogenization from 30 to 180 sec and from 15000 to 20000 rpm followed with 30 sec ultrasonication did not improve the extraction. ASG and AIG constitute about 77% and 23% of the pellet glycogen respectively. Extraction with cold Tris-buffer failed to extract glycogen completely.  Increasing the extent of homogenization followed with ultrasonication also did not improve the extraction. Thus it is necessary to re-examine the previous findings obtained by extraction with cold Tris-buffer.

Stimulated low-frequency Raman scattering in plant virus suspensions

NASA Astrophysics Data System (ADS)

Donchenko, E. K.; Karpova, O. V.; Kudryavtseva, A. D.; Pershin, S. M.; Savichev, V. I.; Strokov, M. A.; Tcherniega, N. V.; Zemskov, K. I.

2017-11-01

The study deals with laser pulse interaction with plant viruses: we investigated tobacco mosaic virus (TMV) and two types of potato viruses (PVX and PVA) in Tris-HCl pH7.5 buffer and in water. We used 20 ns ruby laser pulses for excitation. We employed Fabry-Pérot interferometers to record spectra of the light passing through the sample and reflected from it. For TMV and PVX in Tris-HCl pH7.5 buffer, same as for PVA in water, we observed additional spectral lines corresponding to the stimulated low-frequency Raman scattering (SLFRS). We believe we were the first to measure SLFRS frequency shifts, conversion efficiency and threshold. High conversion efficiency of the scattered light is evidence of laser pulses efficiently exciting gigahertz vibrations in viruses. SLFRS can be used to identify and affect biological nanoparticles.

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

PubMed

Hubbell, H R; Rothblum, L I; Hsu, T C

1979-10-01

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Fish Viruses: Buffers and Methods for Plaquing Eight Agents Under Normal Atmosphere

PubMed Central

Wolf, Ken; Quimby, M. C.

1973-01-01

A universal procedure was sought for plaque assay of eight fish viruses (bluegill myxovirus, channel catfish virus, eel virus, Egtved virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, lymphocystis virus, and the agent of spring viremia of carp (Rhabdovirus carpio), in dish cultures of various fish cells. Eagle minimal essential medium with sodium bicarbonate-CO2 buffer (Earle’s salt solution) was compared with minimal essential medium buffered principally with tris (hydroxymethyl)aminomethane or N-2-hydroxyethylpiperazine-N′-2′-ethanesulfonic acid at a pH or in the range of 7.6 to 8.0 depending upon temperature. Five fish cell lines collectively capable of replicating all fish viruses thus far isolated were tested and quantitatively found to grow comparably well in the three media. Two-phase (gel-liquid) media incorporating the various buffer systems allowed plaquing at 15 to 33 C either in partial pressures of CO2 or in normal atmosphere, but greater efficiency and sensitivity were obtained with the organic buffers, and, overall, the best results were obtained with tris(hydroxymethyl)aminomethane. Epizootiological data, specific fish cell line response, and plaque morphology permit presumptive identification of most of the agents. At proper pH, use of organic buffers obviates the need for CO2 incubators. Images PMID:4349252

Common stock solutions, buffers, and media.

PubMed

2001-05-01

This collection of recipes describes the preparation of buffers and reagents used in Current Protocols in Pharmacology for cell culture, manipulation of neural tissue, molecular biological methods, and neurophysiological/neurochemical measurements. RECIPES: Acid, concentrated stock solutions Ammonium hydroxide, concentrated stock solution EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0) Ethidium bromide staining solution Fetal bovine serum (FBS) Gel loading buffer, 6× LB medium (Luria broth) and LB plates Potassium phosphate buffer, 0.1 M Sodium phosphate buffer, 0.1 M TE (Tris/EDTA) buffer Tris⋅Cl, 1 M.

Basal buffer systems for a newly glycosylated recombinant human interferon-β with biophysical stability and DoE approaches.

PubMed

Kim, Nam Ah; Song, Kyoung; Lim, Dae Gon; Hada, Shavron; Shin, Young Kee; Shin, Sangmun; Jeong, Seong Hoon

2015-10-12

The purpose of this study was to develop a basal buffer system for a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a), termed R27T, to optimize its biophysical stability. The protein was pre-screened in solution as a function of pH (2-11) using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the result, its experimental pI and optimal pH range were 5.8 and 3.6-4.4, respectively. Design of experiment (DoE) approach was developed as a practical tool to aid formulation studies as a function of pH (2.9-5.7), buffer (phosphate, acetate, citrate, and histidine), and buffer concentration (20 mM and 50 mM). This method employed a weight-based procedure to interpret complex data sets and to investigate critical key factors representing protein stability. The factors used were Tm, enthalpy, and relative helix contents which were obtained by DSC and Fourier Transform Infrared spectroscopy (FT-IR). Although the weights changed by three responses, objective functions from a set of experimental designs based on four buffers were highest in 20 mM acetate buffer at pH 3.6 among all 19 scenarios tested. Size exclusion chromatography (SEC) was adopted to investigate accelerated storage stability in order to optimize the pH value with susceptible stability since the low pH was not patient-compliant. Interestingly, relative helix contents and storage stability (monomer remaining) increased with pH and was the highest at pH 4.0. On the other hand, relative helix contents and thermodynamic stability decreased at pH 4.2 and 4.4, suggesting protein aggregation issues. Therefore, the optimized basal buffer system for the novel biobetter was proposed to be 20 mM acetate buffer at pH 3.8±0.2. Copyright © 2015 Elsevier B.V. All rights reserved.

In vitro corrosion of magnesium alloy AZ31 — a synergetic influence of glucose and Tris

NASA Astrophysics Data System (ADS)

Li, Ling-Yu; Liu, Bin; Zeng, Rong-Chang; Li, Shuo-Qi; Zhang, Fen; Zou, Yu-Hong; Jiang, Hongwei George; Chen, Xiao-Bo; Guan, Shao-Kang; Liu, Qing-Yun

2018-05-01

Biodegradable Mg alloys have generated great interest for biomedical applications. Accurate predictions of in vivo degradation of Mg alloys through cost-effective in vivo evaluations require the latter to be conducted in an environment close to that of physiological scenarios. However, the roles of glucose and buffering agents in regulating the in vivo degradation performance of Mg alloys has not been elucidated. Herein, degradation behavior of AZ31 alloy is investigated by hydrogen evolution measurements, pH monitoring and electrochemical tests. Results indicate that glucose plays a content-dependent role in degradation of AZ31 alloy in buffer-free saline solution. The presence of a low concentration of glucose, i.e. 1.0 g/L, decreases the corrosion rate of Mg alloy AZ31, whereas the presence of 2.0 and 3.0 g/L glucose accelerates the corrosion rate during long term immersion in saline solution. In terms of Tris-buffered saline solution, the addition of glucose increases pH value and promotes pitting corrosion or general corrosion of AZ31 alloy. This study provides a novel perspective to understand the bio-corrosion of Mg alloys in buffering agents and glucose containing solutions.

In vitro corrosion of magnesium alloy AZ31 — a synergetic influence of glucose and Tris

NASA Astrophysics Data System (ADS)

Li, Ling-Yu; Liu, Bin; Zeng, Rong-Chang; Li, Shuo-Qi; Zhang, Fen; Zou, Yu-Hong; Jiang, Hongwei George; Chen, Xiao-Bo; Guan, Shao-Kang; Liu, Qing-Yun

2018-06-01

Biodegradable Mg alloys have generated great interest for biomedical applications. Accurate predictions of in vivo degradation of Mg alloys through cost-effective in vivo evaluations require the latter to be conducted in an environment close to that of physiological scenarios. However, the roles of glucose and buffering agents in regulating the in vivo degradation performance of Mg alloys has not been elucidated. Herein, degradation behavior of AZ31 alloy is investigated by hydrogen evolution measurements, pH monitoring and electrochemical tests. Results indicate that glucose plays a content-dependent role in degradation of AZ31 alloy in buffer-free saline solution. The presence of a low concentration of glucose, i.e. 1.0 g/L, decreases the corrosion rate of Mg alloy AZ31, whereas the presence of 2.0 and 3.0 g/L glucose accelerates the corrosion rate during long term immersion in saline solution. In terms of Tris-buffered saline solution, the addition of glucose increases pH value and promotes pitting corrosion or general corrosion of AZ31 alloy. This study provides a novel perspective to understand the bio-corrosion of Mg alloys in buffering agents and glucose containing solutions.

Tris(hydroxymethyl)aminomethane photooxidation on titania based photoanodes and its implication for photoelectrochemical biofuel cells

NASA Astrophysics Data System (ADS)

Filipiak, Marcin S.; Zloczewska, Adrianna; Grzeskowiak, Piotr; Lynch, Robert; Jönsson-Niedziolka, Martin

2015-09-01

In many photoelectrochemical biofuel cells tris(hydroxymethyl)aminomethane (TRIS) is used a buffer. We show that TRIS can be readily photooxidised on titania electrodes. Combining a titania nanotube photoanode in a TRIS buffer with an air-breathing enzymatic biocathode we construct a relatively efficient photoelectrochemical biofuel cell using the TRIS buffer as fuel. This shows both the prospect of using air-breathing bio-cathodes in this kind of cells, but more importantly, shows the need for caution when using TRIS as buffer in photoelectrochemical applications.

Effect of Three Factors in Cheese Production (pH, Salt, and Heat) on Mycobacterium avium subsp. paratuberculosis Viability

PubMed Central

Sung, Nackmoon; Collins, Michael T.

2000-01-01

Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20°C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log10 concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 103 cells of M. avium subsp. paratuberculosis per ml. PMID:10742208

High-performance liquid chromatographic separation of human haemoglobins. Simultaneous quantitation of foetal and glycated haemoglobins.

PubMed

Bisse, E; Wieland, H

1988-12-29

A high-performance liquid chromatographic system, which uses a weak cation exchanger (PolyCATA) together with Bis-Tris buffer (pH 6.47-7.0) and sodium acetate gradients, is described. Samples from adults and newborns were analysed and a clean separation of many minor and major normal and abnormal haemoglobin (Hb) variants was greatly improved. The method allows the separation of minor foetal haemoglobin (HbF) variants and the simultaneous quantitation of HbF and glycated HbA. HbF values correlated well with those obtained by the alkali denaturation method (r = 0.997). The glycated haemoglobin (HbAIc) levels measured in patients with high HbF concentrations correlated with the total glycated haemoglobin determined by bioaffinity chromatography (r = 0.973). The procedure is useful for diagnostic applications and affords an effective and sensitive way of examining blood samples for haemoglobin abnormalities.

Tabun scavengers based on hydroxamic acid containing cyclodextrins.

PubMed

Brandhuber, Florian; Zengerle, Michael; Porwol, Luzian; Bierwisch, Anne; Koller, Marianne; Reiter, Georg; Worek, Franz; Kubik, Stefan

2013-04-28

Arrangement of several hydroxamic acid-derived substituents along the cavity of a cyclodextrin ring leads to compounds that detoxify tabun in TRIS-HCl buffer at physiological pH and 37.0 °C with half-times as low as 3 min.

TRIS buffer in simulated body fluid distorts the assessment of glass-ceramic scaffold bioactivity.

PubMed

Rohanová, Dana; Boccaccini, Aldo Roberto; Yunos, Darmawati Mohamad; Horkavcová, Diana; Březovská, Iva; Helebrant, Aleš

2011-06-01

The paper deals with the characterisation of the bioactive phenomena of glass-ceramic scaffold derived from Bioglass® (containing 77 wt.% of crystalline phases Na(2)O·2CaO·3SiO(2) and CaO·SiO(2) and 23 wt.% of residual glass phase) using simulated body fluid (SBF) buffered with tris-(hydroxymethyl) aminomethane (TRIS). A significant effect of the TRIS buffer on glass-ceramic scaffold dissolution in SBF was detected. To better understand the influence of the buffer, the glass-ceramic scaffold was exposed to a series of in vitro tests using different media as follows: (i) a fresh liquid flow of SBF containing tris (hydroxymethyl) aminomethane; (ii) SBF solution without TRIS buffer; (iii) TRIS buffer alone; and (iv) demineralised water. The in vitro tests were provided under static and dynamic arrangements. SBF buffered with TRIS dissolved both the crystalline and residual glass phases of the scaffold and a crystalline form of hydroxyapatite (HAp) developed on the scaffold surface. In contrast, when TRIS buffer was not present in the solutions only the residual glassy phase dissolved and an amorphous calcium phosphate (Ca-P) phase formed on the scaffold surface. It was confirmed that the TRIS buffer primarily dissolved the crystalline phase of the glass-ceramic, doubled the dissolving rate of the scaffold and moreover supported the formation of crystalline HAp. This significant effect of the buffer TRIS on bioactive glass-ceramic scaffold degradation in SBF has not been demonstrated previously and should be considered when analysing the results of SBF immersion bioactivity tests of such systems. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparison of behavior in muscle fiber regeneration after bupivacaine hydrochloride- and acid anhydride-induced myonecrosis.

PubMed

Akiyama, C; Kobayashi, S; Nonaka, I

1992-01-01

We compared the morphologic characteristics of muscle fiber necrosis and subsequent regeneration after injury induced by intramuscular injections of bupivacaine hydrochloride (BPVC) and a variety of solutions at acid and alkaline pH (acetic anhydride, citric acid buffer, and sodium carbonate buffer). After BPVC injection the necrotic muscle fibers were rapidly invaded by phagocytic cells, followed by active regeneration and very little fibrous scar formation. The regenerating muscle fibers increased rapidly in size and attained complete fiber type differentiation and regained their initial fiber diameter within 1 month. Both alkaline and acid solutions induced muscle fiber necrosis followed by regeneration. Fiber necrosis induced by alkaline buffers and acetic anhydride solutions above pH 5.0 produced changes quite similar to that induced by BPVC. However, injection with 0.1 M acetic anhydride at pH below 4.0 resulted in coagulative necrosis of the injured muscle with very little phagocytic infiltration with poor regenerative activity and dense fibrous tissue scarring. Thus, pH 4.0 appears to be the critical pH determining the type of muscle injury and subsequent poor phagocytic and regenerative activities. This model of acidic acetic anhydride injury may lead to the identification of factors which interfere with regeneration and cause fibrous tissue scarring in human muscular dystrophy.

Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP.

PubMed

Chaudhuri, Gouri; Chatterjee, Saswata; Venu-Babu, P; Ramasamy, K; Thilagaraj, W Richard

2013-02-01

The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as V(max), K(m) and K(cat) under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37 degrees C: V(max), 3.12 and 1.6 micromoles min(-1) unit(-1); K(m), 7.6 x 10(-4) M and 4 x 10(-4) M; and K(cat), 82.98 s(-1) and 42.55 s(-1), respectively. CIAP displayed a high temperature optimum of 45 degrees C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. P(i) generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.

Refractive index of dark-adapted bacteriorhodopsin and tris(hydroxymethyl)aminomethane buffer between 390 and 880 nm.

PubMed

Heiner, Zsuzsanna; Osvay, Károly

2009-08-10

The refractivity of wild-type bacteriorhodopsin (bR(WT)) suspended in tris(hydroxymethyl)aminomethane (TRIS) buffer has been measured in the spectral range of 390-840 nm by the method of angle of minimal deviation with the use of a hollow glass prism. The refractive indices of pure bR(WT) as well as of TRIS buffer have been determined from the concentration dependent refraction values. Sellmeier-type dispersion equations have been fitted for both the TRIS buffer and pure bR(WT).

The Effects of pH on the Growth and Aspect Ratio of Chicken Egg White Lysozyme Crystals Prepared in Different Buffers

NASA Technical Reports Server (NTRS)

Gibson, U. J.; Horrell, E. E.; Kou, Y.; Pusey, Marc

2000-01-01

We have measured the nucleation and aspect ratio of CEWL crystals grown by vapor diffusion in acetate, butyrate, carbonate, succinate, and phosphate buffers in a range of pH spanning the pK(sub a) of these buffers. The nucleation numbers drop off significantly in the vicinity of pK(sub a) for each of the buffers except the phosphate system, in which we used only the pH range around the second titration point(pK2). There is a concomitant increase in the sizes of the crystals. Some typical nucleation number results are shown. These data support and extend other observations. In addition, we have examined changes in aspect ratio which accompany the suppression of nucleation within each buffer system. The length of the face in the [001] direction was measured, and compared to the width of the (110) face in the [110] type directions. We find that while the aspect ratio of the crystals is affected by pH, it is dominated by a correlation with the size of the crystals. Small crystals are longer in the [0011 direction than crystals that are larger (higher pH within a buffer system). This relationship is found to hold independent of the choice of buffer. These results are consistent with those of Judge et al, who used a batch process which resulted in uniform sizing of crystals at each pH. In these experiments, we specifically avoid agitating the protein/salt buffer mixture when combining the two. This permits the formation of a range of sizes at a given pH. The results for a .05 M acetate 5% NaCl buffer are also shown. We will discuss these results in light of a growth model.

Partial Purification and Characterization of Restriction Endonuclease from Neisseria meningitidis.

DTIC Science & Technology

1983-12-01

by centrifugation at 15,000 x g for 10 min. Preparation of Cell-Free Extract The cell pellet was suspended in 10 mL of Tris-HCI buffer pH 7.6 (Tris, 20...free extract (CFE) was obtained by centrifugation at 100,000 x g for I h. To the CFE, glycerol was added to a final concentration of 10% and stored at... extract obtained from N. meningilidis when incubated with A DNA and analyzed by agarose gel electrophoresis did not give a clean fragmentation pattern

Measurement of superoxide dismutase-like activity in peel and pulp of apple from Anshan acres

NASA Astrophysics Data System (ADS)

Zhao, Lijuan

2018-04-01

Pyrogallol autoxidation method was used for measurement of SOD-like activity. They are 259.56 u/g and 148.78 u/g in peel and pulp of apple from Anshan acres. The proper-conditions for this measurement was determined through experiment as following: detection wavelength of spectrophotometer 325nm, buffer system Tris-HAc, concentration of Tris-HAc solution 50 mmoL and pH8.2. The inhibition rate of SOD-L again pyrogallol antioxidation are 92% and 90% in peel and pulp of apple.

Influence of pH, buffers and role of quinolinic acid, a novel iron chelating agent, in the determination of hydroxyl radical scavenging activity of plant extracts by Electron Paramagnetic Resonance (EPR).

PubMed

Fadda, Angela; Barberis, Antonio; Sanna, Daniele

2018-02-01

The Fenton reaction is used to produce hydroxyl radicals for the evaluation of the antioxidant activity of plant extracts. In this paper the parameters affecting the production of hydroxyl radicals and their spin trapping with DMPO were studied. The use of quinolinic acid (Quin) as an Fe(II) ligand was proposed for antioxidant activity determination of Green tea, orange juice and asparagus extracts. Quin, buffers and pH affect the DMPO-OH signal intensity of the EPR spectra. Quin/Fe(II) and low pH enhance the OH generation. Phosphate and Tris-HCl buffers decrease the signal intensity measured in Fe(II)-sulfate and Fe(II)-Quin systems. The extracts were analyzed with Fenton systems containing Fe(II)-sulfate and Fe(II)-Quin with and without buffer. The highest activity was shown with Fe(II)-Quin without buffer, this system being less influenced by pH and chelating agents present in the extracts. This paper will help researchers to better design spin trapping experiments for food matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aluminum elution and precipitation in glass vials: effect of pH and buffer species.

PubMed

Ogawa, Toru; Miyajima, Makoto; Wakiyama, Naoki; Terada, Katsuhide

2015-02-01

Inorganic extractables from glass vials may cause particle formation in the drug solution. In this study, the ability of eluting Al ion from borosilicate glass vials, and tendencies of precipitation containing Al were investigated using various pHs of phosphate, citrate, acetate and histidine buffer. Through heating, all of the buffers showed that Si and Al were eluted from glass vials in ratios almost the same as the composition of borosilicate glass, and the amounts of Al and Si from various buffer solutions at pH 7 were in the following order: citrate > phosphate > acetate > histidine. In addition, during storage after heating, the Al concentration at certain pHs of phosphate and acetate buffer solution decreased, suggesting the formation of particles containing Al. In citrate buffer, Al did not decrease in spite of the high elution amount. Considering that the solubility profile of aluminum oxide and the Al eluting profile of borosilicate glass were different, it is speculated that Al ion may be forced to leach into the buffer solution according to Si elution on the surface of glass vials. When Al ions were added to the buffer solutions, phosphate, acetate and histidine buffer showed a decrease of Al concentration during storage at a neutral range of pHs, indicating the formation of particles containing Al. In conclusion, it is suggested that phosphate buffer solution has higher possibility of forming particles containing Al than other buffer solutions.

An Efficient Method for Co-purification of Eggshell Matrix Proteins OC-17, OC-116, and OCX-36

PubMed Central

2016-01-01

In this study, we improved the eggshell-membrane separation process by separating the shell and membrane with EDTA solution, evaluating effects of three different extraction solutions (acetic acid, EDTA, and phosphate solution), and co-purifying multiple eggshell proteins with two successive ion-exchange chromatography procedures (CM Sepharose Fast Flow and DEAE Sepharose Fast Flow). The recovery and residual rates of eggshell and membrane separated by the modified method with added EDTA solution were 93.88%, 91.15% and 1.01%, 2.87%, respectively. Ovocleidin-116 (OC-116) and ovocalyxin-36 (OCX-36) were obtained by loading 50 mM Na-Hepes, pH 7.5, 2 mM DTT and 350 mM NaCl buffer onto the DEAE-FF column at a flow rate of 1 mL/min, ovocleidin-17 (OC-17) was obtained by loading 100 mM NaCl, 50 mM Tris, pH 8.0 on the CM-FF column at a flow rate of 0.5 mL/min. The purities of OCX-36, OC-17 and OC-116 were 96.82%, 80.15% and 73.22%, and the recovery rates were 55.27%, 53.38% and 36.34%, respectively. Antibacterial activity test suggested that phosphate solution extract exhibited significantly higher activity against the tested bacterial strains than the acetic acid or EDTA extract, probably due to more types of proteins in the extract. These results demonstrate that this separation method is feasible and efficient. PMID:28115888

A hexa-quinoline based C3-symmetric chemosensor for dual sensing of zinc(ii) and PPi in an aqueous medium via chelation induced "OFF-ON-OFF" emission.

PubMed

Sinha, Sanghamitra; Chowdhury, Bijit; Adarsh, Nayarassery N; Ghosh, Pradyut

2018-05-15

A quinoline-based C3-symmetric fluorescent probe (1), N,N',N''-((2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene))tris(1-(quinolin-2-yl)-N-(quinolin-2-ylmethyl)methanamine), has been developed which can selectively detect Zn2+ without the interference of Cd2+via significant enhancement in emission intensity (fluorescence "turn-ON") associated with distinct fluorescence colour changes and very low detection limits (35.60 × 10-9 M in acetonitrile and 29.45 × 10-8 M in 50% aqueous buffer (10 mM HEPES, pH = 7.4) acetonitrile media). Importantly, this sensor is operative with a broad pH window (pH 4-10). The sensing phenomenon has been duly studied through UV-vis, steady-state, and time-resolved fluorescence spectroscopic methods indicating 1 : 3 stoichiometric binding between 1 and Zn2+ which is further corroborated by 1H NMR studies. Density functional theoretical (DFT) calculations provide the optimized molecular geometry and properties of the zinc complex, 1[Zn(ClO4)]33+, which is proposed to be formed in acetonitrile. The results are in line with the solution-state experimental findings. The single crystal X-ray study provides the solid state structure of the trinuclear Zn2+ complex showing solubility in an aqueous buffer (10 mM HEPES, pH = 7.4). Finally, the resulting trinuclear Zn2+ complex has been utilized as a fluorescence "turn-OFF" sensor for the selective detection of pyrophosphate in a 70% aqueous buffer (10 mM HEPES, pH = 7.4) acetonitrile solvent with a nanomolar detection limit (45.37 × 10-9 M).

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography.

PubMed

Chaisuwan, Patcharin; Nacapricha, Duangjai; Wilairat, Prapin; Jiang, Zhengjin; Smith, Norman W

2008-06-01

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.

Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum.

PubMed

Matare, Tapiwa; Nziramasanga, Pasipanodya; Gwanzura, Lovemore; Robertson, Valerie

2017-01-01

The potential of NaHCO 3 versus human serum to induce germ tube formation in Candida albicans was investigated. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO 3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20  μ L inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO 3 concentration. Parallel germ tube formation using human serum was done in test tubes. The optimum concentration of NaHCO 3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO 3 . Tris-maleate buffered NaHCO 3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

Bicarbonate stabilizes isolated D1/D2/cytochrome b559 complex of photosystem 2 against thermoinactivation.

PubMed

Pobeguts, O V; Smolova, T N; Klimov, V V

2012-02-01

It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b(559) complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO(3)(-), with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe(2+), it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.

Characterization of the Polypeptides in Varicella Zoster Virus - Infected Cells

DTIC Science & Technology

1984-03-16

DNA binding proteins.. 127 38. Autoradiogram of guanidine hydrochloride wash of DNA cellulose columns 129 Figure Page 32 39. Autoradiogram of P...of purification was seventy-fold 35 1^ with respect to host proteins and the S-methionine or G- glucosamine labeled virions were subjected to SDS... hydrochloride [pH7.5]. 20 mM EDTA, (2 x STE buffer), was used. For electron microscopy pellets were resuspended in 10 mM Tris- hydrochloride [pH 7.5]. 1 inM

Isolation, Cloning and Expression of the Genes for Microbial Polyurethane Degradation

DTIC Science & Technology

1991-05-31

utilizing lysing enzymes, Gluculase and Novozyme , digest the mycelium and produce protoplasts but also tend to digest the DNA. The detergent procedures did...at -20 ’C. Novozyme Procedure One liter of fresh HAFB-2F-Br culture, grown to saturation, was vacuum filtered through Whatman filter paper, #40...resuspended in buffer (15% sucrose, 50 mM Tris pH 7.6, 50 mM EDTA) at 0.2 g filtrate/ml buffer in 15 ml centrifuge tubes. Novozyme was added at 40 mg/ml and

8-aminoquinoline functionalized silica nanoparticles: a fluorescent nanosensor for detection of divalent zinc in aqueous and in yeast cell suspension.

PubMed

Rastogi, Shiva K; Pal, Parul; Aston, D Eric; Bitterwolf, Thomas E; Branen, A Larry

2011-05-01

Zinc is one of the most important transition metal of physiological importance, existing primarily as a divalent cation. A number of sensors have been developed for Zn(II) detection. Here, we present a novel fluorescent nanosensor for Zn(II) detection using a derivative of 8-aminoquinoline (N-(quinolin-8-yl)-2-(3 (triethoxysilyl)propylamino)acetamide (QTEPA) grafted on silica nanoparticles (SiNPs). These functionalized SiNPs were used to demonstrate specific detection of Zn(II) in tris-HCl buffer (pH 7.22), in yeast cell (Saccharomyces cerevisiae) suspension, and in tap water. The silane QTEPA, SiNPs and final product were characterized using solution and solid state nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption spectroscopy, transmission electron microscopy, elemental analysis, thermogravimetric techniques, and fluorescence spectroscopy. The nanosensor shows almost 2.8-fold fluorescence emission enhancement and about 55 nm red-shift upon excitation with 330 ± 5 nm wavelength in presence of 1 μM Zn(II) ions in tris-HCl (pH 7.22). The presence of other metal ions has no observable effect on the sensitivity and selectivity of nanosensor. This sensor selectively detects Zn(II) ions with submicromolar detection to a limit of 0.1 μM. The sensor shows good applicability in the determination of Zn(II) in tris-HCl buffer and yeast cell environment. Further, it shows enhancement in fluorescence intensity in tap water samples.

Kinetics and equilibria of cyanide binding to prostaglandin H synthase.

PubMed

MacDonald, I D; Dunford, H B

1989-09-01

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector.

PubMed

Zinellu, Angelo; Caria, Marcello A; Tavera, Claudio; Sotgia, Salvatore; Chessa, Roberto; Deiana, Luca; Carru, Ciriaco

2005-07-15

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.

Biphasic reduction model for predicting the impacts of dye-bath constituents on the reduction of tris-azo dye Direct Green-1 by zero valent iron (Fe0).

PubMed

Kumar, Raja; Sinha, Alok

2017-02-01

Influence of common dye-bath additives, namely sodium chloride, ammonium sulphate, urea, acetic acid and citric acid, on the reductive decolouration of Direct Green 1 dye in the presence of Fe 0 was investigated. Organic acids improved dye reduction by augmenting Fe 0 corrosion, with acetic acid performing better than citric acid. NaCl enhanced the reduction rate by its 'salting out' effect on the bulk solution and by Cl - anion-mediated pitting corrosion of iron surface. (NH 4 ) 2 SO 4 induced 'salting out' effect accompanied by enhanced iron corrosion by SO 4 2- anion and buffering effect of NH 4 + improved the reduction rates. However, at 2g/L (NH 4 ) 2 SO 4 concentration, complexating of SO 4 2- with iron oxides decreased Fe 0 reactivity. Urea severely compromised the reduction reaction, onus to its chaotropic and 'salting in' effect in solution, and due to it masking the Fe 0 surface. Decolouration obeyed biphasic reduction kinetics (R 2 >0.993 in all the cases) exhibiting an initial rapid phase, when more than 95% dye reduction was observed, preceding a tedious phase. Maximum rapid phase reduction rate of 0.955/min was observed at pH2 in the co-presence of all dye-bath constituents. The developed biphasic model reckoned the influence of each dye-bath additive on decolouration and simulated well with the experimental data obtained at pH2. Copyright © 2016. Published by Elsevier B.V.

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

PubMed

Gordon, G L; Doelle, H W

1976-08-16

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Antitumour, antimicrobial and catalytic activity of gold nanoparticles synthesized by different pH propolis extracts

NASA Astrophysics Data System (ADS)

Gatea, Florentina; Teodor, Eugenia Dumitra; Seciu, Ana-Maria; Covaci, Ovidiu Ilie; Mănoiu, Sorin; Lazăr, Veronica; Radu, Gabriel Lucian

2015-07-01

The Romanian propolis was extracted in five different media, respectively, in water (pH 6.8), glycine buffer (pH 2.5), acetate buffer (pH 5), phosphate buffer (pH 7.4) and carbonate buffer (pH 9.2). The extracts presented different amounts of flavonoids and phenolic acids, increasing pH leading to higher concentrations of active compounds. Five variants of gold nanoparticles suspensions based on different pH Romanian propolis aqueous extracts were successfully synthesized. The obtained nanoparticles presented dimensions between 20 and 60 nm in dispersion form and around 18 nm in dried form, and different morphologies (spherical, hexagonal, triangular). Fourier transform infrared spectroscopy proved the attachment of organic compounds from propolis extracts to the colloidal gold suspensions and X-ray diffraction certified that the suspensions contain metallic gold. The obtained propolis gold nanoparticles do not exhibit any antibacterial or antifungal activity, but presented different catalytic activities and toxicity on tumour cells.

Mechanism of Action of Ribavirin: An Antiviral Drug of Military Importance,

DTIC Science & Technology

1980-06-01

was suspended in 120 mM Tris, 60 mM sodium ace- tate, 3 mM EDTA (pH 7.4) plus 10% glycerol and 0.1% bromophenol blue. Following electrophoresis, gels ... polyacryl - amide gels . J. Mol. Biol. 26:373-387. 2. Browne, M. J. 1979. Mechanism and specificity of action of ri- bavirin. Antimicrob. Agents...of 10 mM EDTA, 1% sodi- um dodecylsulfate (SDS) and 0.4 N sodium acetate (pH 5.2) then ex- tracted twice with a mixture of 50% phenol, 49% chloroform

Concurrent determination of total serum calcium and magnesium by thermometric titration with ethylenediaminetetraacetate.

PubMed

Callicott, R H; Carr, P W

1976-07-01

Total serum calcium and magnesium may be determined in one thermometric titration, with disodium ethylenediaminetetraacetate as the titrant. A 1-ml serum sample is diluted with 1 ml of tris(hydroxymethyl)aminomethane buffer (pH 8) and titrated at a constant rate with a motorized syringe buret. Results by the thermometric method compared well with those by atomic absorption spectroscopy.

The Effects of Acetate Buffer Concentration on Lysozyme Solubility

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Pusey, Marc L.

1996-01-01

The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have studied the solubility of tetragonal lysozyme over an acetate buffer concentration range of 0.01M to 0.5M as a function of temperature. The lysozyme solubility decreased with increasing acetate concentration from 0.01M to 0.1M. This decrease may simply be due to the net increase in solvent ionic strength. Increasing the acetate concentration beyond 0.1M resulted in an increase in the lysozyme solubility, which reached a peak at - 0.3M acetate concentration. This increase was believed to be due to the increased binding of acetate to the anionic binding sites of lysozyme, preventing their occupation by chloride. In keeping with the previously observed reversal of the Hoffmeister series for effectiveness of anions in crystallizing lysozyme, acetate would be a less effective precipitant than chloride. Further increasing the acetate concentration beyond 0.3M resulted in a subsequent gradual decrease in the lysozyme solubility at all NaCl concentrations.

Functional Analysis of Human NF1 in Drosophila

DTIC Science & Technology

2007-01-01

adjusted to 1 mg/ml. Fifty microlitres of 2 assay buffer (50 mM Tris– acetate buffer at pH 7.5, 20 mM MgCl2, 2 mM dithiothreitol, 10 mM creatine phosphate...200 units/ml creatinine kinase, 0.1 mM cAMP at pH 7.5, 0.2 mg/ml bovine serum albumin, 0.02 mg/ml aprotinin, 0.02 mg/ml pepstatin and fresh 0.2 mg

Automatized sspKa measurements of dihydrogen phosphate and Tris(hydroxymethyl) aminomethane in acetonitrile/water mixtures from 20 to 60°C.

PubMed

Acquaviva, A; Tascon, M; Padró, J M; Gagliardi, L G; Castells, C B

2014-09-01

We measured pKa values of Tris(hydroxymethyl)aminomethane and dihydrogen phosphate; both are commonly used to prepare buffers for reverse-phase liquid chromatography (RPLC), in acetonitrile/water mixtures from 0% to 70% (v/v) (64.6% (w/w)) acetonitrile and at 20, 30, 40, 50, and 60°C. The procedure is based on potentiometric measurements of pH of buffer solutions of variable solvent compositions using a glass electrode and a novel automated system. The method consists in the controlled additions of small volumes of a thermostated solution from an automatic buret into another isothermal solution containing exactly the same buffer-component concentrations, but a different solvent composition. The continuous changes in the solvent composition induce changes in the potentials. Thus, only two sequences of additions are needed: increasing the amount of acetonitrile from pure water and decreasing the content of acetonitrile from 70% (v/v) (64.6% (w/w)). In the procedure with homemade apparatus, times for additions, stirring, homogenization, and data acquisition are entirely controlled by software programmed for this specific routine. This rapid, fully automated method was applied to acquire more than 40 potential data covering the whole composition range (at each temperature) in about two hours and allowed a systematic study of the effect of temperature and acetonitrile composition on acid-base equilibria of two widely used substances to control pH close to 7. The experimental pKa results were fitted to empirical functions between pKa and temperature and acetonitrile composition. These equations allowed predictions of pKa to estimate the pH of mixtures at any composition and temperature, which would be very useful, for instance, during chromatographic method development. Copyright © 2014 Elsevier B.V. All rights reserved.

Plaquing procedure for infectious hematopoietic necrosis virus

USGS Publications Warehouse

Burke, J.A.; Mulcahy, D.

1980-01-01

A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used.

Buffer capacity of biologics--from buffer salts to buffering by antibodies.

PubMed

Karow, Anne R; Bahrenburg, Sven; Garidel, Patrick

2013-01-01

Controlling pH is essential for a variety of biopharmaceutical process steps. The chemical stability of biologics such as monoclonal antibodies is pH-dependent and slightly acidic conditions are favorable for stability in a number of cases. Since control of pH is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Experimentally derived values largely coincide with values calculated from a model that had been proposed in 1922 by van Slyke. As high concentrated protein formulations become more and more prevalent for biologics, the self-buffering potential of proteins becomes of relevance. The current study provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0 and shows that a monoclonal antibody at 50 mg/mL exhibits similar buffer capacity as 6 mM citrate or 14 mM histidine (pH 5.0-6.0). Buffer capacity of antibody solutions scales linearly with protein concentration up to more than 200 mg/mL. At a protein concentration of 220 mg/mL, the buffer capacity resembles the buffer capacity of 30 mM citrate or 50 mM histidine (pH 5.0-6.0). The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40°C. Changes in ionic strength of ΔI=0.15, in contrast, can alter the buffer capacity up to 35%. In conclusion, due to efficient self-buffering by antibodies in the pH range of favored chemical stability, conventional buffer excipients could be dispensable for pH stabilization of high concentrated protein solutions. Copyright © 2013 American Institute of Chemical Engineers.

Studies on the electrochemical behavior of thiazolidine and its applications using a flow-through chronoamperometric sensor based on a gold electrode.

PubMed

Wang, Lai-Hao; Li, Wen-Jie

2011-09-06

The electrochemical behaviors of thiazolidine (tetrahydrothiazole) on gold and platinum electrodes were investigated in a Britton-Robinson buffer (pH 2.77-11.61), acetate buffer (pH 4.31), phosphate buffer solutions (pH 2.11 and 6.38) and methanol or acetonitrile containing various supporting electrolytes. Detection was based on a gold wire electrochemical signal obtained with a supporting electrolyte containing 20% methanol-1.0 mM of phosphate buffer (pH 6.87, potassium dihydrogen phosphate and dipotassium hydrogen phosphate) as the mobile phase. Comparison with results obtained with a commercial amperometric detector shows good agreement. Using the chronoamperometric sensor with the current at a constant potential, and measurements with suitable experimental parameters, a linear concentration from 0.05 to 16 mg L-1 was found. The limit of quantification (LOQ) of the method for thiazolidine was found to be 1 ng.

Effect of Pressure-Induced Changes in the Ionization Equilibria of Buffers on Inactivation of Escherichia coli and Staphylococcus aureus by High Hydrostatic Pressure

PubMed Central

Gayán, Elisa; Condón, Santiago; Álvarez, Ignacio; Nabakabaya, Maria

2013-01-01

Survival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (ΔV°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ΔV° values are approximately 5.0 to 7.0 cm3 mol−1, than in phosphate or dimethyl glutarate (DMG), whose ΔV° values are about −25 cm3 mol−1. However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na2SO4 and CaCl2) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na2SO4 than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes. PMID:23624471

DNA polymerases in the rat pituitary gland. Effect of oestrogens and sulpiride.

PubMed

Jahn, G A; Kalbermann, L E; Machiavelli, G; Szijan, I; Burdman, J A

1980-06-01

Changes in the activity of DNA polymerase and [3H]thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats. The activities of DNA polymerases alpha and beta, extracted in Tris--HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively alpha, while in the phosphate soluble fraction it is a mixture of alpha and beta. The administration of oestrogens to male rats increases [3H]thymidine incorporation and enhances the activity of DNA polymerases in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further increment of [3H]thymidine incorporation and of DNA polymerase activity in the Tris-soluble fraction. In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction. Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase alpha. This activity changes in parallel with [3H]thymidine incorporation into DNA which is an indication of cell proliferation in the gland. This is discussed with respect to a negative feedback mechanism between intracellular prolactin concentration and DNA synthesis in the APG.

A novel enzymatic method for determination of homocysteine using electrochemical hydrogen sulfide sensor.

PubMed

Zhao, Dong; Liu, Tsan-Zon; Chan, Err-Cheng; Fein, Harry; Zhang, Xueji

2007-05-01

Homocysteine is a sulfur-containing compound produced during metabolism process of methionine. Its uptake in human plasma is believed to be the cause of cardiovascular diseases and many other diseases. An electrochemical method was proposed for selective and quantitative measurement of homocysteine by employing hydrogen sulfide sensor coupled with methionine a, g-lyase. The principle of this method is to measure the evolved hydrogen sulfide from the enzymatic reaction between homocysteine and methionine a, g-lyase. The sensitivities of the measurements at different pH values of the tris buffer solutions and at room temperature peaked to 275 pA/mM at pH 6.5 with detection limit of 150 nM (based on 3 s cutoff). The linearity measurements at pH 6.5 were performed for the homocysteine concentrations range from 0.5 to 200 mM, which is wider than the human blood plasma total homocysteine level of 5 to 100 mM, and the regressive analysis of the experiments gave R2=0.9987. The enzyme also showed the fastest response to homocysteine in the tris buffer solution of pH 7.5 with the current approaching its maximum at 134 seconds. The interference tests against several common agents were carried out, and found that cysteine and methionine were the major two species to introduce measurement problem. The solution to this interference problem was explored and discussed thoroughly based on the preliminary tests. The sensitivities of the experiments against several enzyme concentrations were also performed.

Buffer capacity of 4% succinylated gelatin does not provide any advantages over acidic 6% hydroxyethyl starch 130/0.4 for acid-base balance during experimental mixed acidaemia in a porcine model.

PubMed

Esche, V; Russ, M; Melzer, S; Grossmann, B; Boemke, W; Unger, J K

2008-11-01

Four percent gelatine is an alkaline compound due to NH2 groups, whereas 6% hydroxyethyl starch 130/0.4 (HES130) has acidic features. We investigated whether these solutions lead to differences in acid-base balance in pigs during acidaemia and correction of pH. Anaesthetized pigs were randomized to HES130 or gelatine infusion (n = 5 per group). Animals received acid infusion (0.4 M solution of lactic acid and HCl diluted in normal saline) and low tidal volume ventilation (6-7 mL kg(-1), PaCO2 of 80-85 mmHg, pH 7.19-7.24). Measurements were made before and after induction of acidaemia, before and after correction of pH with haemofiltration (continuous venovenous haemofiltration) and tris-hydroxymethylaminomethane infusion. We measured parameters describing acid-base balance according to Stewart's approach, ketone body formation, oxygen delivery, haemodynamics, diuresis and urinary pH. Acid-base balance did not differ significantly between the groups. In HES130-treated pigs, the haemodilution-based drop of haemoglobin (1.4 +/- 1.0 g dL(-1), median +/- SD) was paralleled by an increase in the cardiac output (0.5 +/- 0.4 L min(-1). Lacking increases in cardiac output, gelatine-treated pigs demonstrated a reduction in oxygen delivery (149.4 +/- 106.0 mL min(-1)). Tris-hydroxymethylaminomethane volumes required for pH titration to desired values were significantly higher in the gelatine group (0.7 +/- 0.1 mL kg(-1) h(-1) vs. HES130: 0.5 +/- 0.2 mL kg(-1) h(-1)). The buffer capacity of gelatine did not lead to favourable differences in acid-base balance in comparison to HES130.

A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins

PubMed Central

Tastet, Christophe; Lescuyer, Pierre; Diemer, Hélène; Luche, Sylvie; van Dorsselaer, Alain; Rabilloud, Thierry

2003-01-01

A new, versatile, multiphasic buffer system for high resolution sodium dodecyl sulfatepolyacrylamide gel electrophoresis of proteins in the relative molecular weight Mw range of 300,000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above Mw range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pKa, as the buffering counter ion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6–200 kDa Mw range, with minimal difficulties in the post electrophoretic identification processes. PMID:12783456

IPE 7

NASA Technical Reports Server (NTRS)

1983-01-01

A description is given of the collection and treatment of samples of Prochloron cells. The cells of Prochloron were obtained and prepared in the following way. Colonies of the symbiotic host, the giant didemnid ascidian Lissoclinum patella, were collected at low-tide level on reef-flat sand between Kamori Island and Koror, Palau, Western Caroline Islands. The animal colonies were taken, immersed in sea water, to an 8,000-litre holding tank and kept with constantly running sea water at 30 deg. Individual colonies were picked clean of contaminants, rinsed in sea water buffered with 40 nM or 100 mM Tris buffer at pH 8.4, and squeezed by hand to express the algal cells from the cloacal atria. The algae were received in about an equal volume of the same buffered sea water; this neutralized the acids liberated by the bruised ascidians and thereby maintained the Ph high enough to keep the algal cells green. The Prochloron cells were washed twice with buffered sea water and concentrated by centrifugation at about 50 g for 90 seconds. Microscopic examination revealed that contamination by animal host cells or bacteria was negligible (much less than 1%).

Formulation and stability of an extemporaneous 0.02% chlorhexidine digluconate ophthalmic solution.

PubMed

Lin, Shu-Chiao; Huang, Chih-Fen; Shen, Li-Jiuan; Wang, Hsueh-Ju; Lin, Chia-Yu; Wu, Fe-Lin Lin

2015-12-01

Acanthamoeba keratitis is difficult to treat because Acanthamoeba cysts are resistant to the majority of antimicrobial agents. Despite the efficacy of 0.02% chlorhexidine in treating Acanthamoeba keratitis, a lack of data in the literature regarding the formulation's stability limits its clinical use. The objective of this study was to develop an optimal extemporaneous 0.02% chlorhexidine digluconate ophthalmic formulation for patients in need. With available active pharmaceutical ingredients, 0.02% chlorhexidine digluconate sample solutions were prepared by diluting with BSS Plus Solution or acetate buffer. Influences of the buffer, type of container, and temperature under daily-open condition were assessed based on the changes of pH values and chlorhexidine concentrations of the test samples weekly. To determine the beyond-use date, the optimal samples were stored at 2-8°C or room temperature, and analyzed at time 0 and at Week 1, Week 2, Week 3, Week 4, Week 5, Week 8, Week 12, and Week 24. Despite chlorhexidine exhibiting better stability in acetate buffer than in BSS solution, its shelf-life was < 14 days when stored in a light-resistant low-density polyethylene container. The acetate-buffered 0.02% chlorhexidine digluconate solution stored in light-resistant high-density polyethylene eyedroppers did not exhibit significant changes in pH or strength at any time interval. The acetate-buffered 0.02% chlorhexidine digluconate ophthalmic solution stored in light-resistant high-density polyethylene eyedroppers demonstrated excellent stability at 2-25°C for 6 months after being sealed and for 1 month after opening. This finding will enable us to prepare 0.02% chlorhexidine digluconate ophthalmic solutions based on a doctor's prescription. Copyright © 2014. Published by Elsevier B.V.

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies

PubMed Central

Buchmueller, Karen L.; Weeks, Kevin M.

2004-01-01

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of ∼3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches ∼1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies. PMID:15601995

Tetragonal Chicken Egg White Lysozyme Solubility in Sodium Chloride Solutions

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Judge, Russell A.; Pusey, Marc L.

1998-01-01

The solubility of chicken egg white lysozyme, crystallized in the tetragonal form was measured in sodium chloride solutions from 1.6 to 30.7 C, using a miniature column solubility apparatus. Sodium chloride solution concentrations ranged from 1 to 7% (w/v). The solutions were buffered with 0.1 M sodium acetate buffer with the solubility being measured at pH values in 0.2 pH unit increments in the range pH 4.0 to 5.4, with data also included at pH 4.5. Lysozyme solubility was found to increase with increases in temperature and decreasing salt concentration. Solution pH has a varied and unpredictable effect on solubility.

[Isolation and purification of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5 using chromatography].

PubMed

Ma, Lina; Wu, Dan; Bian, Liujiao

2012-08-01

The Kringle 5 domain of plasminogen is one of the most potent angiogenesis inhibitors known to date, which can inhibit cell proliferation and migration efficiently. In the study, on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5, a two-step chromatographic method, including the use of SP Sepharose Fast Flow cation exchanger and Sephacryl S-100 HR size exclusion chromatography in sequence, was established to separate and purify angiogenesis inhibitor Kringle 5. On the SP Sepharose Fast Flow column, the buffer A consisted of 50.0 mmol/L acetic acid-sodium acetate (pH 5.2), and the buffer B consisted of buffer A with the addition of 0.5 mol/L sodium chloride (pH 5.2); on Sephacryl S-100 HR column, the elution buffer was 5.0 mmol/L phosphate solution (pH 7.0). Through the two-step chromatographic purification process, the purity of the obtained Kringle 5 was more than 98%. In addition, it was found that the obtained Kringle 5 could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively. Finally it is concluded that this method can effectively separate active recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5.

Effect of acetic acid in recycling water on ethanol production for cassava in an integrated ethanol-methane fermentation process.

PubMed

Yang, Xinchao; Wang, Ke; Zhang, Jianhua; Tang, Lei; Mao, Zhonggui

2016-11-01

Recently, the integrated ethanol-methane fermentation process has been studied to prevent wastewater pollution. However, when the anaerobic digestion reaction runs poorly, acetic acid will accumulate in the recycling water. In this paper, we studied the effect of low concentration of acetic acid (≤25 mM) on ethanol fermentation at different initial pH values (4.2, 5.2 or 6.2). At an initial pH of 4.2, ethanol yields increased by 3.0% and glycerol yields decreased by 33.6% as the acetic acid concentration was increased from 0 to 25 mM. Raising the concentration of acetic acid to 25 mM increased the buffering capacity of the medium without obvious effects on biomass production in the cassava medium. Acetic acid was metabolized by Saccharomyces cerevisiae for the reason that the final concentration of acetic acid was 38.17% lower than initial concentration at pH 5.2 when 25 mM acetic acid was added. These results confirmed that a low concentration of acetic acid in the process stimulated ethanol fermentation. Thus, reducing the acetic acid concentration to a controlled low level is more advantageous than completely removing it.

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

PubMed

Zsolnai, A; Orbán, L; Chrambach, A

1993-03-01

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

The effect of pH and buffer concentration on anode biofilms of Thermincola ferriacetica.

PubMed

Lusk, Bradley G; Parameswaran, Prathap; Popat, Sudeep C; Rittmann, Bruce E; Torres, Cesar I

2016-12-01

We assessed the effects of pH and buffer concentration on current production and growth of biofilms of Thermincola ferriacetica - a thermophilic, Gram-positive, anode-respiring bacterium (ARB) - grown on anodes poised at a potential of -0.06V vs. SHE in microbial electrolysis cells (MECs) at 60°C. T. ferriacetica generated current in the pH range of 5.2 to 8.3 with acetate as the electron donor and 50mM bicarbonate buffer. Maximum current density was reduced by ~80% at pH5.2 and ~14% at 7.0 compared to pH8.3. Increasing bicarbonate buffer concentrations from 10mM to 100mM resulted in an increase in the current density by 40±6%, from 6.8±1.1 to 11.2±2.7Am(-2), supporting that more buffer alleviated pH depression within T. ferriacetica biofilms. Confocal laser scanning microscopy (CLSM) images indicated that higher bicarbonate buffer concentrations resulted in larger live biofilm thicknesses: from 68±20μm at 10mM bicarbonate to >150μm at 100mM, supporting that buffer availability was a strong influence on biofilm thickness. In comparison to mesophilic Geobacter sulfurreducens biofilms, the faster transport rates at higher temperature and the ability to grow at relatively lower pH allowed T. ferriacetica to produce higher current densities with lower buffer concentrations. Published by Elsevier B.V.

Highly efficient treatment of aerobic vaginitis with simple acidic buffered gels: The importance of pH and buffers on the microenvironment of vaginas.

PubMed

Sun, Xiaodong; Qiu, Haiying; Jin, Yiguang

2017-06-15

Aerobic vaginitis (AV) leads to uterus deep infection or preterm birth. Antibacterial agents are not optimal therapeutics of AV. Here, we report a series of temperature-sensitive in situ forming acidic buffered gels for topical treatment of AV, involving lactate, acetate, and citrate gels at pH 3.5, 5.0, and 6.5. AV rat models were prepared following vaginal infection with Staphylococcus aureus and Escherichia coli. In vitro/in vivo studies of the buffered gels were performed compared with ofloxacin gels and blank gels. All the buffered gels showed the lower in vitro antibacterial activities than ofloxacin gels but the better in vivo anti-S. aureus effects and similar anti-E. coli effects. The buffered gels improved Lactobacillus growth in the vaginas. Both the healthy rat vaginal pH and the pH of rat vaginas treated with the buffered gels were about 6.5 though the AV rat models or ones treated with ofloxacin gels still remained at the high pH more than 7.0. After treatments with the buffered gels, the vaginal smears changed to a clean state nearly without aerobic bacteria, the vaginal tissues were refreshed, and the immunoreactions were downregulated. The acidic buffered gels bring rapid decrease of local vaginal pH, high antibacterial activities, improvement of probiotics, and alleviation of inflammation. They are simple, highly efficient, and safe anti-AV formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

THE FLOCCULATION MAXIMUM (pH) OF FIBRINOGEN AND SOME OTHER BLOOD-CLOTTING REAGENTS. (RELATIVE TURBIDIMETRY WITH THE EVELYN PHOTOELECTRIC COLORIMETER).

PubMed

Ferguson, J H

1942-03-20

By means of a novel adaptation of the Evelyn photoelectric colorimeter to the measurement of relative turbidities, the question of the flocculation maximum (F.M.) in acetate buffer solutions of varying pH and salt content has been studied on (a) an exceptionally stable prothrombin-free fibrinogen and its solutions after incipient thermal denaturation and incomplete tryptic proteolysis, (b) plasma, similarly treated, (c) prothrombin, thrombin, and (brain) thromboplastin solutions. All the fibrinogens show a remarkable uniformity of the precipitation pattern, viz. F.M. =4.7 (+/-0.2) pH in salt-containing buffer solutions and pH = 5.3 (+/-0.2) in salt-poor buffer (N/100 acetate). The latter approximates the isoelectric point (5.4) obtained by cataphoresis (14). There is no evidence that denaturation or digestion can produce any "second maximum." The data support the view that fibrin formation (under the specific influence of thrombin) is intrinsically unrelated to denaturation and digestion phenomena, although all three can proceed simultaneously in crude materials. A criticism is offered, therefore, of Wöhlisch's blood clotting theory. Further applications of the photoelectric colorimeter to coagulation problems are suggested, including kinetic study of fibrin formation and the assay of fibrinogen, with a possible sensitivity of 7.5 mg. protein in 100 cc. solution.

Influence of cell culture medium composition on in vitro dissolution behavior of a fluoride-containing bioactive glass.

PubMed

Shah, Furqan A; Brauer, Delia S; Wilson, Rory M; Hill, Robert G; Hing, Karin A

2014-03-01

Bioactive glasses are used clinically for bone regeneration, and their bioactivity and cell compatibility are often characterized in vitro, using physiologically relevant test solutions. The aim of this study was to show the influence of varying medium characteristics (pH, composition, presence of proteins) on glass dissolution and apatite formation. The dissolution behavior of a fluoride-containing bioactive glass (BG) was investigated over a period of one week in Eagle's Minimal Essential Medium with Earle's Salts (MEM), supplemented with either, (a) acetate buffer, (b) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, (c) HEPES + carbonate, or (d) HEPES + carbonate + fetal bovine serum. Results show pronounced differences in pH, ion release, and apatite formation over 1 week: Despite its acidic pH (pH 5.8 after BG immersion, as compared to pH 7.4-8.3 for HEPES-containing media), apatite formation was fastest in acetate buffered (HEPES-free) MEM. Presence of carbonate resulted in formation of calcite (calcium carbonate). Presence of serum proteins, on the other hand, delayed apatite formation significantly. These results confirm that the composition and properties of a tissue culture medium are important factors during in vitro experiments and need to be taken into consideration when interpreting results from dissolution or cell culture studies. Copyright © 2013 Wiley Periodicals, Inc.

Delayed release film coating applications on oral solid dosage forms of proton pump inhibitors: case studies.

PubMed

Missaghi, Shahrzad; Young, Cara; Fegely, Kurt; Rajabi-Siahboomi, Ali R

2010-02-01

Formulation of proton pump inhibitors (PPIs) into oral solid dosage forms is challenging because the drug molecules are acid-labile. The aim of this study is to evaluate different formulation strategies (monolithic and multiparticulates) for three PPI drugs, that is, rabeprazole sodium, lansoprazole, and esomeprazole magnesium, using delayed release film coating applications. The core tablets of rabeprazole sodium were prepared using organic wet granulation method. Multiparticulates of lansoprazole and esomeprazole magnesium were prepared through drug layering of sugar spheres, using powder layering and suspension layering methods, respectively. Tablets and drug-layered multiparticulates were seal-coated, followed by delayed release film coating application, using Acryl-EZE(R), aqueous acrylic enteric system. Multiparticulates were then filled into capsules. The final dosage forms were evaluated for physical properties, as well as in vitro dissolution testing in both compendial acid phase, 0.1N HCl (pH 1.2), and intermediate pH, acetate buffer (pH 4.5), followed by phosphate buffer, pH 6.8. The stability of the delayed release dosage forms was evaluated upon storage in accelerated conditions [40 degrees C/75% relative humidity] for 3 months. All dosage forms demonstrated excellent enteric protection in the acid phase, followed by rapid release in their respective buffer media. Moreover, the delayed release dosage forms remained stable under accelerated stability conditions for 3 months. Results showed that Acryl-EZE enteric coating systems provide excellent performance in both media (0.1N HCl and acetate buffer pH 4.5) for monolithic and multiparticulate dosage forms.

Two new glucose 6-phosphate dehydrogenase variants associated with congenital nonspherocytic hemolytic anemia found in Japan: GD(-) Tokushima and GD(-) Tokyo.

PubMed

Miwa, S; Ono, J; Nakashima, K; Abe, S; Kageoka, T

1976-01-01

Two new variants of glucose 6-phosphate dehydrogenase (G6PD) deficiency associated with chronic nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Tokushima was found in a 17-years-old male whose erythrocytes contained 4.4% of normal enzyme activity. Partially purified enzyme revealed a main band of normal electrophoretic mobility with additional two minor bands of different mobility; normal Km G6P, and Km NADP five-to sixfold higher than normal; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; marked thermal instability; a normal pH curve; and normal Ki NADPH. The hemolytic anemia was moderate to severe. Gd(-) Tokyo was characterized from a 15-year-old male who had chronic nonspherocytic hemolytic anemia of mild degree. The erythrocytes contained 3% of normal enzyme activity, and partially purified enzyme revealed slow electrophoretic mobility (90% of normal for both a tris-hydrochloride buffer system and a tris-EDTA-borate buffer system, and 70% of normal for a phosphate buffer system); normal Km G6P and Km NADP; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; greatly increased thermal instability; a normal pH curve; and normal Ki NADPH. These two variants are clearly different from hitherto described G6PD variants, including the Japanese variants Gd(-) Heian and Gd(-) Kyoto. The mothers of both Gd(-) Tokushima and Gd(-) Tokoyo were found to be heterozygote by an ascorbate-cyanide test.

Effect of various halide salts on the incompatibility of cyanocobalamin and ascorbic acid in aqueous solution.

PubMed

Ichikawa, Makoto; Ide, Nagatoshi; Shiraishi, Sumihiro; Ono, Kazuhisa

2005-06-01

Combination of cyanocobalamin (VB12) and ascorbic acid (VC) has been widely seen in pharmaceutical products and dietary supplements. However, VB12 has been reported that its behavior in stability in aqueous solution is quite different when VC is mixed. In the present study, we examined the stabilities of these vitamins in acetate buffer (pH 4.8) using high performance liquid chromatography. Degradation of VB12 was not observed in the absence of VC in the buffer. However, when VC was mixed in the VB12 solution, VB12 concentrations decreased in accordance with VC degradation. VB12 and VC degradations were inhibited by adding sodium halides to acetate buffer at pH 4.8. These stabilization effects were also observed in the range from pH 3.5 to 5.3 and by adding potassium, magnesium, and calcium halides. Furthermore, our data demonstrated that increases in the halide anion concentrations and atomic number (Cl-

Generation of Free Radicals during Cold Injury and Rewarming

DTIC Science & Technology

1988-01-01

a mixture of sodium citrate (0.05 M) and sodium acetate (0.03 M) (pH 4.5); 50 ; 1 of 70% perchloric acid was then added to the mixture. The resultant...products of salicylic acid were eluted with buffer (degassed and filtered) containing 0.03 M sodium acetate and 0.05 M sodium citrate 6 (pH 4.5) at a...Malonaldehyde was measured as an index for lipid peroxidation. Plasma (0.5 ml) was added to 0.5 ml ice-cold perchloric acid (15%) and then treated with

Determination of suxamethonium in a pharmaceutical formulation by capillary electrophoresis with contactless conductivity detection (CE-C(4)D).

PubMed

Nussbaumer, Susanne; Fleury-Souverain, Sandrine; Rudaz, Serge; Bonnabry, Pascal; Veuthey, Jean-Luc

2009-02-20

A simple method based on capillary electrophoresis with a capacitively coupled contactless conductivity detector (CE-C(4)D) was developed for the determination of suxamethonium (SUX) in a pharmaceutical formulation. A hydro-organic mixture, consisting of 100mM Tris-acetate buffer at pH 4.2 and acetonitrile (90:10, v/v), was selected as background electrolyte (BGE). The applied voltage was 30kV, and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 50 microm and a total length of 64.5cm. Under these conditions, a complete separation between SUX, sodium ions and the main degradation products (choline) was achieved in less than 4min. The presence of acetonitrile in the BGE allowed a reduction of SUX adsorption on the capillary wall. The CE-C(4)D method was validated, and trueness values between 98.8% and 101.1% were obtained with repeatability and intermediate precision values of 0.7-1.3% and 1.2-1.6%, respectively. Therefore, this method was found appropriate for controlling pharmaceutical formulations containing suxamethonium and degradation products.

CE of phytosiderophores and related metal species in plants.

PubMed

Xuan, Yue; Scheuermann, Enrico B; Meda, Anderson R; Jacob, Peter; von Wirén, Nicolaus; Weber, Günther

2007-10-01

Phytosiderophores (PS) and the closely related substance nicotianamine (NA) are key substances in metal uptake into graminaceous plants. Here, the CE separation of these substances and related metal species is demonstrated. In particular, the three PS 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), and NA, are separated using MES/Tris buffer at pH 7.3. Moreover, three Fe(III) species of the different PS are separated without any stability problems, which are often present in chromatographic analyses. Also divalent metal species of Cu, Ni, and Zn with the ligands DMA and NA are separated with the same method. By using a special, zwitterionic CE capillary, even the separation of two isomeric Fe(III) chelates with the ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) is possible (i.e., meso-Fe(III)-EDDHA and rac-Fe(III)-EDDHA), and for fast separations of NA and respective divalent and trivalent metal species, a polymer CE microchip with suppressed EOF is described. The proposed CE method is applicable to real plant samples, and enables to detect changes of metal species (Cu-DMA, Ni-NA), which are directly correlated to biological processes.

A method for early determination of meat ultimate pH.

PubMed

Young, O A; West, J; Hart, A L; van Otterdijk, F F H

2004-02-01

A patented method of rapidly determining the ultimate pH from approximate glycolytic potential of muscles of slaughtered animals has been devised. The method is based on the rapid hydrolysis of muscle glycogen to glucose by the enzyme amyloglucosidase and subsequent measurement of the liberated glucose. In acetate buffer at pH 4.5, glucose concentration can be determined in 30 s with domestic meters for diabetes control. The meter response differed from that of glucose in blood, but was linear with concentration. In slurries comprising homogenised meat in acetate buffer and added glucose, a similar linear response was obtained. Amyloglucosidase was capable of rapidly hydrolysing glycogen to glucose in such slurries. In the 24 h following slaughter, a decrease in glycogen, as determined by glucose, occurred in parallel with the decline in pH. At the same time, lactate progressively accumulated as expected. Values for the approximate glycolytic potential and (by calibration) ultimate pH, were obtained on prerigor muscle within 7 min of muscle sampling in an industrial environment. The method is suitable for on-line application in beef abattoirs particularly those employing hot boning where ultimate must be known at the grading point.

Mechanism of Indole-3-acetic Acid Conjugation

PubMed Central

Goren, Raphael; Bukovac, Martin J.; Flore, James A.

1974-01-01

Formation of indole-3-acetic acid-aspartate in detached primary leaves of cowpea (Vigna sinensis Endl.) floating on 14C-indole-3-acetic acid (3 μc; 3.15 μm, phosphate-citrate buffer, pH 4.75), almost doubled when leaves were pretreated with 31.5 μm12C-indole-3-acetic acid for 17 hr and then transferred to 14C-indole-3-acetic acid for 4 hours as compared with leaves preincubated in buffer only. When leaves were preincubated with ethylene (11.0 and 104 μl/l) instead of 12C-indole-3-acetic acid, no induction of indole-3-acetylaspartic acid formation was observed, and the rate of indole-3-acetylaspartic acid formation decreased as compared with control leaves. Rhizobitoxine (1.87 μm) inhibited indole-3-acetic acid-induced ethylene production but did not prevent the formation of indole-3-acetylaspartic acid. In view of the similarity of these results and those previously obtained with α-naphthaleneacetic acid, it is concluded that ethylene has no role in the auxin-induced indole-3-acetylaspartic acid formation in cowpea leaves. PMID:16658669

Physicochemical parameters influencing coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13.

PubMed

Min, K R; Zimmer, M N; Rickard, A H

2010-11-01

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.

THE FLOCCULATION MAXIMUM (pH) OF FIBRINOGEN AND SOME OTHER BLOOD-CLOTTING REAGENTS. (RELATIVE TURBIDIMETRY WITH THE EVELYN PHOTOELECTRIC COLORIMETER)

PubMed Central

Ferguson, John H.

1942-01-01

By means of a novel adaptation of the Evelyn photoelectric colorimeter to the measurement of relative turbidities, the question of the flocculation maximum (F.M.) in acetate buffer solutions of varying pH and salt content has been studied on (a) an exceptionally stable prothrombin-free fibrinogen and its solutions after incipient thermal denaturation and incomplete tryptic proteolysis, (b) plasma, similarly treated, (c) prothrombin, thrombin, and (brain) thromboplastin solutions. All the fibrinogens show a remarkable uniformity of the precipitation pattern, viz. F.M. =4.7 (±0.2) pH in salt-containing buffer solutions and pH = 5.3 (±0.2) in salt-poor buffer (N/100 acetate). The latter approximates the isoelectric point (5.4) obtained by cataphoresis (14). There is no evidence that denaturation or digestion can produce any "second maximum." The data support the view that fibrin formation (under the specific influence of thrombin) is intrinsically unrelated to denaturation and digestion phenomena, although all three can proceed simultaneously in crude materials. A criticism is offered, therefore, of Wöhlisch's blood clotting theory. Further applications of the photoelectric colorimeter to coagulation problems are suggested, including kinetic study of fibrin formation and the assay of fibrinogen, with a possible sensitivity of 7.5 mg. protein in 100 cc. solution. PMID:19873299

Internal acid buffering in San Joaquin Valley fog drops and its influence on aerosol processing

NASA Astrophysics Data System (ADS)

Collett, Jeffrey L.; Hoag, Katherine J.; Rao, Xin; Pandis, Spyros N.

Although several chemical pathways exist for S(IV) oxidation in fogs and clouds, many are self-limiting: as sulfuric acid is produced and the drop pH declines, the rates of these pathways also decline. Some of the acid that is produced can be buffered by uptake of gaseous ammonia. Additional internal buffering can result from protonation of weak and strong bases present in solution. Acid titrations of high pH fog samples (median pH=6.49) collected in California's San Joaquin Valley reveal the presence of considerable internal acid buffering. In samples collected at a rural location, the observed internal buffering could be nearly accounted for based on concentrations of ammonia and bicarbonate present in solution. In samples collected in the cities of Fresno and Bakersfield, however, significant additional, unexplained buffering was present over a pH range extending from approximately four to seven. The additional buffering was found to be associated with dissolved compounds in the fogwater. It could not be accounted for by measured concentrations of low molecular weight ( C1- C3) carboxylic acids, S(IV), phosphate, or nitrophenols. The amount of unexplained buffering in individual fog samples was found to correlate strongly with the sum of sample acetate and formate concentrations, suggesting that unmeasured organic species may be important contributors. Simulation of a Bakersfield fog episode with and without the additional, unexplained buffering revealed a significant impact on the fog chemistry. When the additional buffering was included, the simulated fog pH remained 0.3-0.7 pH units higher and the amount of sulfate present after the fog evaporated was increased by 50%. Including the additional buffering in the model simulation did not affect fogwater nitrate concentrations and was found to slightly decrease ammonium concentrations. The magnitude of the buffering effect on aqueous sulfate production is sensitive to the amount of ozone present to oxidize S(IV) in these high pH fogs.

Protein Buffering in Model Systems and in Whole Human Saliva

PubMed Central

Lamanda, Andreas; Cheaib, Zeinab; Turgut, Melek Dilek; Lussi, Adrian

2007-01-01

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and α-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva. PMID:17327922

Adherence of Enterohemorrhagic Escherichia coli to Human Epithelial Cells: The Role of Intimin

DTIC Science & Technology

1995-04-28

1994). Contamination of salad vegetables (Abdul-Raouf et al., 1993), raw milk (MacDonald et al., 1988), and unpasteurized apple cider (Besser et...membranes were blocked with 5% nonfat dried milk (Carnation Company, los Angeles, Cali!.) in Tris-buffered saline, pH 7.2 with 0.1% Tween-20 (v/v) (1B5-T...then ove~aid with a 1 :5000 dilution of either horseradish peroxidase-conjugated goat anti-mouse Ig (BMB), donkey anti-rabbit Ig (Amersham), or sheep

A Receptor-Coupled Evanescent Biosensor

DTIC Science & Technology

1990-05-01

fibers ....................................... 18 6. The effects of various concentrations of d-TC (0), carbamyl- choline (&), and a𔃾GT (0) on binding of...affinity gel washed with the homogenization buffer containing 0.1% Triton X-100. The affinity gel was then mixed with 50 mL of 1 M carbamy- choline for 4 h...at 23*C, then filtered, and the filtrate, containing carbamyl- choline and the nAChR protein, was dialyzed against 5 mM Tris pH 7.2 to remove the drug

Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.

PubMed

Ablinger, Elisabeth; Hellweger, Monika; Leitgeb, Stefan; Zimmer, Andreas

2012-10-15

In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions. Copyright © 2012. Published by Elsevier B.V.

In Vitro and in Vivo Evaluation of Native Glucagon and Glucagon Analog (MAR-D28) during Aging: Lack of Cytotoxicity and Preservation of Hyperglycemic Effect

PubMed Central

Ward, W Kenneth; Massoud, Ryan G; Szybala, Cory J; Engle, Julia M; El Youssef, Joseph; Carroll, Julie M; Roberts, Charles T; DiMarchi, Richard D

2010-01-01

Background For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen® would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation. PMID:21129325

Development of Hybrid pH sensor for long-term seawater pH monitoring.

NASA Astrophysics Data System (ADS)

Nakano, Y.; Egashira, T.; Miwa, T.; Kimoto, H.

2016-02-01

We have been developing the in situ pH sensor (Hybrid pH sensor: HpHS) for the long-term seawater pH monitoring. We are planning to provide the HpHS for researchers and environmental consultants for observation of the CCS (Carbon dioxide Capture and Storage) monitoring system, the coastal environment monitoring system (e.g. Blue Carbon) and ocean acidification. The HpHS has two types of pH sensors (i.e. potentiometric pH sensor and spectrophotometric pH sensor). The spectrophotometric pH sensor can measure pH correctly and stably, however it needs large power consumption and a lot of reagents in a long period of observation. The pH sensor used m-cresol purple (mCP) as an indicator of pH (Clayton and Byrne, 1993 and Liu et al., 2011). We can choose both coefficients before deployment. On the other hand, although the potentiometric pH sensor is low power consumption and high-speed response (within 10 seconds), drifts in the pH of the potentiometric measurements may possibly occur for a long-term observation. The HpHS can measure in situ pH correctly and stably combining advantage of both pH sensors. The HpHS consists of an aluminum pressure housing with optical cell (main unit) and an aluminum silicon-oil filled, pressure-compensated vessel containing pumps and valves (diaphragm pump and valve unit) and pressure-compensated reagents bags (pH indicator, pure water and Tris buffer or certified reference material: CRM) with an ability to resist water pressure to 3000m depth. The main unit holds system control boards, pump drivers, data storage (micro SD card), LED right source, photodiode, optical cell and pressure proof windows. The HpHS also has an aluminum pressure housing that holds a rechargeable lithium-ion battery or a lithium battery for the power supply (DC 24 V). The HpHS is correcting the value of the potentiometric pH sensor (measuring frequently) by the value of the spectrophotometric pH sensor (measuring less frequently). It is possible to calibrate in situ with Tris buffer or CRM on the spectrophotometric pH sensor. Therefore, the drifts in the value of potentiometric pH measurements can be compensated using the pH value obtained from the spectrophotometric pH measurements. Thereby, the sensor can measure accurately the value of pH over a long period of time with low power consumption.

Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition.

PubMed

Chen, Tse-Hsien; Misra, Tarun Kumar; Liu, Chuen-Ying

2008-04-01

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.

Test of the mechanism of UV-induced K/sup +/ efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, T.M.; Huerta, A.J.

1987-04-01

UV radiation and certain plant pathogens stimulate a net efflux of K/sup +/ from cultured plant cells. Many aspects of the efflux are uncertain, including the counterion(s) involved. In the case of UV irradiation of rose cells, Murphy and Wilson suggest a coordinate loss of K/sup +/ and HCO/sub 3//sup -/; in contrast, Atkinson et al. suggest that treatment of tobacco cells with Erwinia pectate lyase introduces a counterflux of K/sup +/ and H/sup +/. In respiring cells, the cytoplasm and medium are buffered by respiratory CO/sub 2/, and it is difficult to distinguish between the two mechanisms. Still, themore » two models predict different influences of external pH on the rate of K/sup +/ flux. The authors have found that increasing pH from 4 to 8 by use of MES-TRIS buffer, pH state, or controlled external CO/sub 2/ concentration does not significantly decrease the rate of UV-induced K/sup +/ efflux. This evidence does not support the application of the K/sup +//H/sup +/ counterflux model to the case of the UV-irradiated rose cells.« less

Effects of extraction and high-performance liquid chromatographic conditions on the determination of lutein in spinach.

PubMed

Simonovska, Breda; Vovk, Irena; Glavnik, Vesna; Cernelič, Katarina

2013-02-08

A major factor in the direct determination of lutein in spinach extracts proved to be obtaining reproducible and stable chromatography of lutein. This was achieved on a C30 column with the mobile phase acetone-0.1M triethylammonium acetate (TEAA) buffer (pH 7) 9:1 (v/v). Extraction of 10mg of lyophilized spinach with 10 mL of extraction solvent (ethanol, acetone, ethanol-ethyl acetate 1:1 (v/v), methanol-THF 1:1 (v/v)) for 15 min with magnetic stirring under nitrogen resulted in equal yields of lutein. The yields were enhanced by addition of 15% of 1M TEAA buffer pH 7 to all four extraction solvents. As confirmed by recovery experiments, no loss of lutein occurred during the extraction. The relative standard deviation from triplicate extractions was less than 5%. The addition of 15% TEAA pH 7 to acetone enhanced the extraction yield of lutein also from unlyophilized spinach. The content of lutein in different spinach samples ranged from 5 to 15 mg/100g of fresh weight. The first separation is reported of all the carotenoids and chlorophylls on a C18 core-shell column and the addition of 15% of 1M TEAA buffer pH 7 to acetone also enhanced the extraction yield of β-carotene compared to the yield produced by pure acetone. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of degree of esterification of pectin and calcium amount on drug release from pectin-based matrix tablets.

PubMed

Sungthongjeen, Srisagul; Sriamornsak, Pornsak; Pitaksuteepong, Tasana; Somsiri, Atawit; Puttipipatkhachorn, Satit

2004-02-12

The aim of this work was to assess the effect of 2 formulation variables, the pectin type (with different degrees of esterification [DEs]) and the amount of calcium, on drug release from pectin-based matrix tablets. Pectin matrix tablets were prepared by blending indomethacin (a model drug), pectin powder, and various amounts of calcium acetate and then tableting by automatic hydraulic press machine. Differential scanning calorimetry, powder x-ray diffraction, and Fourier transformed-infrared spectroscopy studies of the compressed tablets revealed no drug-polymer interaction and the existence of drug with low crystallinity. The in-vitro release studies in phosphate buffer (United States Pharmacopeia) and tris buffer indicated that the lower the DE, the greater the time for 50% of drug release (T50). This finding is probably because of the increased binding capacity of pectin to calcium. However, when the calcium was excluded, the pectins with different DEs showed similar release pattern with insignificant difference of T50. When the amount of calcium acetate was increased from 0 to 12 mg/tablet, the drug release was significantly slower. However, a large amount of added calcium (ie, 24 mg/tablet) produced greater drug release because of the partial disintegration of tablets. The results were more pronounced in phosphate buffer, where the phosphate ions induced the precipitation of calcium phosphate. In conclusion, both pectin type and added calcium affect the drug release from the pectin-based matrix tablets.

Biochemical characteristics of glucose-6-phosphate dehydrogenase variants among the Malays of Singapore with report of a new non-deficient (GdSingapore) and three deficient variants.

PubMed

Saha, N; Hong, S H; Wong, H A; Jeyaseelan, K; Tay, J S

1991-12-01

Biochemical characteristics of one non-deficient fast G6PD variant (GdSingapore) and six different deficient variants (three new, two Mahidol, one each of Indonesian and Mediterranean) were studied among the Malays of Singapore. The GdSingapore variant had normal enzyme activity (82%) and fast electrophoretic mobilities (140% in TEB buffer, 160% in phosphate and 140% in Tris-HCl buffer systems respectively). This variant is further characterized by normal Km for G6P; utilization of analogues (Gal6P, 2dG6P; dAmNADP), heat stability and pH optimum. The other six deficient G6PD variants had normal electrophoretic mobility in TEB buffer with enzyme activities ranging from 1 to 12% of GdB+. The biochemical characteristics identity them to be 2 Mahidol, 1 Indonesian and 1 Mediterranean variants and three new deficient variants.

Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase.

PubMed Central

Cammack, R; Barber, M J; Bray, R C

1976-01-01

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres. PMID:183752

Isolation of Active Mitochondria From Tomato Fruit 1

PubMed Central

Ku, Han San; Pratt, Harlan K.; Spurr, Arthur R.; Harris, William M.

1968-01-01

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl2, 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 × g for 15 minutes. The supernatant is then centrifuged at 11,000 × g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl2, 10 mm tris-HCl, 10 mm KH2PO4 and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and `Honey Dew' melon. Images PMID:16656857

Amphiphilic polymeric micelle as pseudostationary phase in electrokinetic chromatography for analysis of eight corticosteroids in cosmetics.

PubMed

Xu, Xiaojin; Ni, Xinjiong; Cao, Yuhua; Zhuo, Xiaolu; Yang, Xiaoxiao; Cao, Guangqun

2014-03-01

Amphiphilic polymeric micelle, as a novel pseudostationary phase in EKC was used to determine eight kinds of corticosteroids namely hydrocortisone, prednisolone, hydrocortisone acetate, prednisone, cortisone acetate, prednisolone acetate, dexamethasone, and triamcinolone acetonide in cosmetics. Amphiphilic random copolymer poly(methyl methacrylate-co-methacrylic acid) (P(MMA-co-MAA)) was micellizated via neutralization in alkaline aqueous solution. The influences of the molar ratio of monomer MMA to MAA, the concentration of polymer and pH on the polymeric micelle microstructure and EKC performances were investigated. As molar ratio of MMA to MAA in P(MMA-co-MAA) increased, both CMC and environmental polarity of the inner core in polymeric micelle decreased dramatically. With increasing monomer ratio, the size of polymeric micelles increased firstly, and then decreased, finally increased again. ζ potential of the micelle had a slight decline trend. As increment of polymer concentration, the size of the polymeric micelle increased steadily. By optimizing the monomer ratio, the polymer concentration, and pH of the running buffer, as well as operation conditions such as separation voltage and temperature, the eight analytes could be separated within 16.5 min using 7.5 mg/mL polymer with the monomer ratio of 7:3 dissolved in pH 9.2 borax buffer as the running buffer. The method has been used for analysis of corticosteroids in cosmetic samples with simple extraction; the recoveries for eight analytes were between 85.9 and 106%. This method was of accuracy, repeatability, pretreatment simplicity, and could be applied to the quality control of cosmetics. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Permanganate Fixation of Plant Cells

PubMed Central

Mollenhauer, Hilton H.

1959-01-01

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO4 at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO4 at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO4, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO4 at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO4. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO4 or OsO4. PMID:14423414

Some characteristics of fructose 1,6-diphosphatase activity in rat liver

NASA Technical Reports Server (NTRS)

Ashman, P. U.; Lampkin, S. L.; Dillon, L.; Parks, R.

1974-01-01

A reliable assay for hepatic fructose 1,6-diphosphatase in the rat was developed. It was found that the greatest enzymic activity and highest protein levels were eluted from the colored portion of the homogenate. When the substrate concentration was 0.01M, the enzyme had optimal activity when incubated with 0.01M MgSO4 for 10 min. at 37 C in 0.05M Tris-HC1 buffer, pH 7.5. Specificity for the substrate, fructose 1,6-diphosphate, was obtained at substrate concentration of 0.01M.

The Role of Newly Discovered Exotoxin (S Toxin) in Pseudomonas aeruginosa Infections

DTIC Science & Technology

1979-08-01

sodium or potassium phosphate 6.0-8.0 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) 6.5-8.5 tris 7.0-9.5 sodium borate 7.5-9.5 sodium...was found to be variable with respect to whether sodium or potassium phosphate buffer was used. With sodium phosphate, virtually all the enzyme...activity bound was eluted between 20-100.2M phosphate at pH 6.8. With the potassium salt, elution occurs at 400-?00mM KP04. Since very little protein was

Effects of pH buffering agents on the anaerobic hydrolysis acidification stage of kitchen waste.

PubMed

Wang, Yaya; Zang, Bing; Gong, Xiaoyan; Liu, Yu; Li, Guoxue

2017-10-01

This study investigated effects of initial pH buffering agents on the lab-scale anaerobic hydrolysis acidification stage of kitchen waste (KW). Different cheap, available and suitable buffering agents (NaOH(s), NaOH(l), CaO(s)-NaOH, KOH(l)-NaOH, K 2 HPO 4 (s)-KOH, Na 2 CO 3 (s)-NaOH) were added under optimal adjusting mode (first two days: per 16h, after: one time per day) which was obtained in previous work. The effects of buffering agents were evaluated according to indexes of pH, VFAs, NH 4 + -N, TS, VS, VS/TS, TS and VS removal rate. The results showed treatment 5 with adding K 2 HPO 4 -KOH buffering agents had the most stable pH (6.7-7.0). Also treatment 5, 2, 4 and 6 provided stable pH ranging in 5-8. Among the treatments, treatment 6 with adding Na 2 CO 3 as initial buffering agents and 10mol/L NaOH as regulator was chosen as the optimal mode for highest VFAs content (44.05g/L) with high acetic acid and butyrate acid proportion (42.64%), TS and VS removal rate (44.84% and 58.67%, respectively), low VS/TS ratio (58.55), fewer adding dosage and low adjusting frequency. The VFAs content of treatment 6 at the end of hydrolysis acidification stage could be used for methanogenic phase of anaerobic two-phase digestion. Thus, treatment 6 (adding Na 2 CO 3 as initial buffering agents and 10mol/L NaOH as regulator) with highest VFAs content and TS and VS removal rate could be considered using in anaerobic hydrolysis acidification stage pH adjustment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stability of sonicated aqueous suspensions of phospholipids under air.

PubMed

Almog, R; Forward, R; Samsonoff, C

1991-12-01

The stability of phospholipids in liposomal aqueous suspension against oxidative degradation in air was investigated using spectrophotometric indices, glutathione peroxidase reactivity and thin layer chromatography. Zwitterionic phospholipid was found to be susceptible to degradation via oxidation of polyunsaturated hydrocarbon chains and ester hydrolysis, producing oxidized lysophosphatide and free fatty acid derivatives. These products were characterized as hydroperoxides based on their reactivity with the selenium-dependent glutathione peroxidase isolated from human erythrocytes. Lecithin in Tris buffer was more resistant to hydrolysis than in water. The sonication of 8.0 mM of soybean phosphatidylcholine (SB-PC) suspension in 0.1 M Tris (pH 7.5) in the presence of air produced relatively high concentration of conjugated diene hydroperoxide, but a small amount of hydrolyzed products. Anionic phospholipids, such as egg-phosphatidylglycerol (egg-PG), demonstrated higher resistance to air oxidation than the zwitterionic lecithin, but its oxidation was promoted by sonication.

HPLC-based lipophilicity of pyrrolyl-acetic acid ARIs: Relationships with biological activity.

PubMed

Chrysanthakopoulos, Marios; Nicolaou, Ioannis; Demopoulos, Vassilis J; Tsantili-Kakoulidou, Anna

2010-01-01

Reversed phase HPLC was used to assess the lipophilicity of a series pyrrolyl-acetic acid derivatives with aldose reductase inhibitory activity. The pH conditions were adjusted at 3.0 to investigate the behavior of the neutral species and at pH 7.4, at which the ionized form predominates, using phosphate and MOPS buffer. Retention was monitored in absence and in presence of different amounts of n-octanol in the mobile phase in order to explore the chromatographic conditions which best reproduce the octanol-water partition or distribution coefficients. The effect of n-octanol in retention was systematically studied and its role in lipophilicity assessment was evaluated. Nevertheless rather moderate regression equations were obtained, which deviated significantly from the ideal 1:1 correlation. No significant effect of buffer was observed. The appropriateness of retention factors to be used in correlation with aldose reductase inhibitory activity was further evaluated and compared to the efficiency of the corresponding octanol-water logP values.

Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.

PubMed

Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

2013-12-01

Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI(-)) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed and stored properly. Copyright © 2013 Elsevier B.V. All rights reserved.

SALICYLATE PROCESS FOR THORIUM SEPARATION FROM RARE EARTHS

DOEpatents

Cowan, G.A.

1959-08-25

The separation of thorium from rare earths is accomplished by forming an aqueous solution of salts of thorium and rare earths and sufficient acetate buffer to provide a pH of between 2 and 5, adding an ammonium salicylate to the aqueous buffered solution, contacting the resultant solution with a substantially water-immiscible organic solvent mixture of an ether and an ester, and separating the solvent extract phase containing thorium salicylate from the aqueous phase containing the rare earths.

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

PubMed Central

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

Aggregation behavior of fullerenes in aqueous solutions: a capillary electrophoresis and asymmetric flow field-flow fractionation study.

PubMed

Astefanei, Alina; Núñez, Oscar; Galceran, Maria Teresa; Kok, Wim Th; Schoenmakers, Peter J

2015-10-01

In this work, the electrophoretic behavior of hydrophobic fullerenes [buckminsterfullerene (C60), C70, and N-methyl-fulleropyrrolidine (C60-pyrr)] and water-soluble fullerenes [fullerol (C60(OH)24); polyhydroxy small gap fullerene, hydrated (C120(OH)30); C60 pyrrolidine tris acid (C60-pyrr tris acid); and (1,2-methanofullerene C60)-61-carboxylic acid (C60CHCOOH)] in micellar electrokinetic capillary chromatography (MECC) was evaluated. The aggregation behavior of the water-soluble compounds in MECC at different buffer and sodium dodecyl sulfate (SDS) concentrations and pH values of the background electrolyte (BGE) was studied by monitoring the changes observed in the electrophoretic pattern of the peaks. Broad and distorted peaks that can be attributed to fullerene aggregation were obtained in MECC which became narrower and more symmetric by working at low buffer and SDS concentrations (below the critical micelle concentration, capillary zone electrophoresis (CZE) conditions). For the characterization of the suspected aggregates formed (size and shape), asymmetrical flow field-flow fractionation (AF4) and transmission electron microscopy (TEM) were used. The results showed that the increase in the buffer concentration promoted the aggregation of the particles, while the presence of SDS micelles revealed multiple peaks corresponding to particles of different aggregation degrees. Furthermore, MECC has been applied for the first time for the analysis of C60 in two different cosmetic products (i.e., anti-aging serum and facial mask).

Buffer Effects in the Solubility, Nucleation and Growth of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Gibson, Ursula J.

1999-01-01

The growth of protein crystals is important for determination of their three-dimensional structure, which relates to their biochemical functions and to the practical goal of designing pharmaceuticals to modify that function. While many proteins have been successfully crystallized by a variety of methods, there is still limited understanding of the process of nucleation and growth of even the simplest proteins. Chicken egg-white lysozyme (CEWL) is readily crystallized under a variety of conditions, and studies underway at MSFC are designed to elucidate the mechanisms by which the crystals nucleate and grow. We have investigated the effect of buffer choice on the solubility, nucleation and growth of CEWL. CEWL was purified by dialysis against a .05M phosphate buffer and chromatographic separation from contaminants in a sepharose column. Solubility studies were made as a function of buffer concentration for phosphate and formate buffers, and the nucleation and growth of crystals at 10 C was studied as a function of pH for oxalate, succinate, formate, butyrate, carbonate, phosphate and acetate buffer solutions. The solubility data support the conclusion that there is a solubility minimum as a function of buffer concentration for amphiphilic molecules, while no minimum is observed for a phosphate buffer. Nucleation is suppressed at pH greater than pKa for all buffers except phosphate. The aspect ratio of the (110) faces is shown to be a function of crystal size, rather than pH.

Solubility of lead and copper in biochar-amended small arms range soils: influence of soil organic carbon and pH.

PubMed

Uchimiya, Minori; Bannon, Desmond I

2013-08-14

Biochar is often considered a strong heavy metal stabilizing agent. However, biochar in some cases had no effects on, or increased the soluble concentrations of, heavy metals in soil. The objective of this study was to determine the factors causing some biochars to stabilize and others to dissolve heavy metals in soil. Seven small arms range soils with known total organic carbon (TOC), cation exchange capacity, pH, and total Pb and Cu contents were first screened for soluble Pb and Cu concentrations. Over 2 weeks successive equilibrations using weak acid (pH 4.5 sulfuric acid) and acetate buffer (0.1 M at pH 4.9), Alaska soil containing disproportionately high (31.6%) TOC had nearly 100% residual (insoluble) Pb and Cu. This soil was then compared with sandy soils from Maryland containing significantly lower (0.5-2.0%) TOC in the presence of 10 wt % (i) plant biochar activated to increase the surface-bound carboxyl and phosphate ligands (PS450A), (ii) manure biochar enriched with soluble P (BL700), and (iii) unactivated plant biochars produced at 350 °C (CH350) and 700 °C (CH500) and by flash carbonization (corn). In weak acid, the pH was set by soil and biochar, and the biochars increasingly stabilized Pb with repeated extractions. In pH 4.9 acetate buffer, PS450A and BL700 stabilized Pb, and only PS450A stabilized Cu. Surface ligands of PS450A likely complexed and stabilized Pb and Cu even under acidic pH in the presence of competing acetate ligand. Oppositely, unactivated plant biochars (CH350, CH500, and corn) mobilized Pb and Cu in sandy soils; the putative mechanism is the formation of soluble complexes with biochar-borne dissolved organic carbon. In summary, unactivated plant biochars can inadvertently increase dissolved Pb and Cu concentrations of sandy, low TOC soils when used to stabilize other contaminants.

Identification of a Novel Esterase from Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All- trans-Astaxanthin.

PubMed

Lu, Ping; Gao, Xinwei; Dong, Hao; Liu, Zhen; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

2018-03-21

Astaxanthin is a pigment with various functions. Free astaxanthin is obtained mainly through saponification methods, which could result in many byproducts. Enzymatic methods using lipases have been used in a few cases, while there are no reports on the use of esterases for the production of free astaxanthin. Herein we present the screening and identification of a novel esterase (Est3-14) from a marine mud metagenomic library. Est3-14 is pH-sensitive and keeps good stability in alkaline buffers (residual activity 94%, pH 8.0, 4 °C, and 36 h). Meanwhile, Est3-14 keeps a good stability in the medium temperature condition (residual activity 56.7%, pH 8.0, 40 °C, and 84 h). Est3-14 displayed high hydrolysis activity to prepare free all- trans-astaxanthin in biphasic systems. Furthermore, under optimal conditions (0.5 mL ethanol, 6 mL 0.1 M Tris-HCl buffer, pH 8.0, 0.5% (w/v) H. pluvialis oil, 40 °C), the hydrolytic conversion ratio was 99.3% after 36 h.

ELECTRODEPOSITION OF PLUTONIUM

DOEpatents

Wolter, F.J.

1957-09-10

A process of electrolytically recovering plutonium from dilute aqueous solutions containing plutonium ions comprises electrolyzing the solution at a current density of about 0.44 ampere per square centimeter in the presence of an acetate-sulfate buffer while maintaining the pH of the solution at substantially 5 and using a stirred mercury cathode.

Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

PubMed Central

Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F.

2013-01-01

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions. PMID:23534985

Development of a colorimetric microfluidic pH sensor for autonomous seawater measurements.

PubMed

Rérolle, Victoire M C; Floquet, Cedric F A; Harris, Andy J K; Mowlem, Matt C; Bellerby, Richard R G J; Achterberg, Eric P

2013-07-05

High quality carbonate chemistry measurements are required in order to fully understand the dynamics of the oceanic carbonate system. Seawater pH data with good spatial and temporal coverage are particularly critical to apprehend ocean acidification phenomena and their consequences. There is a growing need for autonomous in situ instruments that measure pH on remote platforms. Our aim is to develop an accurate and precise autonomous in situ pH sensor for long term deployment on remote platforms. The widely used spectrophotometric pH technique is capable of the required high-quality measurements. We report a key step towards the miniaturization of a colorimetric pH sensor with the successful implementation of a simple microfluidic design with low reagent consumption. The system is particularly adapted to shipboard deployment: high quality data was obtained over a period of more than a month during a shipboard deployment in northwest European shelf waters, and less than 30 mL of indicator was consumed. The system featured a short term precision of 0.001 pH (n=20) and an accuracy within the range of a certified Tris buffer (0.004 pH). The quality of the pH system measurements have been checked using various approaches: measurements of certified Tris buffer, measurement of certified seawater for DIC and TA, comparison of measured pH against calculated pH from pCO2, DIC and TA during the cruise in northwest European shelf waters. All showed that our measurements were of high quality. The measurements were made close to in situ temperature (+0.2°C) in a sampling chamber which had a continuous flow of the ship's underway seawater supply. The optical set up was robust and relatively small due to the use of an USB mini-spectrometer, a custom made polymeric flow cell and an LED light source. The use of a three wavelength LED with detection that integrated power across the whole of each LED output spectrum indicated that low wavelength resolution detectors can be used instead of the current USB mini spectrophotometer. Artefacts due to the polychromatic light source and inhomogeneity in the absorption cell are shown to have a negligible impact on the data quality. The next step in the miniaturization of the sensor will be the incorporation of a photodiode as detector to replace the spectrophotometer. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Comparison of immunoexpression of 2 antibodies for estrogen receptors (1D5 and 6F11) in breast carcinomas using different antigen retrieval and detection methods.

PubMed

Vassallo, J; Pinto, G A; Alvarenga, J M; Zeferino, L C; Chagas, C A; Metze, K

2004-06-01

The importance of in situ immunodetection of hormone receptors for therapy planning and prognostic evaluation in patients with breast carcinoma is well established. Sensitive detection methods are of utmost importance, especially in poorly fixed tissues, which are not uncommon in routine pathologic practice. The purpose of the present study is to compare immunoexpression of estrogen receptors in 20 cases of invasive ductal carcinoma using two antibodies, 1D5 and 6F11, and to verify the effect of different antigen retrieval solutions and detection systems. Immunoperoxidase was performed on paraffin sections using 1D5 and 6F11 as primary antibodies. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 8.9). Detection was achieved using the following systems: EnVision, EnVision Plus, and labeled streptavidin-biotin peroxidase complex. Reaction was semiquantified from 0 to 4. There were no differences between the two markers, 1D5 and 6F11, except when 6F11 was used with EnVision and citrate buffer, in which case weaker reactivity was observed. Only in this combination (6F11/EnVision) was EDTA buffer significantly better than citrate. Labeled streptavidin-biotin peroxidase complex presented the best results, followed by EnVision Plus.

Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1 ▿

PubMed Central

Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.; Heald, Steve; Dohnalkova, Alice C.; Sybirna, Kateryna; Bottin, Hervé; Squier, Thomas C.; Zachara, John M.; Fredrickson, James K.

2011-01-01

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that has been implicated in H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H2ase in trans restored the mutant's ability to produce H2 at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H2ase coupled H2 oxidation to reduction of Tc(VII)O4− and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated reduction of Tc(VII)O4− but not methyl viologen. Under the conditions tested, all Tc(VII)O4− used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O4− was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2·nH2O, which was also the product of Tc(VII)O4− reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H2ase catalyzes Tc(VII)O4− reduction directly by coupling to H2 oxidation. PMID:21724888

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection.

PubMed

Yeh, Hsin-Hua; Yang, Yuan-Han; Ko, Ju-Yun; Chen, Su-Hwei

2006-07-07

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.

Lethality and injuring the effect of compression and decompression rates of high hydrostatic pressure on Escherichia coli O157:H7 in different matrices

NASA Astrophysics Data System (ADS)

Syed, Qamar Abbas; Buffa, Martin; Guamis, Buenaventura; Saldo, Jordi

2013-03-01

The effect of compression and decompression rates of high hydrostatic pressure (HHP) on Escherichia coli O157:H7 was investigated. Samples of orange juice, skimmed milk and Tris buffer were inoculated with E. coli O157:H7 and subjected to 600 MPa for 3 min at 4°C with fast, medium and slow compression and decompression. Analyses immediately after HHP treatment revealed that E. coli in milk and juice treated with fast compression suffered more than slow compression rates. Slow decompression resulted in higher inactivation of E. coli in all matrices. After overnight storage, highest stress-recovery (1.19 log cfu/mL) was observed in Tris buffer. Healthy cells were<1 log cfu/mL in milk and buffer samples, but no growth was detected in orange juice for any of the treatments immediately after HHP. After 15 days at 4°C, E. coli cells in skimmed milk and Tris buffer recovered significantly, whereas the recovery of sublethally injured cells was inhibited in orange juice.

Redox Cycling, pH Dependence, and Ligand Effects of Mn(III) in Oxalate Decarboxylase from Bacillus subtilis.

PubMed

Twahir, Umar T; Ozarowski, Andrew; Angerhofer, Alexander

2016-11-29

This contribution describes electron paramagnetic resonance (EPR) experiments on Mn(III) in oxalate decarboxylase of Bacillus subtilis, an interesting enzyme that catalyzes the redox-neutral dissociation of oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized, although the N-terminal Mn(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions, in agreement with the general view that it represents the active site. Significantly, Mn(III) was observed in untreated OxDC in succinate and acetate buffers, while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR-visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn(III) are affected by small carboxylate ligands that can enter the active site and have been recorded for formate, acetate, and succinate. The results from a previous report [Zhu, W., et al. (2016) Biochemistry 55, 429-434] could therefore be reinterpreted as evidence of formate-bound Mn(III) after the enzyme is allowed to turn over oxalate. The pH dependence of the Mn(III) EPR signal compares very well with that of enzymatic activity, providing strong evidence that the catalytic reaction of oxalate decarboxylase is driven by Mn(III), which is generated in the presence of dioxygen.

Spectrophotometric study on binding of 2-thioxanthone acetic acid with ct-DNA.

PubMed

Ataci, Nese; Ozcelik, Elif; Arsu, Nergis

2018-06-02

Thioxanthone and its derivatives are the most remarkable molecules due to their vast variety of application such as radiation curing that is, until using them as a therapeutic drug. Therefore, in this study it was intended to use 2-Thioxanthone acetic acid with and without NaCl in Tris HCl buffer solution (pH:7.0) to represent the interaction with ct-DNA. The UV-vis absorption spectra of TXCH 2 COOH in the presence of ct-DNA showed hypochromism and the intrinstic binding constant (K b ) was determined as 6 × 10 3  L mol -1 . The fluoresence intensity of TXCH 2 COOH with ct-DNA clearly increased up to 101% which indicated that the fluorescence intensity was very sensitive to ct-DNA concentration. The binding constant (K) and the values of number of binding sites (n) and were calculated as 1.8 × 10 3  L mol -1 and 0.69, respectively. When the quenching constants (K sv ) of free TXCH 2 COOH and TXCH 2 COOH, which were bonded with ct-DNA were compared, slightly changed values of Ksv were seen. Moreover, displacement assay with Hoechst 33,258 and viscosity measurements in the presence and absence of NaCl salt also confirmed the binding mode which noted the electrostatic interaction following groove binding between TXCH 2 COOH and ct-DNA. Last but not least, the salt effect was examined on ct-DNA binding with TXCH 2 COOH. The results of the experiments indicated that the groove binding was strengthened by NaCl whereas in the high NaCl concentration, the binding ability of TXCH 2 COOH to ct-DNA was inversely affected. Copyright © 2018 Elsevier B.V. All rights reserved.

Pleiotrophin as a Growth Factor and Therapeutic Target in Breast Cancer

DTIC Science & Technology

1997-10-01

novel phospholipase A2 related gene. Nucl Acid Res 21:135-143. 11. Gattoni-Celli, S., K . Kirsch, S. Kalled , and K . J. Isselbacher. 1986. Expression...clone (G11-F7) is enlarged. Genomic Southern blot probes (a,b,c) and restriction sites are shown (B=BamHI, H=HindIII, Sc=ScaI, K =KpnI). 10WJ 3fr 4&V...otherwise in 25 mM Tris pH8.3/200 mM glycine/20% methanol. The membrane was blocked in PBS (phospate-buffered saline )/0.1% Tween 20/5% powdered milk and

Optimized conditions for chelation of yttrium-90-DOTA immunoconjugates.

PubMed

Kukis, D L; DeNardo, S J; DeNardo, G L; O'Donnell, R T; Meares, C F

1998-12-01

Radioimmunotherapy (RIT) with 90Y-labeled immunoconjugates has shown promise in clinical trials. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) binds 90Y with extraordinary stability, minimizing the toxicity of 90Y-DOTA immunoconjugates arising from loss of 90Y to bone. However, reported 90Y-DOTA immunoconjugate product yields have been typically only < or =50%. Improved yields are needed for RIT with 90Y-DOTA immunoconjugates to be practical. (S) 2-[p-(bromoacetamido)benzyl]-DOTA (BAD) was conjugated to the monoclonal antibody Lym-1 via 2-iminothiolane (2IT). The immunoconjugate product, 2IT-BAD-Lym-1, was labeled in excess yttrium in various buffers over a range of concentrations and pH. Kinetic studies were performed in selected buffers to estimate radiolabeling reaction times under prospective radiopharmacy labeling conditions. The effect of temperature on reaction kinetics was examined. Optimal radiolabeling conditions were identified and used in eight radiolabeling experiments with 2IT-BAD-Lym-1 and a second immunoconjugate, DOTA-peptide-chimeric L6, with 248-492 MBq (6.7-13.3 mCi) of 90Y. Ammonium acetate buffer (0.5 M) was associated with the highest uptake of yttrium. On the basis of kinetic data, the time required to chelate 94% of 90Y (four half-times) under prospective radiopharmacy labeling conditions in 0.5 M ammonium acetate was 17-148 min at pH 6.5, but it was only 1-10 min at pH 7.5. Raising the reaction temperature from 25 degrees C to 37 degrees C markedly increased the chelation rate. Optimal radiolabeling conditions were identified as: 30-min reaction time, 0.5 M ammonium acetate buffer, pH 7-7.5 and 37 degrees C. In eight labeling experiments under optimal conditions, a mean product yield (+/- s.d.) of 91%+/-8% was achieved, comparable to iodination yields. The specific activity of final products was 74-130 MBq (2.0-3.5 mCi) of 90Y per mg of monoclonal antibody. The immunoreactivity of 90Y-labeled immunoconjugates was 100%+/-11%. The optimization of 90Y-DOTA chelation conditions represents an important advance in 90Y RIT because it facilitates the dependable and cost-effective preparation of 90Y-DOTA pharmaceuticals.

Ultrasonication as a potential tool to predict solute crystallization in freeze-concentrates.

PubMed

Ragoonanan, Vishard; Suryanarayanan, Raj

2014-06-01

We hypothesize that ultrasonication can accelerate solute crystallization in freeze-concentrates. Our objective is to demonstrate ultrasonication as a potential predictive tool for evaluating physical stability of excipients in frozen solutions. The crystallization tendencies of lyoprotectants (trehalose, sucrose), carboxylic acid buffers (citric, tartaric, malic, and acetic) and an amino acid buffer (histidine HCl) were studied. Aqueous solutions of buffers, lyoprotectants and mixtures of the two were cooled from room temperature to -20°C and sonicated to induce solute crystallization. The crystallized phases were identified by X-ray diffractometry (laboratory or synchrotron source). Sonication accelerated crystallization of trehalose dihydrate in frozen trehalose solutions. Sonication also enhanced solute crystallization in tartaric (200 mM; pH 5), citric (200 mM pH 4) and malic (200 mM; pH 4) acid buffers. At lower buffer concentrations, longer annealing times following sonication were required to facilitate solute crystallization. The time for crystallization of histidine HCl progressively increased as a function of sucrose concentration. The insonation period required to effect crystallization also increased with sucrose concentration. Sonication can substantially accelerate solute crystallization in the freeze-concentrate. Ultrasonication may be useful in assessing the crystallization tendency of formulation constituents used in long term frozen storage and freeze-drying.

The influence of diesel-truck exhaust particles on the kinetics of the atmospheric oxidation of dissolved sulfur dioxide by oxygen.

PubMed

Meena, Vimlesh Kumar; Dhayal, Yogpal; Saxena, Deepa; Rani, Ashu; Chandel, C P Singh; Gupta, K S

2016-09-01

The automobile exhausts are one of the major sources of particulate matter in urban areas and these particles are known to influence the atmospheric chemistry in a variety of ways. Because of this, the oxidation of dissolved sulfur dioxide by oxygen was studied in aqueous suspensions of particulates, obtained by scraping the particles deposited inside a diesel truck exhaust pipe (DEP). A variation in pH showed the rate to increase with increase in pH from 5.22 to about ∼6.3 and to decrease thereafter becoming very slow at pH = 8.2. In acetate-buffered medium, the reaction rate was higher than the rate in unbuffered medium at the same pH. Further, the rate was found to be higher in suspension than in the leachate under otherwise identical conditions. And, the reaction rate in the blank reaction was the slowest. This appears to be due to catalysis by leached metal ions in leachate and due to catalysis by leached metal ions and particulate surface both in suspensions. The kinetics of dissolved SO2 oxidation in acetate-buffered medium as well as in unbuffered medium at pH = 5.22 were defined by rate law: k obs  = k 0 + k cat [DEP], where k obs and k 0 are observed rate constants in the presence and the absence of DEP and k cat is the rate constant for DEP-catalyzed pathway. At pH = 8.2, the reaction rate was strongly inhibited by DEP in buffered and unbuffered media. Results suggest that the DEP would have an inhibiting effect in those areas where rainwater pH is 7 or more. These results at high pH are of particular significance to the Indian subcontinent, because of high rainwater pH. Conversely, it indicates the DEP to retard the oxidation of dissolved SO2 and control rainwater acidification.

Fission Yeast Model Study for Dissection of TSC Pathway

DTIC Science & Technology

2010-04-01

prepared as follows. A total of 1010 cells were incubated at 37! for 1 hr in spheroplasts buffer [50 mm citrate–phosphate (pH 5.6) and 1.2 m sorbitol ...potassium acetate, and 0.1 m sorbitol ] containing 0.4 mm phenylmethyl- sulfonyl fluoride and 13 protease inhibitor cocktail (Nacalai Tesque) and downed

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

PubMed

Saxena, Ashima; Luo, Chunyuan; Doctor, Bhupendra P

2008-10-01

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

In vitro digestion method for estimation of copper bioaccessibility in Açaí berry.

PubMed

Ruzik, Lena; Wojcieszek, Justyna

Copper is an essential trace element for humans and its deficiency can lead to numerous diseases. A lot of mineral supplements are available to increase intake of copper. Unfortunately, only a part of the total concentration of elements is available for human body. Thus, the aim of the study was to determine bioaccessibility of copper in Açai berry, known as a "superfood" because of its antioxidant qualities. An analytical methodology was based on size exclusion chromatography (SEC) coupled to a mass spectrometer with inductively coupled plasma (ICP MS) and on capillary liquid chromatography coupled to tandem mass spectrometer with electrospray ionization (µ-HPLC-ESI MS/MS). To extract various copper compounds, berries were treated with the following buffers: ammonium acetate, Tris-HCl, and sodium dodecyl sulfate (SDS). The best extraction efficiency of copper was obtained for SDS extract (88 %), while results obtained for Tris-HCl and ammonium acetate were very similar (47 and 48 %, respectively). After SEC-ICP-MS analysis, main signal was obtained for all extracts in the region of molecular mass about 17 kDa. A two-step model simulated gastric (pepsin) and gastrointestinal (pancreatin) digestion was used to obtain the knowledge about copper bioaccessibility. Copper compounds present in Açai berry were found to be highly bioaccessible. The structures of five copper complexes with amino acids such as aspartic acid, tyrosine, phenylalanine, were proposed after µ-HPLC-ESI MS/MS analysis. Obtained results show that copper in enzymatic extracts is bound by amino acids and peptides what leads to better bioavailability of copper for human body.

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

PubMed

Habibi-Moini, S; D'mello, A P

2001-03-14

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

Enzymatic and chemical synthesis of new anticoagulant peptides.

PubMed

Origone, Anabella; Bersi, Grisel; Illanes, Andrés; Sturniolo, Héctor; Liggieri, Constanza; Guzmán, Fanny; Barberis, Sonia

2018-06-08

In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris - HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the above mentioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136% and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS.

PubMed

Wojcieszek, Justyna; Ruzik, Lena

2016-03-01

Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris-HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium allows confirming the presence of vitamin B12 and probably selenomethionine in the fraction bioaccessible by human body (obtained during enzymatic extraction). It should be noted that the presence of small seleno-compounds in Cape gooseberry was performed for the first time. The results show that the combination of SEC and ICP MS could provide a simple method for separating of soluble element species. Copyright © 2015 Elsevier GmbH. All rights reserved.

Assessment and kinetics of soil phosphatase in Brazilian Savanna systems.

PubMed

Ferreira, Adão S; Espíndola, Suéllen P; Campos, Maria Rita C

2016-05-31

The activity and kinetics of soil phosphatases are important indicators to evaluate soil quality in specific sites such as the Cerrado (Brazilian Savanna). This study aimed to determine the activity and kinetic parameters of soil phosphatase in Cerrado systems. Soil phosphatase activity was assessed in samples of native Cerrado (NC), no-tillage (NT), conventional tillage (CT) and pasture with Brachiaria brizantha (PBb) and evaluated with acetate buffer (AB), tris-HCl buffer (TB), modified universal buffer (MUB) and low MUB. The Michaelis-Menten equation and Eadie-Hofstee model were applied to obtain the kinetic parameters of soil phosphatase using different concentrations of p-nitrophenol phosphate (p-NPP). MUB showed the lowest soil phosphatase activity in all soils whereas AB in NC and NT presented the highest. Low MUB decreased interferences in the assessment of soil phosphatase activity when compared to MUB, suggesting that organic acids interfere on the soil phosphatase activity. In NC and NT, soil phosphatase activity performed with TB was similar to AB and low MUB. Km values from the Michaels-Menten equation were higher in NC than in NT, which indicate a lower affinity of phosphatase activity for the substrate in NC. Vmax values were also higher in NC than in NT. The Eadie-Hofstee model suggests that NC had more phosphatase isoforms than NT. The study showed that buffer type is of fundamental importance when assessing soil phosphatase activity in Cerrado soils.

Secretin stimulates HCO3(-) and acetate efflux but not Na+/HCO3(-) uptake in rat pancreatic ducts.

PubMed

Novak, I; Christoffersen, B C

2001-03-01

Pancreatic ducts secrete HCO3(-), but transport mechanisms are unresolved and possibly vary between species. Our aim was to study the intracellular pH (pHi) regulation and thus H+/HCO3- transport in rat pancreatic ducts. Of particular interest was the Na+/HCO3(-) cotransporter, thought to be important in HCO3(-) -transporting epithelia. pHi was measured with BCECF in freshly isolated intralobular ducts. A reduction in extracellular Na+ concentration or application of HOE 694 (1 microM) decreased pHi by 0.1 to 0.6 pH units, demonstrating Na+/H+ exchanger activity. A reduction in extracellular Cl- concentration or addition of H2DIDS (10 microM) increased pHi by 0.1 to 0.5 pH units, demonstrating Cl-/ HCO(3)- (OH ) exchanger activity. In experimental acidosis, extracellular HCO3(-)/CO2 buffer did not increase the rate of pHi recovery, indicating that provision of HCO3(-) by the Na+/HCO3(-) cotransporter was not apparent. Most importantly, Na+/HCO3(-) cotransport was not stimulated by secretin (1 nM). In contrast, in experimental alkalosis the pHi recovery was increased in HCO3(-)/CO2 buffer, possibly due to Na+/HCO3(-) cotransport in the efflux mode. Secretin (1 nM) and carbachol (1 microM) stimulated HCO3(-) efflux, which can account for the observed HCO3(-) concentrations in rat pancreatic juice. Acetate and HCO3(-) buffers were handled similarly, indicating similar transport mechanisms in pancreatic ducts.

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells.

PubMed

Chen, Gunng-Shinng; Lee, Shiao-Pieng; Huang, Shu-Fu; Chao, Shih-Chi; Chang, Chung-Yi; Wu, Gwo-Jang; Li, Chung-Hsing; Loh, Shih-Hurng

2018-06-01

Homeostasis of intracellular pH (pH i ) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na + -H + exchanger (NHE), Na + -HCO 3 - co-transporter (NBC), Cl - /HCO 3 - exchanger (AE) and Cl - /OH - exchanger (CHE) have been identified to co-regulate pH i homeostasis. However, functional and biological pH i -regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pH i changes. NH 4 Cl and Na + -acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH i -regulators were detected by Western blot technique. The resting pH i was no significant difference between that in HEPES-buffered (nominal HCO 3 - -free) solution or CO 2 /HCO 3 -buffered system (7.42 and 7.46, respectively). The pH i recovery following the induced-intracellular acidosis was blocked completely by removing [Na + ] o , while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pH i recovery was inhibited entirely by removing [Na + ] o , while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO 2 /HCO 3 -buffered system solution, the pH i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl - ] o . Western blot analysis showed the isoforms of pH i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. We demonstrate for the first time that resting pH i is significantly higher than 7.2 and meditates functionally by two Na + -dependent acid extruders (NHE and NBC), two Cl - -dependent acid loaders (CHE and AE) and one Na + -independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Separation and structural analysis of saponins in a bark extract from Quillaja saponaria Molina.

PubMed

Nord, L I; Kenne, L

1999-07-20

Six major saponins were isolated from a bark extract from Quillaja saponaria Molina. Solid-phase extraction, followed by a two-step reversed-phase HPLC separation procedure with phosphate and ammonium acetate buffers of different pH values, was used. The compounds were characterised using NMR spectroscopy, mass spectrometry and chemical methods.

Formulation and comparative in vitro evaluation of various dexamethasone-loaded pH-sensitive polymeric nanoparticles intended for dermal applications.

PubMed

Sahle, Fitsum Feleke; Gerecke, Christian; Kleuser, Burkhard; Bodmeier, Roland

2017-01-10

pH-sensitive nanoparticles have a great potential for dermal and transfollicular drug delivery. In this study, pH-sensitive, dexamethasone-loaded Eudragit ® L 100, Eudragit ® L 100-55, Eudragit ® S 100, HPMCP-50, HPMCP-55 and cellulose acetate phthalate nanoparticles were prepared by nanoprecipitation and characterized. The pH-dependent swelling, erosion, dissolution and drug release kinetics were investigated in vitro using dynamic light scattering and Franz diffusion cells, respectively. Their toxicity potential was assessed by the ROS and MTT assays. 100-700nm nanoparticles with high drug loading and entrapment efficiency were obtained. The nanoparticles bear no toxicity potential. Cellulose phthalates nanoparticles were more sensitive to pH than acrylates nanoparticles. They dissolved in 10mM pH 7.5 buffer and released>80% of the drug within 7h. The acrylate nanoparticles dissolved in 40mM pH 7.5 buffer and released 65-70% of the drug within 7h. The nanoparticles remained intact in 10 and 40mM pH 6.0 buffers (HPMCP nanoparticles dissolved in 40mM pH 6.0 buffer) and released slowly. The nanoparticles properties could be modulated by blending the different polymers. In conclusion, various pH-sensitive nanoparticles that could release differently on the skin surface and dissolve and release in the hair follicles were obtained. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of microwave-assisted extraction of analgesic and anti-inflammatory drugs from human plasma and urine using response surface experimental designs.

PubMed

Fernández, Purificación; Fernández, Ana M; Bermejo, Ana M; Lorenzo, Rosa A; Carro, Antonia M

2013-04-01

The performance of microwave-assisted extraction and HPLC with photodiode array detection method for determination of six analgesic and anti-inflammatory drugs from plasma and urine, is described, optimized, and validated. Several parameters affecting the extraction technique were optimized using experimental designs. A four-factor (temperature, phosphate buffer pH 4.0 volume, extraction solvent volume, and time) hybrid experimental design was used for extraction optimization in plasma, and three-factor (temperature, extraction solvent volume, and time) Doehlert design was chosen to extraction optimization in urine. The use of desirability functions revealed the optimal extraction conditions as follows: 67°C, 4 mL phosphate buffer pH 4.0, 12 mL of ethyl acetate and 9 min, for plasma and the same volume of buffer and ethyl acetate, 115°C and 4 min for urine. Limits of detection ranged from 4 to 45 ng/mL in plasma and from 8 to 85 ng/mL in urine. The reproducibility evaluated at two concentration levels was less than 6.5% for both specimens. The recoveries were from 89 to 99% for plasma and from 83 to 99% for urine. The proposed method was successfully applied in plasma and urine samples obtained from analgesic users. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immobilisation of an antibacterial drug to Ti6Al4V components fabricated using selective laser melting

NASA Astrophysics Data System (ADS)

Vaithilingam, Jayasheelan; Kilsby, Samuel; Goodridge, Ruth D.; Christie, Steven D. R.; Edmondson, Steve; Hague, Richard J. M.

2014-09-01

Bacterial infections from biomedical implants and surgical devices are a major problem in orthopaedic, dental and vascular surgery. Although the sources of contaminations that lead to bacterial infections are known, it is not possible to control or avoid such infections completely. In this study, an approach to immobilise Ciprofloxacin® (an antibacterial drug) to phosphonic acid based self-assembled monolayers (SAMs) adsorbed on a selectively laser melted (SLM) Ti6Al4V structure, has been presented. X-ray photoelectron spectroscopy (XPS) and static water contact angle measurements confirmed the attachment of SAMs and the drug. Results showed that Ciprofloxacin® is highly stable under the oxidative conditions used in this study. In-vitro stability was estimated by immersing the Ciprofloxacin® immobilised substrates in 10 mM of Tris-HCl buffer (pH-7.4) for 42 days. The Tris-HCl buffer was analysed using UV-vis spectrophotometry at 7, 14, 28 and 42 day time intervals to determine the release of the immobilised drug. The drug was observed to release in a sustained manner. 50% of the drug was released after 4 weeks with approximately 40% of the drug remaining after 6 weeks. Antibacterial susceptibility tests revealed that the immobilised drug was therapeutically active upon its release. This study demonstrates the potential to use self-assembled monolayers to modify SLM fabricated surfaces with therapeutics.

Complexation of buffer constituents with neutral complexation agents: part I. Impact on common buffer properties.

PubMed

Riesová, Martina; Svobodová, Jana; Tošner, Zdeněk; Beneš, Martin; Tesařová, Eva; Gaš, Bohuslav

2013-09-17

The complexation of buffer constituents with the complexation agent present in the solution can very significantly influence the buffer properties, such as pH, ionic strength, or conductivity. These parameters are often crucial for selection of the separation conditions in capillary electrophoresis or high-pressure liquid chromatography (HPLC) and can significantly affect results of separation, particularly for capillary electrophoresis as shown in Part II of this paper series (Beneš, M.; Riesová, M.; Svobodová, J.; Tesařová, E.; Dubský, P.; Gaš, B. Anal. Chem. 2013, DOI: 10.1021/ac401381d). In this paper, the impact of complexation of buffer constituents with a neutral complexation agent is demonstrated theoretically as well as experimentally for the model buffer system composed of benzoic acid/LiOH or common buffers (e.g., CHES/LiOH, TAPS/LiOH, Tricine/LiOH, MOPS/LiOH, MES/LiOH, and acetic acid/LiOH). Cyclodextrins as common chiral selectors were used as model complexation agents. We were not only able to demonstrate substantial changes of pH but also to predict the general complexation characteristics of selected compounds. Because of the zwitterion character of the common buffer constituents, their charged forms complex stronger with cyclodextrins than the neutral ones do. This was fully proven by NMR measurements. Additionally complexation constants of both forms of selected compounds were determined by NMR and affinity capillary electrophoresis with a very good agreement of obtained values. These data were advantageously used for the theoretical descriptions of variations in pH, depending on the composition and concentration of the buffer. Theoretical predictions were shown to be a useful tool for deriving some general rules and laws for complexing systems.

Optimalization of Poly(neutral red) Coated-wire Electrode for Determination of Citrate in Soft Drinks

PubMed Central

Broncová, Gabriela; Shishkanova, Tatiana V.; Krondak, Martin; Volf, Radko; Král, Vladimír

2008-01-01

This report presents an optimization of potentiometric measurements with citrate-selective electropolymerized poly(neutral red) electrodes. The optimal background electrolyte for these measurements is a TRIS buffer with nitrate at pH 8.5. The electrodes described here exhibit stable and reproducible near-Nernstian response to citrates with a low detection limit of 6 × 10-6 M. Electrodes polymerized from sulfuric acid and acetonitrile are compared in detail. Simple and sensitive method for quantification of citrate in real-life samples by potentiometry with poly(neutral red) electrodes are presented. Data from potentiometric measurements of citrate are compared with capillary electrophoresis. PMID:27879724

The effect of buffering dairy cow diets with limestone, calcareous marine algae, or sodium bicarbonate on ruminal pH profiles, production responses, and rumen fermentation.

PubMed

Cruywagen, C W; Taylor, S; Beya, M M; Calitz, T

2015-08-01

Six ruminally cannulated Holstein cows were used to evaluate the effect of 2 dietary buffers on rumen pH, milk production, milk composition, and rumen fermentation parameters. A high concentrate total mixed ration [35.2% forage dry matter (DM)], formulated to be potentially acidotic, was used to construct 3 dietary treatments in which calcareous marine algae (calcified remains of the seaweed Lithothamnium calcareum) was compared with limestone (control) and sodium bicarbonate plus limestone. One basal diet was formulated and the treatment diets contained either 0.4% of dietary DM as Acid Buf, a calcified marine algae product (AB treatment), or 0.8% of dietary DM as sodium bicarbonate and 0.37% as limestone (BC treatment), or 0.35% of dietary DM as limestone [control (CON) treatment]. Cows were randomly allocated to treatments according to a double 3×3 Latin square design, with 3 treatments and 3 periods. The total experimental period was 66 d during which each cow received each treatment for a period of 15 d before the data collection period of 7 d. Rumen fluid was collected to determine volatile fatty acids, lactic acid, and ammonia concentrations. Rumen pH was monitored every 10min for 2 consecutive days using a portable data logging system fitted with in-dwelling electrodes. Milk samples were analyzed for solid and mineral contents. The effect of treatment on acidity was clearly visible, especially from the period from midday to midnight when rumen pH dropped below 5.5 for a longer period of time (13 h) in the CON treatment than in the BC (8.7 h) and AB (4 h) treatments. Daily milk, 4% fat-corrected milk, and energy-corrected milk yields differed among treatments, with AB being the highest, followed by BC and CON. Both buffers increased milk fat content. Treatment had no effect on milk protein content, but protein yield was increased in the AB treatment. Total rumen volatile fatty acids and acetate concentrations were higher and propionate was lower in the AB treatment than in CON. The molar proportion of acetate was higher in AB than in CON, but that of propionate was lower in both buffer treatments than in CON. The acetate:propionate ratio was increased in the AB and BC treatments compared with CON. Lactic acid concentration was higher in the CON treatment than in the buffer treatments. Treatment had no effect on rumen ammonia concentrations. Results indicated that buffer inclusion in high concentrate diets for lactating dairy cows had a positive effect on milk production and milk composition. Calcareous marine algae, at a level of 90 g/cow per day, had a greater effect on rumen pH, milk production and milk composition, and efficiency of feed conversion into milk than sodium bicarbonate at a level of 180 g/cow per day. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

pH-based fiber optic biosensors for use in clinical and biotechnological applications

NASA Astrophysics Data System (ADS)

Mueller, Cord; Hitzmann, Bernd; Schubert, Florian; Scheper, Thomas

1995-05-01

The development of pH-based fiber optic biosensors and their uses in clinical and biotechnological applications are described. Based on a pH-sensitive optode, different biosensors for urea, penicillin, glucose and creatinine were developed. A multichannel modular fluorimeter was used to measure signals from up to three optodes simultaneously. The pH value and the buffer capacity are critical factors for biosensors based on pH probes and influence the biosensor signal. A flow injection analysis (FIA) system is used to eliminate the latter influences. With this integrated system, samples can be analyzed sequentially by the injection of a defined volume of each sample into a continuously flowing buffer stream that transports the samples to the sensors. The complex signal is transformed and analyzed by a computer system. Characteristic features of the FIA peak give information about the buffer capacity in the solution. With the help of intelligent computing (neural networks) it is possible to recognize these features and relate them to the respective buffer capacity to obtain more accurate values. Various applications of these biosensors are discussed. The pH optode is also used to monitor enzymatic reactions in non aqueous solvents. In this case the production of acetic acid can be detected on line.

A new capillary electrophoresis buffer for determining organic and inorganic anions in electroplating bath with surfactant additives.

PubMed

Sun, H; Lau, K M; Fung, Y S

2010-05-07

Monitoring of trace impurities in electroplating bath is needed to meet EU requirements for WEEE and RoHS and for quality control of electrodeposits. Methods using IC and 100% aqueous CE buffer were found producing non-repeatable results attributed to interference of surfactants and major methanesulphonate anion. A new CE buffer containing 1.5mM tetraethylenepentaamine, 3mM 1,3,5-benzenetricarboxylic acid and 15 mM Tris in 20% (v/v) methanol at pH=8.4 was shown to enhance the separation window, reduce interaction between buffer and bath constituents, and give satisfactory repeatability with baseline separation for 14 organic and inorganic anions within 14 min, good repeatability for migration time (0.32-0.57% RSD), satisfactory peak area and peak height (2.9-4.5 and 3-4.7% respectively), low detection limit (S/N=2, 20-150 ppb), and wide working ranges (0.1-100 ppm). The CE buffer with 20% (v/v) methanol has demonstrated its capability for identifying anion impurities causing problem in aged tin bath and the use of only 10-fold dilution to produce reliable results for quality assessment in plating bath containing high surfactant additives. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase.

PubMed

Li, Yuwei; Yang, Xiaolan; Wang, Deqiang; Hu, Xiaolei; Yuan, Mei; Pu, Jun; Zhan, Chang-Guo; Yang, Zhaoyong; Liao, Fei

2016-08-01

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t 0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t 0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t 0.5 of PAAS was ~14 days in 50 mM Tris-HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.

Shelf-life extension of cod fillets with an acetate buffer spray prior to packaging under modified atmospheres.

PubMed

Boskou, G; Debevere, J

2000-01-01

Fresh cod fillets (Gadus morhua) were sprayed with a 10% acetate buffer (pH 5.6), packed with an industrial gas-flushing packaging machine under modified atmospheres (50% CO2--45% O2--5% N2, 2 cm3/1 g gas/product ratio) and stored at 7 degrees C for 12 days. Control cod fillets were directly packed and stored under the same conditions. A reduction of the aerobic plate counts was observed immediately after the cod fillets had been sprayed. During storage under modified atmospheres, there was complete inhibition of H2S-producing bacteria and Enterobacteriaceae in the treated cod fillets. Production of total volatile bases and trimethylamine (TMA) was inhibited in treated fillets for 10 days' storage under modified atmospheres. Inhibition of TMA production can be attributed to growth inhibition of H2S-producing bacteria, inhibition of the trimethylamine oxide (TMAO)-dependent metabolism of TMAO-reducing bacteria and the stable pH during storage. The shelf-life, at 7 degrees C, of treated cod fillets, based on cooked flavour score, was almost 12 days, ca 8 days more than shelf-life of the control fillets.

Capillary-Channeled Polymer (C-CP) Fibers as a Stationary Phase for Sample Clean-Up of Protein Solutions for Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Manard, Benjamin T.; Marcus, R. Kenneth

2012-08-01

Capillary-channeled polymer (C-CP) fibers are employed in a micropipette tip format to affect a stationary phase for the solid phase extraction (SPE) of proteins from buffer solutions prior to MALDI-MS analysis. Proteins readily adsorb to the polypropylene (PP) C-CP fibers while buffer species are easily washed off the tips using DI-H2O. Elution of the solutes is achieved with an aliquot of 50:50 ACN:H2O, which is compatible with the subsequent spotting on the MALDI target with the matrix solution. Lysozyme and cytochrome c are used as test species, with a primary buffer composition of 100 mM Tris-HCl. In this case, direct MALDI-MS produces no discernible protein signals. SPE on the C-CP fibers yields high fidelity mass spectra for 1 μL sample volumes. Limits of detection for cytochrome c in 100 mM Tris-HCl are on the order of 40 nM. Extraction of cytochrome c from buffer concentrations of up to 1 M Tris-HCl, provides signal recoveries that are suppressed by only ~50 % versus neat protein solutions. Finally, extraction of 3.1 μM cytochrome c from a synthetic urine matrix exhibits excellent recovery.

Hormetic effect of ionic liquid 1-ethyl-3-methylimidazolium acetate on bacteria

DOE PAGES

Nancharaiah, Y. V.; Francis, A. J.

2015-02-19

The biological effect of ionic liquids (ILs) is one of the highly debated topics as they are being contemplated for various industrial applications. 1-ethyl-2-methylimidazolium acetate ([EMIM][Ac]) showed remarkable hormesis on anaerobic Clostridium sp. and aerobic Psueudomonas putida. Bacterial growth was stimulated at up to 2.5 g L -1 and inhibited at > 2.5 g L -1 of ([EMIM][Ac]). The growth of Clostridium sp. and P. putida were higher by 0.4 and 4-fold respectively, in the presense of 0.5 g L -1 of ([EMIM][Ac]). Assessment of the effect of [EMIM][Ac] under different growth conditions showed that the hormesis of [EMIM][Ac] wasmore » mediated via regulation of medium pH. Hormetic effect of [EMIM][Ac] was evident only in medium with poor buffering capacity and in the presence of a fermentable substrate as the carbon source. The hormetic effect of [EMIM][Ac] on bacterial growth is most likely associated with the buffering capacity of acetate anion. These observations have implications in ILs toxicity studies and ecological risk assessment.« less

Low-Level Effects of VX Vapor Exposure on Pupil Size and Cholinesterase Levels in Rats

DTIC Science & Technology

2005-03-01

Longwood, FL) along with 25 tL of 2.08 mM sodium lauryl sulfate in a pH 7.2 phosphate buffer (30 mM). The plate was read at 536 nm and 37°C using a...Hemoglobin Determination by Using Sodium Lauryl Sulfate (SLS). Clin. Biochem. 15(1) 83-88 (1982). Prins, J., "Product and Process Comparison," Chapter 7...standard VX-G were eluted with I mL ethyl acetate that was collected and dried over anhydrous sodium sulfate . The ethyl acetate was removed from the

Use of the short-term inflammatory response in the mouse peritoneal cavity to assess the biological activity of leached vitreous fibers.

PubMed Central

Donaldson, K; Addison, J; Miller, B G; Cullen, R T; Davis, J M

1994-01-01

We used a special-purpose glass microfiber sample, Johns-Manville Code 100/475, to study the effects of various acid and alkali treatments on biological activity as assessed by inflammation in the mouse peritoneal cavity, the leaching of Si, and the phase contrast optical microscopy (PCOM) fiber number. We used mild and medium treatments with oxalic acid and Tris buffer and harsh treatment with concentrated HCl and NaOH. Mild oxalic acid and Tris treatment for 2 weeks had no effect on any of the end-points, but prolonging the mild oxalic acid treatment time to 2 months reduced the biological activity and the fiber number. Medium oxalic acid treatment reduced the biological activity and the fiber number and caused a loss of Si. Medium Tris alkali treatment reduced the PCOM-countable fibers and the biological activity but did not cause a substantial loss of Si. Harsh treatment with strong HCl did not affect the fiber number or cause leaching but the biological activity was reduced; strong NaOH reduced the fiber number and biological activity, and caused marked leaching of Si. The medium oxalic acid conditions (pH 1.4) were more acid than those found in lung cells but produced the same effects (reduction in fiber number and biological activity) as the more physiological mild treatment (pH 4.0), when prolonged. This study suggests that medium oxalic acid treatment can be used as a short-term assay to compare loss of Si, reduction in fiber number, and change in biological activity of vitreous fibers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7882922

Application and comparison of high performance liquid chromatography and high speed counter-current chromatography in enantioseparation of (±)-2-phenylpropionic acid.

PubMed

Tong, Shengqiang; Zheng, Ye; Yan, Jizhong

2013-03-15

High performance liquid chromatography (HPLC) and high speed counter-current chromatography (HSCCC) were applied and compared in enantioseparation of 2-phenylpropionic acid (2-PPA) when hydroxypropyl-β-cyclodextrin (HP-β-CD) was used as chiral mobile phase additive. For HPLC, the enantioseparation was achieved on ODS C(18) reverse phase column and the mobile phase was 25 mmol L(-1) HP-β-CD aqueous buffer solution (pH 4.0, adjusted with triethylamine): methanol: glacial acetic acid (85:15:0.5 (v/v/v)). For HSCCC, the two-phase solvent system was composed of n-hexane-ethyl acetate-0.1 mol L(-1) phosphate buffer solution pH2.67 (5:5:10 for isocratic elution and 8:2:10 for recycling elution (v/v/v)) added with 0.1 mol L(-1) HP-β-CD. The key parameters, such as a substitution degree of HP-β-CD, the concentration of HP-β-CD, pH value of the aqueous phase and the temperature were optimized for both separation methods. Using the optimum conditions a complete HSCCC enantioseparation of 40 mg of 2-propylpropionic acid in a recycling elution mode gave 15-18 mg of (+)-2-PPA and (-)-2-PPA enantiomers with 95-98% purity and 85-93% recovery. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection.

PubMed

Solínová, Veronika; Kasicka, Václav; Koval, Dusan; Barth, Tomislav; Ciencialová, Alice; Záková, Lenka

2004-08-25

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).

Corrosion and stability study of Bacillus thuringiensis var. kurstaki starch industry wastewater-derived biopesticide formulation.

PubMed

Gnepe, Jean R; Tyagi, Rajeshwar D; Brar, Satinder K; Valéro, José R; Surampalli, Rao Y

2014-01-01

Biopesticides are usually sprayed on forests by using planes made up of aluminum alloy. Bioval derived from starch industry wastewater (SIW) in suspension form was developed as stable anticorrosive biopesticide formulation. In this context, various anticorrosion agents such as activated charcoal, glycerin, ethylene glycol, phytic acid, castor oil and potassium silicate were tested as anticorrosive agents. There was no corrosion found in Bioval formulation where potassium silicate (0.5% w/v) was added and compared with Foray 76 B, as an industrial standard, when stored over 6 months. In relation to other parameters, the anticorrosion formulation of Bioval+buffer+KSi reported excellent zeta potential (-33.19 ± 4 mV) and the viscosity (319.13 ± 32 mPa.s) proving it's stability over 6 months, compared to the standard biopesticide Foray 76 B (-36.62 ± 4 mV potential zeta, pH 4.14 ± 0.1 and 206 ± 21 mPa.s viscosity). Metal analysis of the different biopesticides showed that Bioval+buffer+KSi has no corrosion (5.11 ± 0.5 mg kg(-1) of Al and 13.53 ± 1.5 mg kg(-1) of Fe) on the aluminum alloy due to the contribution of sodium acetate buffer at pH 5. The bioassays reported excellent results for Bioval+Buffer+KSi (2.95 ± 0.3 × 10(9) CFU mL(-1) spores and 26.6 ± 2.7 × 10(9) IU L(-1) Tx) compared with initial Bioval (2.46 ± 0.3 × 10(9) CFU mL(-1) spores and 23.09 ± 3 × 10(9) IU L(-1) Tx) and Foray 76 B (2.3 ± 0.2 × 10(9) CFU mL(-1) spores and 19.950 ± 2.1 UI L(-1) Tx) which was due to the break-up of the external chitinous membrane due to abrasive action of potassium silicate after ingestion by insects. The contribution of sodium acetate buffer and potassium silicate (0.5% and at pH = 5) as anticorrosion agent in the Bioval allowed production of an efficient biopesticide with a reduced viscosity and favorable pH as compared to Foray 76 B which enhanced the entomotoxic potential against spruce budworm (SB) larvae (Lepidoptera: Choristoneura fumiferana).

Tested Demonstrations: Buffer Capacity of Various Acetic Acid-Sodium Acetate Systems: A Lecture Experiment.

ERIC Educational Resources Information Center

Donahue, Craig J.; Panek, Mary G.

1985-01-01

Background information and procedures are provided for a lecture experiment which uses indicators to illustrate the concept of differing buffer capacities by titrating acetic acid/sodium acetate buffers with 1.0 molar hydrochloric acid and 1.0 molar sodium hydroxide. A table with data used to plot the titration curve is included. (JN)

The effect of pH control and 'hydraulic flush' on hydrolysis and Volatile Fatty Acids (VFA) production and profile in anaerobic leach bed reactors digesting a high solids content substrate.

PubMed

Cysneiros, Denise; Banks, Charles J; Heaven, Sonia; Karatzas, Kimon-Andreas G

2012-11-01

The effect of hydraulic flush and pH control on hydrolysis, Volatile Fatty Acids (VFA) production and profile in anaerobic leach bed reactors was investigated for the first time. Six reactors were operated under different regimes for two consecutive batches of 28days each. Buffering at pH ∼6.5 improved hydrolysis (Volatile Solid (VS) degradation) and VFA production by ∼50%. Butyric and acetic acid were dominant when reactors were buffered, while only butyric acid was produced at low pH. Hydraulic flush enhanced VS degradation and VFA production by ∼15% and ∼32%, respectively. Most Probable Number (MPN) of cellulolytic microorganisms indicated a wash out when hydraulic flush was applied, but pH control helped to counteract this. The highest VS degradation (∼89%), VFA yield (0.84kgCODkg(-1)VS(added)) and theoretical methane potential (0.37m(3)CH(4)kg(-1)VS(added)) were obtained when pH control and hydraulic flush were applied, and therefore, these conditions are recommended. Copyright © 2012 Elsevier Ltd. All rights reserved.

Purification and Characterization of [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Liang; Belchik, Sara M.; Plymale, Andrew E.

2011-08-02

The γ-proteobacterium Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that was implicated in both H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned into a protein expression vector. The resulting plasmid was transformed into a MR-1 mutant deficient in H2 formation. Expression of MR-1 [NiFe]-H2ase in trans restored the mutant’s ability to produce H2 at 37% of that for wild type. Following expression, MR-1 [NiFe]-H2ase was purified to near homogeneity. The purified MR-1 [NiFe]-H2ase could couplemore » H2 oxidation to reduction of Tc(VII) and methyl viologen directly. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated Tc(VII) but not methyl viologen reductions. Under the conditions tested, Tc(VII) reduction was complete in Tris buffer but not in HEPES buffer. The reduced Tc(IV) was soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc(IV) precipitates formed in HEPES buffer were packed with crystallites. Although X-ray absorption near-edge spectroscopy measurements confirmed that the reduction products found in both buffers were Tc(IV), extended X-ray adsorption fine-structure measurements revealed that these products were very different. While the product in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O2•nH2O. These results shows for the first time that MR-1 [NiFe]-H2ase is a bidirectional enzyme that catalyzes both H2 formation and oxidation as well as Tc(VII) reduction directly by coupling H2 oxidation.« less

Effect of buffer at nanoscale molecular recognition interfaces - electrostatic binding of biological polyanions.

PubMed

Rodrigo, Ana C; Laurini, Erik; Vieira, Vânia M P; Pricl, Sabrina; Smith, David K

2017-10-19

We investigate the impact of an over-looked component on molecular recognition in water-buffer. The binding of a cationic dye to biological polyanion heparin is shown by isothermal calorimetry to depend on buffer (Tris-HCl > HEPES > PBS). The heparin binding of self-assembled multivalent (SAMul) cationic micelles is even more buffer dependent. Multivalent electrostatic molecular recognition is buffer dependent as a result of competitive interactions between the cationic binding interface and anions present in the buffer.

Determination of carmine food dye (E120) in foodstuffs by stripping voltammetry.

PubMed

Alghamdi, Ahmad H; Alshammery, Hamed M; Abdalla, Mohamed A; Alghamdi, Ali F

2009-01-01

The behavior of the food colorant agent carmine (E120) was studied by square-wave adsorptive stripping voltammetry (SW-AdSV) at the hanging mercury drop electrode. It was observed that carmine gave a sensitive stripping voltammetric peak at -350 mV in pH 3 acetate buffer. The cyclic voltammetric technique was also used to characterize the electrochemical reduction process of carmine. The adsorptive voltammetric signal was evaluated with respect to various experimental conditions, and the optimized values were supporting electrolyte, acetate buffer; buffer acidity, pH 3; dye concentration, 3 x 10(-7) M; accumulation time, 150 s; accumulation potential, -0.2 V; scan rate, 300 mV/s; pulse amplitude, 185 mV; SW frequency, 20 Hz; working electrode area, 0.6 mm2; and convection rate, 2600 rpm. The SW-AdSV peak currents depended linearly on the concentration of carmine from 5 x 10(-8) to 1.25 x 10(-7) mol/L (r = 0.99). A detection limit of 1.43 x 10(-9) mol/L with an RSD of 2.2% and a mean recovery of 97.9% were obtained. Possible interferences by several substances usually present in food products such as food additive dyes (E102, E100, E123, E127, and E129), artificial sweeteners, preservatives, and antioxidants were also evaluated. The proposed electrochemical procedure was successfully applied to the determination of carmine food dye in spiked commercially available ice cream and soft drinks.

Comparison of phosphorus forms in three extracts of dairy feces by solution 31P NMR analysis

USDA-ARS?s Scientific Manuscript database

Using solution 31P NMR spectroscopy, we compared three extractants, deionized water, sodium acetate buffer (pH 5.0) with fresh sodium dithionite (NaAc-SD), and 0.25 M NaOH-0.05 M EDTA (NaOH-EDTA), for the profile of P compounds in two dairy fecal samples. Phosphorus extracted was 35% for water, and...

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Apparent electric charge of protein molecules. Human thyroxine - binding proteins.

PubMed

Hocman, G; Sadlon, J

1977-01-01

1. By comparison of electrophoretic mobilities of two different charged particles under the same conditions the net elementary electrostatic charge of one particle could be calculated when the charge of the other is known. 2. The electrophoretic mobility of human thyroxine - binding globulin does not depend upon the concentration of Tris - HCl buffer in the range 0.05 to 0.20 molar. The value of this mobility is 0.078 and 0.083 cm2 vol(-1) hour(-1) at pH 7.0 and 8.6, respectively. 3. The net elementary electrostatic charge of the human thyroxine - binding globulin appears to be approximately 22 negative elementary electrostatic units in mild alkaline solutions.

Metal ion promoted hydrogels for bovine serum albumin adsorption: Cu(II) and Co(II) chelated poly[(N-vinylimidazole)-maleic acid].

PubMed

Pekel, Nursel; Salih, Bekir; Güven, Olgun

2005-05-10

Poly[(N-vinylimidazole)-maleic acid] (poly(VIm-MA)), copolymeric hydrogels were prepared by gamma-irradiating ternary mixtures of N-vinylimidazole-maleic acid-water in a (60)Co-gamma source. Cu(II) and Co(II) ions were chelated within the gels at pH=5.0. The maximum adsorption capacity of the gels were 3.71 mmol/g dry gel for Cu(II) and 1.25 mmol/g dry gel for Co(II) at pH=5.0. The swelling ratios of the gels were 1200% for poly(VIm-MA), 60 and 45% for Cu(II) and Co(II)-chelated poly(VIm-MA) gels at pH=5.0 in acetate buffer solution. These affinity gels with different swelling ratios for plain poly(VIm-MA), Cu(II)-, and Co(II)-chelated poly(VIm-MA), in acetate and phosphate buffers were used in the bovine serum albumin (BSA) adsorption/desorption studies in batch reactor. The maximum BSA adsorption capacities of the gels were 0.38 g/g dry gel for plain, 0.88 g/g dry gel for Cu(II)-chelated poly(VIm-MA) and 1.05 g/g dry gel for Co(II)-chelated poly(VIm-MA) gels. Adsorption capacity of BSA by the gels was reduced dramatically by increasing the ionic strength adjusted with NaCl. More than 95% of BSA were desorbed in 10 h in desorption medium containing 0.1M of EDTA for metal ion-chelated gels at pH=4.7.

Calcium manganese(IV) oxides: biomimetic and efficient catalysts for water oxidation.

PubMed

Najafpour, Mohammad Mahdi; Pashaei, Babak; Nayeri, Sara

2012-04-28

CaMnO(3) and Ca(2)Mn(3)O(8) were synthesized and characterized by SEM, XRD, FTIR and BET. Both oxides showed oxygen evolution activity in the presence of oxone, cerium(IV) ammonium nitrate and H(2)O(2). Oxygen evolution from water during irradiation with visible light (λ > 400 nm) was also observed upon adding these manganese oxides to an aqueous solution containing tris(2,2'-bipyridyl) ruthenium(II), as photosensitizer, and chloro pentaammine cobalt(III) chloride, as electron acceptor, in an acetate buffer. The amounts of dissolved manganese and calcium from CaMnO(3) and Ca(2)Mn(3)O(8) in the oxygen evolving reactions were reported and compared with other (calcium) manganese oxides. Proposed mechanisms of oxygen evolution and proposed roles for the calcium ions are also considered. This journal is © The Royal Society of Chemistry 2012

CSE-MECC two-dimensional capillary electrophoresis analysis of proteins in the mouse tumor cell (AtT-20) homogenate

PubMed Central

Chen, Xingguo; Fazal, Md. Abul; Dovichi, Norman J.

2007-01-01

Two-dimensional capillary electrophoresis was used for the separation of proteins and biogenic amines from the mouse AtT-20 cell line. The first-dimension capillary contained a TRIS-CHES-SDS-dextran buffer to perform capillary sieving electrophoresis, which is based on molecular weight of proteins. The second-dimension capillary contained a TRIS-CHES-SDS buffer for micel1ar electrokinetic capillary chromatography. After a 61 seconds preliminary separation, fractions from the first-dimension capillary were successively transferred to the second-dimension capillary, where they further separated by MECC. The two-dimensional separation required 60 minutes. PMID:17637850

Sodium Is Not Essential for High Bioactivity of Glasses

PubMed Central

Chen, Xiaojing; Chen, Xiaohui; Brauer, Delia S.; Wilson, Rory M.; Law, Robert V.; Hill, Robert G.; Karpukhina, Natalia

2017-01-01

This study aims to demonstrate that excellent bioactivity of glass can be achieved without the presence of an alkali metal component in glass composition. In vitro bioactivity of two sodium-free glasses based on the quaternary system SiO2-P2O5-CaO-CaF2 with 0 and 4.5 mol% CaF2 content was investigated and compared with the sodium containing glasses with equivalent amount of CaF2. The formation of apatite after immersion in Tris buffer was followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), 31P and 19F solid state MAS-NMR. The dissolution study was completed by ion release measurements in Tris buffer. The results show that sodium free bioactive glasses formed apatite at 3 hours of immersion in Tris buffer, which is as fast as the corresponding sodium containing composition. This signifies that sodium is not an essential component in bioactive glasses and it is possible to make equally degradable bioactive glasses with or without sodium. The results presented here also emphasize the central role of the glass compositions design which is based on understanding of structural role of components and/or predicting the network connectivity of glasses. PMID:29271977

Sodium Is Not Essential for High Bioactivity of Glasses.

PubMed

Chen, Xiaojing; Chen, Xiaohui; Brauer, Delia S; Wilson, Rory M; Law, Robert V; Hill, Robert G; Karpukhina, Natalia

2017-12-01

This study aims to demonstrate that excellent bioactivity of glass can be achieved without the presence of an alkali metal component in glass composition. In vitro bioactivity of two sodium-free glasses based on the quaternary system SiO 2 -P 2 O 5 -CaO-CaF 2 with 0 and 4.5 mol% CaF 2 content was investigated and compared with the sodium containing glasses with equivalent amount of CaF 2 . The formation of apatite after immersion in Tris buffer was followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), 31 P and 19 F solid state MAS-NMR. The dissolution study was completed by ion release measurements in Tris buffer. The results show that sodium free bioactive glasses formed apatite at 3 hours of immersion in Tris buffer, which is as fast as the corresponding sodium containing composition. This signifies that sodium is not an essential component in bioactive glasses and it is possible to make equally degradable bioactive glasses with or without sodium. The results presented here also emphasize the central role of the glass compositions design which is based on understanding of structural role of components and/or predicting the network connectivity of glasses.

Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

PubMed Central

Kirkpatrick, Christopher; Maurer, Lisa M.; Oyelakin, Nikki E.; Yoncheva, Yuliya N.; Maurer, Russell; Slonczewski, Joan L.

2001-01-01

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins. PMID:11591692

Micro-apparatus for rapid determinations of protein solubilities

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Munson, Sibyl

1991-01-01

We have developed a column-based micro-technique for rapid determinations of protein solubilities. While retaining a large crystal surface area, the column dead volume has been reduced to equal to or less than 5 micro liters. The technique was tested with tetragonal lysozyme (pH 4.5, 0.1 M acetate, 3.0 percent NaCl, 5-25 C) and column volumes of about 60, 300, and 900 micro liters. Identical solubility data were obtained, indicating that equilibration was obtained even in the smallest columns. In addition, solubility data for Br- and I- salts of lysozyme (pH 4.5, 0.1 M acetate buffer, 0.5 M salt concentrations) were obtained. It appears that the technique can be further miniaturized. The limit in further reducing the crystalline column volume is determined by the minimum solution sample size needed to determine the protein concentration.

[Study on preparation of sagittatoside B with epimedin B converted from cellulase].

PubMed

Xu, Feng-Juan; Sun, E; Zhang, Zhen-Hai; Cui, Li; Jia, Xiao-Bin

2014-01-01

To prepare sagittatoside B with epimedin B Hydrolyzed from cellulase. With the conversion ratio as the index, the effects of pH value, temperature, reaction time, dosage of enzyme and concentration of substrates on the conversion ratio were detected. L9 (3(4)) orthogonal design was adopted to optimize the preparation process. Hydrolyzed products were identified by MS, 1H-NMR, and 13C-NMR. The results showed that the optimum reaction conditions for the enzymatic hydrolysis were that the temperature was 50 degrees C, the reaction medium was pH 5.6 acetic acid-sodium acetate buffer solution, the concentration of substrates was 20 g x L(-1), the mass ratio between enzyme and substrate was 3: 5, and the relative molecular mass of the reaction product was 646.23. NMR data proved that the product was sagittatoside B. The process is simple and reliable under mild reaction conditions, thus suitable for industrial production.

DNA adsorption onto glass surfaces

NASA Astrophysics Data System (ADS)

Carlson, Krista Lynn

Streaming potential measurements were performed on microspheres of silica, lime silicate (SLS) and calcium aluminate (CA) glasses containing silica and iron oxide (CASi and CAFe). The silicate based glasses exhibited acidic surfaces with isoelectric points (IEP) around a pH of 3 while the calcium aluminates displayed more basic surfaces with IEP ranging from 8--9.5. The surface of the calcium aluminate microspheres containing silica reacted with the background electrolyte, altering the measured zeta potential values and inhibiting electrolyte flow past the sample at ˜ pH 4 due to formation of a solid plug. DNA adsorption experiments were performed using the microspheres and a commercially available silicate based DNA isolation filter using a known quantity of DNA suspended in a chaotropic agent free 0.35 wt% Tris(hydroxymethyl)aminomethane (Tris) buffer solution. The microspheres and commercial filter were also used to isolate DNA from macrophage cells in the presence of chaotropic agents. UV absorbance at ˜260 nm and gel electrophoresis were used to quantify the amount and size of the DNA strands that adsorbed to the microsphere surfaces. In both experiments, the 43--106 microm CAFe microspheres adsorbed the largest quantity of DNA. However, the 43--106 microm SLS microspheres isolated more DNA from the cells than the <43 microm CAFe microspheres, indicating that microsphere size contributes to isolation ability. The UV absorbance of DNA at ˜260 nm was slightly altered due to the dissolution of the calcium aluminate glasses during the adsorption process. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) determined that calcium and aluminum ions leached from the CA and CAFe microsphere surfaces during these experiments. Circular dichroism (CD) spectroscopy showed that the leached ions had no effect on the conformation of the DNA, and therefore would not be expected to interfere in downstream applications such as DNA replication. The 0.35 wt% Tris solution completely inhibited the formation of the hydrated crystalline layer that develops when the calcium aluminate glassess are incubated in deionized water. A Tris concentration of 0.24 wt% allowed for the formation of both hexagonal and cubic hydrates, however they were severely distorted and present in low amounts such that they were undectable by XRD.

2D spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism.

PubMed

Wang, Xin; Ando, Eri; Takahashi, Daishi; Arakawa, Takahiro; Kudo, Hiroyuki; Saito, Hirokazu; Mitsubayashi, Kohji

2010-08-15

A novel 2-dimensional spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism has been developed, which employed a low level light CCD camera to detect chemiluminescence (CL) generated by catalytic reactions of standard gaseous ethanol and expired gaseous ethanol after oral administration. First, the optimization of the substrates for visualization and the concentration of luminol solution for CL were investigated. The cotton mesh and 5.0 mmol L(-1) luminol solution were selected for further investigations and this system is useful for 0.1-20.0 mmol L(-1) of H(2)O(2) solution. Then, the effect of pH condition of Tris-HCl buffer solution was also evaluated with CL intensity and under the Tris-HCl buffer solution pH 10.1, a wide calibration range of standard gaseous ethanol (30-400 ppm) was obtained. Finally, expired air of 5 healthy volunteers after oral administration was measured at 15, 30, 45, 60, 75, 90, 105 and 120 min after oral administration, and this system showed a good sensitivity on expired gaseous ethanol for alcohol metabolism. The peaks of expired gaseous ethanol concentration appeared within 30 min after oral administration. During the 30 min after oral administration, the time variation profile based on mean values showed the absorption and distribution function, and the values onward showed the elimination function. The absorption and distribution of expired gaseous ethanol in 5 healthy volunteers following first-order absorption process were faster than the elimination process, which proves efficacious of this system for described alcohol metabolism in healthy volunteers. This system is expected to be used as a non-invasive method to detect VOCs as well as several other drugs in expired air for clinical purpose. Copyright 2010 Elsevier B.V. All rights reserved.

Effects of bran pre-hydration on functional characteristics and bread baking quality of bran and flour blends

USDA-ARS?s Scientific Manuscript database

The effect of bran pre-hydration on the composition and bread baking quality was determined using bran and flour of two wheat varieties. Bran was hydrated in sodium acetate buffer (50 mM, pH 5.3) to 50% moisture at 25 or 55°C for 1.5 or 12 h. The soluble sugar content in bran increased with pre-hydr...

Voltammetric analysis of N-containing drugs using the hanging galinstan drop electrode (HGDE).

PubMed

Channaa, H; Surmann, P

2009-03-01

The electrochemical behaviour of several N-containing voltammetric active drugs such as 1,4-benzodiazepines (chlordiazepoxide, nitrazepam and diazepam) as well as one nitro-compound (nitrofurantoin) and one azo-compound (phenazopyridine) is described using a new kind of liquid electrode, the hanging galinstan drop electrode. Concentrations of 10(-5) - 10(-8) mol L(-1) are generally measurable. Differential pulse and adsorptive stripping voltammograms are recorded in different supporting electrolytes, like 0.1 M KNO3, acetate buffer solution pH = 4.6 and phosphate buffer solution pH = 7.0. The effects of varying the starting potentials, U(start) for DPV and accumulation times, t(acc) for AdSV are considered. Briefly, it is shown that the novel galinstan electrode is suitable for reducing several functional groups in organic substances, here presented for N-oxide-, azomethine-, nitro- and azo-groups.

Conditions affecting transformation of a group H streptococcus.

PubMed

Schlissel, H J; Sword, C P

1966-11-01

Schlissel, Harvey J. (The University of Kansas, Lawrence), and C. P. Sword. Conditions affecting transformation of a group H streptococcus. J. Bacteriol. 92:1357-1363. 1966.-A defined transforming medium (DTM) containing buffer and 5 to 10 mug per ml of deoxyribonucleic acid was developed to study the physical and chemical requirements for optimal transformation in streptococcal strain SBE. Optimal transformation in DTM occurred at pH 7.5 and 7.0 in 0.07 m sodium phosphate buffer and 0.05 m tris(hydroxymethyl)aminomethane buffer, respectively. In the presence of either a monovalent or a divalent cation, transformation was stimulated maximally by Mn(+2) (10(-3)m) and K(+) (0.05 m). Other cations tested (Na(+), Mg(+2), Ca(+2)) were less stimulatory. A mixture of K(+) and Mn(+2) stimulated transformation to a level higher than either cation alone. Kinetic studies showed that the stimulating effect of cations was greatest during the early part of the transformation reaction and decreased with time. Transformation was inhibited by Cu(+2) (10(-5)m) and Mn(+2) (10(-2)m). Ethylenediaminetetraacetic acid (EDTA) inhibited transformation at 10(-5)m. The inhibition by EDTA could be overcome by Mn(+2) during the early part of the transformation reaction.

Efficient extraction of vaccines formulated in aluminum hydroxide gel by including surfactants in the extraction buffer

PubMed Central

Zhu, Daming; Huang, Shuhui; McClellan, Holly; Dai, Weili; Syed, Najam R; Gebregeorgis, Elizabeth; Mullen, Gregory E. D.; Long, Carole; Martin, Laura B.; Narum, David; Duffy, Patrick; Miller, Louis H.; Saul, Allan

2011-01-01

Efficient antigen extraction from vaccines formulated on aluminum hydroxide gels is a critical step for the evaluation of the quality of vaccines following formulation. It has been shown in our laboratory that the efficiency of antigen extraction from vaccines formulated on Alhydrogel decreased significantly with increased storage time. To increase antigen extraction efficiency, the present study determined the effect of surfactants on antigen recovery from vaccine formulations. The Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated on Alhydrogel and stored at 2-8 °C for three years was used as a model in this study. The AMA1 on Alhydrogel was extracted in the presence or absence of 30 mM sodium dodecyl sulfate (SDS) or 20 mM cetylpyridinium chloride in the extraction buffer (0.60 M citrate, 0.55 M phosphate, pH 8.5) using our standard antigen extraction protocols. Extracted AMA1 antigen was analyzed by 4-20% Tris-glycine SDS-PAGE followed by silver staining or western blotting. The results showed that inclusion of SDS or cetylpyridinium chloride in extraction buffer increased the antigen recovery dramatically and can be used for efficient characterization of Alhydrogel vaccines. PMID:22107848

Membrane optode for uranium(VI) ions preconcentration and quantification based on a synergistic combination of 4-(2-thiazolylazo)-resorcinol with 8-hydroxyquinaldine

NASA Astrophysics Data System (ADS)

Kalyan, Y.; Pandey, A. K.; Naidu, G. R. K.; Reddy, A. V. R.

2009-12-01

A membrane optode was developed utilizing the 8-hydroxyquinaldine (HQ) facilitated preconcentration of UO 22+ ions and subsequent colored complex formation of UO 22+ with 4-(2-thiazolylazo)-resorcinol (TAR) in optode matrix. The composition of the membrane optode was optimized by scanning several extractants immobilized in different plasticized polymer matrices. It was observed that the chelating agent HQ along with an indicator TAR immobilized in the tri-(2-ethylhexyl)phosphate (TEHP) plasticized cellulose triacetate matrix (CTA) was best suited as an optode for the UO 22+ ions in aqueous samples. On sorption of UO 22+ in the optode matrix, TAR changes color of the optode from yellow to magenta having a maximum absorbance ( λmax) at 546 nm. The uptake of UO 22+ ions in the optode was found to be pH dependent and was maximum (>90%) at pH above 3. The acetate buffer (0.1 mol L -1 sodium acetate + 0.1 mol L -1 acetic acid) was found to be necessary for the stable response. The optimum equilibration time for the optode (2 cm × 1 cm) was found to be 30 min in 10 mL aqueous sample containing acetate buffer (pH 4.75). The equilibration time was found to increase with increase in aqueous sample volume. The optode response was found to be linear in the UO 22+ ions concentration range of 0.01-0.11 μmol L -1 in tap water as well as aqueous solutions containing 0.1 mol L -1 NaCl or NaNO 3. The tolerance to the presence of several cations and anions in the determination of UO 22+ ion was studied. It was observed that the optode in the presence of buffer can tolerate presence of large amounts of interfering cations (Ce 4+, V 4+, Eu 3+, Al 3+, Fe 3+, Ni 2+, Cd 2+, Co 2+, Pb 2+, Hg 2+, Cu 2+ and Th 4+ ions) without hindering the sorption of UO 22+ ions in the optode matrix. The present work indicated that 50 ppb UO 22+ ions in 100 mL sample can easily be quantified using this optode. The optode was found to be fully reversible, can readily be regenerated by equilibrating it with 0.1 mol L -1 HNO 3 and reusable up to three cycles. The applicability of the developed optode in real samples was studied by determining uranium in the ground water samples spiked with a known quantity of UO 22+ ions.

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen.

PubMed

Nawaz, Saima; Mullen, Gregory E D; Sunassee, Kavitha; Bordoloi, Jayanta; Blower, Philip J; Ballinger, James R

2017-10-25

Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68 Ge/ 68 Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4-6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 μg conjugate with gallium-68 for 5 min without post-labelling purification. 68 Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa.

Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

PubMed

Jandaruang, Jinda; Siritapetawee, Jaruwan; Songsiriritthigul, Chomphunuch; Preecharram, Sutthidech; Azuma, Taoka; Dhiravisit, Apisak; Fukumori, Yoshihiro; Thammasirirak, Sompong

2014-08-01

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.

Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.

2012-07-11

The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB{sub BR}) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB{sub BR} buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB{sub BR} in each buffer. While both sets of conditions supported crystal growth in space group P2{sub 1}2{sub 1}2{sub 1}, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 {angstrom} formore » crystal form 1 and a = 34.6, b = 98.3, c = 99.0 {angstrom} for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 {angstrom} resolution. A complete data set has been collected to 1.3 {angstrom} resolution with an overall R{sub merge} value of 0.04 and an R{sub merge} value of 0.33 in the highest resolution shell.« less

Low Molecular Weight Chitosan–Insulin Polyelectrolyte Complex: Characterization and Stability Studies

PubMed Central

Al-Kurdi, Zakieh I.; Chowdhry, Babur Z.; Leharne, Stephen A.; Al Omari, Mahmoud M. H.; Badwan, Adnan A.

2015-01-01

The aim of the work reported herein was to investigate the effect of various low molecular weight chitosans (LMWCs) on the stability of insulin using USP HPLC methods. Insulin was found to be stable in a polyelectrolyte complex (PEC) consisting of insulin and LMWC in the presence of a Tris-buffer at pH 6.5. In the presence of LMWC, the stability of insulin increased with decreasing molecular weight of LMWC; 13 kDa LMWC was the most efficient molecular weight for enhancing the physical and chemical stability of insulin. Solubilization of insulin-LMWC polyelectrolyte complex (I-LMWC PEC) in a reverse micelle (RM) system, administered to diabetic rats, results in an oral delivery system for insulin with acceptable bioactivity. PMID:25830681

Resonance light scattering technique for the determination of protein with rutin and cetylpyridine bromide system.

PubMed

Liu, Yang; Yang, Jinghe; Liu, Shufang; Wu, Xia; Su, Benyu; Wu, Tao

2005-02-01

A new resonance light scattering (RLS) assay of protein is presented. In Tris-NaOH (pH = 10.93) buffer, the RLS of rutin-cetylpyridine bromide (CPB) system can be greatly enhanced by protein, including bovine serum albumin (BSA) and human serum albumin (HSA). The enhanced RLS intensities are in proportion to the concentration of proteins in the range of 5 x 10(-9) to 2.5 x 10(-6) g ml(-1) for BSA and 2.5 x 10(-8) to 3.5 x 10(-6) g ml(-1) for HSA. The detection limits (S/N = 3) are 3.0 ng ml(-1) for BSA and 10.0 ng ml(-1) for HSA. Samples are determined satisfactorily.

Effects of organic acids on thermal inactivation of acid and cold stressed Enterococcus faecium.

PubMed

Fernández, Ana; Alvarez-Ordóñez, Avelino; López, Mercedes; Bernardo, Ana

2009-08-01

In this study the adaptative response to heat (70 degrees C) of Enterococcus faecium using fresh and refrigerated (at 4 degrees C for up to 1 month) stationary phase cells grown in Brain Heart Infusion (BHI) buffered at pH 7.4 (non-acid-adapted cells) and acidified BHI at pH values of 6.4 and 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells) was evaluated. In all cases, the survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetic. The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, whereas the subsequent cold storage of cells reduced E. faecium thermal tolerance. Fresh acid-adapted cells showed t(2.5)-values (time needed to obtain an inactivation level of 2.5 log10 cycles) ranging from 2.57 to 9.51 min, while non-acid-adapted cells showed t(2.5)-values of 1.92 min. The extent of increased heat tolerance varied with the acid examined, resulting in the following order: citric > or = acetic > malic > or = lactic > hydrochloric > or = ascorbic. In contrast, cold storage progressively decreased E. faecium thermal resistance. The t(2.5) values found at the end of the period studied were about 2-3-fold lower than those corresponding to non-refrigerated cells, although this decrease was more marked (about 5-fold) when cells were grown in buffered BHI and BHI acidified at pH 5.4 with hydrochloric acid. These findings highlight the need for a better understanding of microbial response to various preservation stresses in order to increase the efficiency of thermal processes and to indicate the convenience of counterbalancing the benefits of the hurdle concept.

Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube.

PubMed

Kan, Hyo; Tsukagoshi, Kazuhiko

2017-07-01

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of individual SCFA on the epithelial barrier of sheep rumen under physiological and acidotic luminal pH conditions.

PubMed

Greco, Gabriele; Hagen, Franziska; Meißner, Svenja; Shen, Zanming; Lu, Zhongyan; Amasheh, Salah; Aschenbach, Jörg R

2018-02-15

The objective of this study was to investigate whether individual short-chain fatty acids (SCFA) have a different potential to either regulate the formation of the ruminal epithelial barrier (REB) at physiological pH or to damage the REB at acidotic ruminal pH. Ruminal epithelia of sheep were incubated in Ussing chambers on their mucosal side in buffered solutions (pH 6.1 or 5.1) containing no SCFA (control), 30 mM of either acetate, propionate or butyrate, or 100 mM acetate. Epithelial conductance (Gt), short-circuit current (Isc), and fluorescein flux rates were measured over 7 h. Thereafter, mRNA and protein abundance, as well as localization of the tight junction proteins claudin (Cldn)-1, -4, -7, and occludin were analyzed. At pH 6.1, butyrate increased Gt and decreased Isc, with additional decreases in claudin-7 mRNA and protein abundance (each P < 0.05) and disappearance of Cldn-7 immunosignals from the stratum corneum. By contrast, the mRNA abundance of Cldn-1 and/or Cldn-4 were upregulated by 30 mM propionate, 30 mM butyrate, or 100 mM acetate (P < 0.05), however, without coordinated changes in protein abundance. At luminal pH 5.1, neither Gt, Isc, nor TJ protein abundance was altered in the absence of SCFA; only fluorescein flux rates were slightly increased (P < 0.05) and fluorescein signals were no longer restricted to the stratum corneum. The presence of acetate, propionate, or butyrate at pH 5.1 increased fluorescein flux rates and Gt, and decreased Isc (each P < 0.05). Protein abundance of Cldn-1 was decreased in all SCFA treatments but 30 mM butyrate; abundance of Cldn -4 and -7 was decreased in all SCFA treatments but 30 mM acetate; and abundance of occludin was decreased in all SCFA treatments but 30 mM propionate (each P < 0.05). Immunofluorescence staining of SCFA-treated tissues at pH 5.1 showed disappearance of Cldn-7, discontinuous pattern for Cldn-4 and blurring of occludin and Cldn-1 signals in tight junction complexes. The fluorescein dye appeared to freely diffuse into deeper cell layers. The strongest increase in Gt and consistent decreases in the abundance and immunosignals of tight junction proteins were observed with 100 mM acetate at pH 5.1. We conclude that SCFA may contribute differently to the REB formation at luminal pH 6.1 with possible detrimental effects of butyrate at 30 mM concentration. At luminal pH 5.1, all SCFA elicited REB damage with concentration appearing more critical than SCFA species.

Method and compositions for detecting of bloodstains using fluorescin-fluorescein reaction

DOEpatents

Di Benedetto, John; Kyle, Kevin; Boan, Terry; Marie, Charlene

2004-02-17

A method, compositions and kit are set forth for detecting blood stains. A reactant solution includes fluorescin solubilized (reduced) in acetic acid in ethanol. The solution may be buffered to a pH of approximately 9. After spraying the reactant solution on the suspected area an oxidizer is applied to promote the fluorescin to fluorescein reaction with the blood. The reacted fluorescein is then detected through luminescence for capture by photography.

Kinetic and Mechanistic Study of the pH-Dependent Activation (Epoxidation) of Prodrug Treosulfan Including the Reaction Inhibition in a Borate Buffer.

PubMed

Romański, Michał; Ratajczak, Whitney; Główka, Franciszek

2017-07-01

A prodrug treosulfan (T) undergoes a pH-dependent activation to epoxide derivatives. The process seems to involve an intramolecular Williamson reaction (IWR) but clear kinetic evidence is lacking. Moreover, a cis-diol system present in the T structure is expected to promote complexation with boric acid. As a result, the prodrug epoxidation would be inhibited; however, this phenomenon has not been investigated. In this article, the effect of pH on the kinetics of T conversion to its monoepoxide was studied from a mechanistic point of view. Also, the influence of boric acid on the reaction kinetics was examined. The rate constants observed for the activation of T (k obs ) in acetate, phosphate, and carbonate buffers satisfied the equation logk obs  = -7.48 + 0.96 pH. The reaction was inhibited in the excess of boric acid over T, and the k obs decreased with increasing borate buffer concentration. The experimental results were consistent with the inhibition model that included the formation of a tetrahedral, anionic T-boric acid monoester. To conclude, in nonborate buffers, the T activation to (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate follows IWR mechanism. A borate buffer changes the reaction kinetics and complicates kinetic analysis. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Biogeochemistry of the sulfur oxidizer Thiomicrospira thermophila

NASA Astrophysics Data System (ADS)

Houghton, J.; Fike, D. A.; Wills, E.; Foustoukos, D.

2013-12-01

Near-seafloor hydrothermal environments such as diffuse flow venting or subsurface mixing are characterized by rapidly changing conditions and steep chemical and thermal gradients. Microorganisms living in these environments can take advantage of these changes by switching among metabolic pathways rather than specializing. We present reaction stoichiometry and rates for T. thermophila grown in a closed system both at ambient and elevated pressure (50 bars) that demonstrate substantial metabolic flexibility, shifting between up to 5 different sulfur cycling reactions over a 24 hour period. Based on the stoichiometry between S2O3 consumed and SO4 produced, three reactions are sulfur oxidation and two are disproportionation, which has not previously been demonstrated for Thiomicrospira strains. Reactants include S2O3, elemental S (both polymeric S chains and S8 rings), HS-, and O2, while products include polymeric elemental S, SO4, HS-, and polysulfides. The presence of μmolal concentrations of HS- has been confirmed during the time series only when stoichiometry predicts disproportionation. Production of HS- in the presence of elemental S results in abiotic conversion to polysulfides, keeping the sulfide concentrations low in solution. The transition from oxidation to disproportionation appears to be triggered by a depletion in dissolved oxygen and the rate of reaction is a second order function of S2O3 and O2 concentrations. Growth was tested at conditions spanning their pH tolerance (5.0 - 8.0) using a citrate buffer (pH 5.0), unbuffered media (initial pH 7.0), and Tris buffer (pH 8.0). The highest rates are observed at pH 8.0 with rates decreasing as a function of pH. The lowest rate occurs at pH 5.0 and exhibits pseudo-first order behavior over a 24 hour period, likely due to a long lag and very slow growth. Repeat injections after the culture is acclimated to the experimental conditions result in very high pseudo-first order rates due to rapid consumption of all available thiosulfate prior to oxygen depletion. Results from high-pressure closed system experiments (at 50 bars, buffered at pH 5.0) exhibit comparable rates to the corresponding ambient pressure condition. Future work will address the effect of dissolved O2 on sulfur disproportionation using continuous culturing of T. thermophila at deep-sea pressure conditions (>200 bar).

Determination of Novel Strategies for Hastening Corneal Wound Healing and Reducing Tissue Inflammation

DTIC Science & Technology

2011-10-01

blocking buffer, 5% fat -free milk in 0.1% Tris-buffered solution/Tween-20, for 1 hour at room temperature and then probed overnight at 5°C with...and blotting onto Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked with blocking buffer, 5% fat -free milk in 1x PBS buffer...distribution unlimited 13. SUPPLEMENTARY NOTES The aim of this study is to uncover novel transient receptor potential protein vanilloid-1 (TRPV1

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

NASA Astrophysics Data System (ADS)

Kabadi, Pradeep G.; Sankaran, Praveen Kallamvalliillam; Palanivelu, Dinesh V.; Adhikary, Laxmi; Khedkar, Anand; Chatterjee, Amarnath

2016-10-01

We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.

PubMed

Xu, Zhihao; Li, Jason; Zhou, Joe X

2012-01-01

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.

Identification of different domains of calpain and calpastatin from chicken blood and their role in post-mortem aging of meat during holding at refrigeration temperatures.

PubMed

Biswas, A K; Tandon, S; Beura, C K

2016-06-01

The aim of this study was to develop a simple, specific and rapid analytical method for accurate identification of calpain and calpastatin from chicken blood and muscle samples. The method is based on liquid-liquid extraction technique followed by casein Zymography detection. The target compounds were extracted from blood and meat samples by tris buffer, and purified and separated on anion exchange chromatography. It has been observed that buffer (pH 6.7) containing 50 mM tris-base appears to be excellent extractant as activity of analytes was maximum for all samples. The concentrations of μ-, m-calpain and calpastatin detected in the extracts of blood, breast and thigh samples were 0.28-0.55, 1.91-2.05 and 1.38-1.52 Unit/g, respectively. For robustness, the analytical method was applied to determine the activity of calpains (μ and m) in eighty postmortem muscle samples. It has been observed that μ-calpain activity in breast and thigh muscles declined very rapidly at 48 h and 24 h, respectively while activity of m-calpain remained stable. Shear force values were also declined with the increase of post-mortem aging showing the presence of ample tenderness of breast and thigh muscles. Finally, it is concluded that the method standardized for the detection of calpain and calpastatin has the potential to be applied to identify post-mortem aging of chicken meat samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology.

PubMed

Hu, Shih-Hao; Kuo, Chia-Hung; Chang, Chieh-Ming J; Liu, Yung-Chuan; Chiang, Wen-Dee; Shieh, Chwen-Jen

2012-01-01

A peptide, N-Ac-Phe-Tyr-NH(2) , with angiotensin I-converting enzyme (ACE) inhibitor activity was synthesized by an α-chymotrypsin-catalyzed condensation reaction of N-acetyl phenylalanine ethyl ester (N-Ac-Phe-OEt) and tyrosinamide (Tyr-NH(2) ). Three kinds of solvents: a Tris-HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic-aqueous solvent (Tris-HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N-Ac-Phe-Tyr-NH(2) could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N-Ac-Phe-Tyr-NH(2) , so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Partial purification and kinetic characterization of acid phosphatase from garlic seedling.

PubMed

Yenigün, Begüm; Güvenilir, Yüksel

2003-01-01

The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40-80% saturation with (NH4)2SOm4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10- 15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4 degrees C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50 degrees C. The enzyme was stable at pH 4.0-10.0 and 40-60 degrees C. Activation energy was between 10 and 20 kcal, and as Michaelis Menten coefficients, Vm values were 100 and 20 mM/s and Km values were 21.27 and 8.33 mM for paranitrophenylphosphate and paranitrophenyl, respectively. Studies of the effect of metal ions on enzyme activity showed both an activating and a deactivating effect. While Cu, Mo, and Mn showed strong inhibitory effects, Na, Ca, and K were the significant activators of acid phosphatase.

[Key factors in the control of electroosmosis with external radial electric field in CE].

PubMed

Zhu, Y; Chen, Y

1999-11-01

Direct control of electroosmosis flow (EOF) by external radial electric field was performed at room temperature using a home-made field-modulated capillary electrophoresis (CE) system. The EOF was monitored at 206 nm by using DMSO as a probe. To apply a radial electric field across the CE capillary wall, the capillary was cased with a wide column. Both of the concentric space and the capillary bore were then filled with an identical running buffer and applied with an axial electric field of 150 V/cm but starting from different levels. All of the tubes used were made of fused silica with polyimide over-coating (from the Yongnian Optical Fiber Work, Hebei, P. R. China). The size of the CE capillaries adopted was 25-100 microns i.d. (375 microns o.d.) x 28.5/45 cm (effective/total length), and that of the casing column 400 microns i.d. x 32 cm. To investigate the fundamentals of the external EOF control when using the flexible fused silica capillaries, various parameters have been inspected such as pH, buffer composition, additives and capillary wall feature etc.. As expected, to well control both of the magnitude and direction of the electroosmosis, the buffer pH should be kept below 4 and the buffer concentration below 50 mmol/L. However, buffers below 1 mmol/L should be avoided because such a diluted running buffer may result in poor CE separation. Weak electrolytes like citric acid, tartaric acid and acetic acid were found to be capable of generating better EOF control than the strong electrolytes such as phosphate and chlorides. This is possibly due to the formation of looser electric double layer with the weak rather than the strong electrolytes. Some wall coatings like calix arene and its derivatives can evidently improve the EOF control even at pH 5. This reveals an exciting way to expend the controllable pH range. In addition, narrow-bore capillaries were demonstrated to be better than wide-bore tubes. Other conditions such as buffer additives and capillary rinse procedure were shown to have only negligible influence on the control.

Bioactivity of Sodium Free Fluoride Containing Glasses and Glass-Ceramics

PubMed Central

Chen, Xiaojing; Chen, Xiaohui; Brauer, Delia S.; Wilson, Rory M.; Hill, Robert G.; Karpukhina, Natalia

2014-01-01

The bioactivity of a series of fluoride-containing sodium-free calcium and strontium phosphosilicate glasses has been tested in vitro. Glasses with high fluoride content were partially crystallised to apatite and other fluoride-containing phases. The bioactivity study was carried out in Tris and SBF buffers, and apatite formation was monitored by XRD, FTIR and solid state NMR. Ion release in solutions has been measured using ICP-OES and fluoride-ion selective electrode. The results show that glasses with low amounts of fluoride that were initially amorphous degraded rapidly in Tris buffer and formed apatite as early as 3 h after immersion. The apatite was identified as fluorapatite by 19F MAS-NMR after 6 h of immersion. Glass degradation and apatite formation was significantly slower in SBF solution compared to Tris. On immersion of the partially crystallised glasses, the fraction of apatite increased at 3 h compared to the amount of apatite prior to the treatment. Thus, partial crystallisation of the glasses has not affected bioactivity significantly. Fast dissolution of the amorphous phase was also indicated. There was no difference in kinetics between Tris and SBF studies when the glass was partially crystallised to apatite before immersion. Two different mechanisms of apatite formation for amorphous or partially crystallised glasses are discussed. PMID:28788139

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography-electrospray tandem mass spectrometry.

PubMed

Dubois, M; Fluchard, D; Sior, E; Delahaut, P

2001-04-05

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC-MS-MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C18 column (125 x 3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate-acetonitrile as the mobile phase at a flow-rate of 0.7 ml min(-1). Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC-MS-MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL-2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.

Affinity of microbial transglutaminase to αs1-, β-, and acid casein under atmospheric and high pressure conditions.

PubMed

Menéndez, Orquídea; Schwarzenbolz, Uwe; Partschefeld, Claudia; Henle, Thomas

2009-05-27

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for β-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of β-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

Preparation and Storage of High-Titer Lactic Streptococcus Bacteriophages1

PubMed Central

Nyiendo, J.; Seidler, Ramon J.; Sandine, W. E.; Elliker, P. R.

1974-01-01

Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 × 109 to 4 × 1011 plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at -22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months. PMID:16349981

Spectroscopic analysis on the resveratrol-DNA binding interactions at physiological pH

NASA Astrophysics Data System (ADS)

Zhang, Shufang; Sun, Xuejun; Jing, Zhihong; Qu, Fengli

2011-11-01

The interaction of resveratrol with calf thymus deoxyribonucleic acid (ctDNA) under physiological conditions (Tris-HCl buffer solutions, pH 7.4) was studied by spectroscopy, fluorescence spectroscopy and viscosity measurement method, respectively. Results indicated that a complex of resveratrol with ctDNA was formed with a binding constant of K17 °C = 5.49 × 10 3 L mol -1 and K37 °C = 1.90 × 10 4 L mol -1. The fluorescence quenching mechanism of acridine orange (AO)-ctDNA by resveratrol was shown to be a static quenching type. The thermodynamic parameters of the complex were calculated by a double reciprocal method: ΔHms=4.64×10 J mol, ΔSms=231.8 J K mol and ΔGms=-2.54×10 J mol (37 °C). Spectroscopic techniques together with viscosity determination provided evidences of intercalation mode of binding for the interaction between resveratrol and ctDNA.

Stability-indicating chromatographic methods for the determination of some oxicams.

PubMed

Taha, Elham Anwer; Salama, Nahla Nour; Abdel Fattah, Laila el-Said

2004-01-01

Two sensitive and selective methods were developed for the determination of some oxicams, namely, lornoxicam (LOX), tenoxicam (TEX), and meloxicam (MEX), in the presence of their alkaline degradation products. The first method is based on the thin-layer chromatographic separation of the 3 drugs from their alkaline degradation products, followed by densitometric measurement of the intact drug spots for LOX, TEX, and MEX at 380, 370, and 364 nm, respectively. The developing systems used for separation are ethyl acetate-methanol-26% ammonia (17 + 3 + 0.35, v/v/v) for LOX and TEX and chloroform-n-hexane-96.0% acetic acid (18 + 1 + 1, v/v/v) for MEX. The linear ranges were 0.25-6.0 microg/spot for LOX and TEX and 0.5-10 microg/spot for MEX, with mean recoveries of 99.80 +/- 1.32, 100.57 +/- 1.34, and 100.71 +/- 1.57%, respectively. The second method is based on the liquid chromatographic separation of the 3 drugs from their alkaline degradation products on a reversed-phase C18 column, using mobile phases of methanol-acetonitrile-acetate buffer, pH 4.6 (4.5 + 0.5 + 5.0, v/v/v) for LOX and MEX and methanol-acetonitrile-acetate buffer, pH 4.6 (1.9 + 0.1 + 3.0, v/v/v) for TEX at ambient temperature. Quantification is achieved by UV detection at 280 nm, based on peak area. The linear ranges were 0.5-20 microg/mL for LOX and TEX and 1.25-50 microg/mL for MEX, with mean recoveries of 99.81 +/- 1.01, 98.90 +/- 1.61, and 100.86 +/- 1.55%, respectively. The methods were validated according to guidelines of the International Conference on Harmonization. The developed methods were successfully applied to the determination of LOX, TEX, and MEX in bulk powder, laboratory-prepared mixtures containing different percentages of degradation products, and pharmaceutical dosage forms.

Relationship between intracellular pH and proton mobility in rat and guinea-pig ventricular myocytes.

PubMed

Swietach, Pawel; Vaughan-Jones, Richard D

2005-08-01

Intracellular H+ ion mobility in eukaryotic cells is low because of intracellular buffering. We have investigated whether Hi+ mobility varies with pHi. A dual microperfusion apparatus was used to expose guinea-pig or rat myocytes to small localized doses (3-5 mm) of ammonium chloride (applied in Hepes-buffered solution). Intracellular pH (pHi) was monitored confocally using the fluorescent dye, carboxy-SNARF-1. Local ammonium exposure produced a stable, longitudinal pHi gradient. Its size was fed into a look-up table (LUT) to give an estimate of the apparent intracellular proton diffusion coefficient (D(app)H). LUTs were generated using a diffusion-reaction model of Hi+ mobility based on intracellular buffer diffusion. To examine the pHi sensitivity of D(app)H, whole-cell pHi was initially displaced using a whole-cell ammonium or acetate prepulse, before locally applying the low dose of ammonium. In both rat and guinea-pig, D(app)H decreased with pHi over the range 7.5-6.5. In separate pipette-loading experiments, the intracellular diffusion coefficient for carboxy-SNARF-1 (a mobile-buffer analogue) exhibited no significant pHi dependence. The pHi sensitivity of D(app)H is thus likely to be governed by the mobile fraction of intrinsic buffering capacity. These results reinforce the buffer hypothesis of Hi+ mobility. The pHi dependence of D(app)H was used to characterize the mobile and fixed buffer components, and to estimate D(mob) (the average diffusion coefficient for intracellular mobile buffer). One consequence of a decline in Hi+ mobility at low pHi is that it will predispose the myocardium to pHi nonuniformity. The physiological relevance of this is discussed.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

2006-08-01

Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

Granulocyte elastase, beta-thromboglobulin, and C3d during acetate or bicarbonate hemodialysis with Hemophan compared to a cellulose acetate membrane.

PubMed

Stegmayr, B G; Esbensen, K; Gutierrez, A; Lundberg, L; Nielsen, B; Stroemsaeter, C E; Wehle, B

1992-01-01

Twenty-two patients were dialysed in a cross-over design using Hemophan or cellulose acetate membranes. The dialysate buffer was acetate (n = 12) or bicarbonate (n = 10). Blood was sampled at 0, 15, 60 and 180 min and mean values were adjusted for changes in total protein in each sample. At 15 min during dialysis a decrease in leukocytes and platelets occurred with both membranes, irrespective of the buffer (Wilcoxon, p less than 0.006). During dialysis, increases were found in granulocyte elastase inhibitor complex (E- alpha 1-PI), beta-thromboglobulin and C3d. beta 2-microglobulin was not significantly changed in blood after dialysis with Hemophan or cellulose acetate membranes with bicarbonate buffer. Side effects were more pronounced at 180 min during dialysis with bicarbonate in patients using cellulose acetate than with Hemophan (p = 0.021, n = 8). Hemophan seemed to be more favourable than cellulose acetate membranes in regard to leukopenia and E- alpha 1-PI. The dialysate buffer may also alter membrane biocompatibility.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

PubMed

Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline; Barisani-Asenbauer, Talin

2017-06-01

To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal–Limbal and Human Conjunctival Epithelial Cells

PubMed Central

Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline

2017-01-01

Purpose: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal–limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. Methods: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT–based assay. The morphology of cells was also investigated. Results: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. Conclusions: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface. PMID:28399036

Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition.

PubMed

Roach, Thomas; Sedoud, Arezki; Krieger-Liszkay, Anja

2013-10-01

Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris-acetate-phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris-acetate-phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less (1)O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of (1)O2 originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA(-)) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB(-)) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to (1)O2 production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting photosystem II charge recombination events and photoinhibition. Copyright © 2013 Elsevier B.V. All rights reserved.

Analytical Measurement of Discrete Hydrogen Sulfide Pools in Biological Specimens

PubMed Central

Shen, Xinggui; Peter, Elvis A.; Bir, Shyamal; Wang, Rui; Kevil, Christopher G.

2015-01-01

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors including pharmaco-therapeutic manipulation. Amongst the challenges facing the field is the accurate measurement of biologically active H2S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bonafide H2S. The complexity of analytical H2S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in different forms, including a free form, an acid labile pool and as bound sulfane sulfur. Here we describe a new protocol to discretely measure specific H2S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping and derivatization of H2S. Acid-labile H2S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM DTPA, in an enclosed system to contain volatilized H2S. Volatilized H2S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H2S measurement performed in an analogous manner to the one described above. The acid labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H2S measurement from the acid liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method protocol allows very sensitive and accurate measurement of the three primary biological pools of H2S including free, acid labile, and bound sulfane sulfur in various biological specimens. PMID:22561703

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

PubMed

Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

2015-08-15

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

Dual functionalized graphene oxide serves as a carrier for delivering oligohistidine- and biotin-tagged biomolecules into cells.

PubMed

Jana, Batakrishna; Mondal, Goutam; Biswas, Atanu; Chakraborty, Indrani; Saha, Abhijit; Kurkute, Prashant; Ghosh, Surajit

2013-11-01

A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cullin 5 Expression in the Rat: Cellular and Tissue Distribution, and Changes in Response to Water Deprivation and Hemorrhagic Shock

DTIC Science & Technology

2003-02-28

of Health p53 tumor suppressor PBS phosphate buffered saline PCO2 partial pressure of carbon dioxide PO2 partial pressure of oxygen PCR...buffered saline TTBS tween-20 tris buffered saline TonEBP tonicity-response enhancer binding protein TSNRP TriService Nursing Research Program...growth and metabolism (Hochstrasser, 1995; Deshaies, 1999). Although traditionally seen as no more than a means of eliminating no longer needed

Spectrophotometric Calibration of pH Electrodes in Seawater Using Purified m-Cresol Purple

PubMed Central

2012-01-01

This work examines the use of purified meta-cresol purple (mCP) for direct spectrophotometric calibration of glass pH electrodes in seawater. The procedures used in this investigation allow for simple, inexpensive electrode calibrations over salinities of 20–40 and temperatures of 278.15–308.15 K without preparation of synthetic Tris seawater buffers. The optimal pH range is ∼7.0–8.1. Spectrophotometric calibrations enable straightforward, quantitative distinctions between Nernstian and non-Nernstian electrode behavior. For the electrodes examined in this study, both types of behavior were observed. Furthermore, calibrations performed in natural seawater allow direct determination of the influence of salinity on electrode performance. The procedures developed in this study account for salinity-induced variations in liquid junction potentials that, if not taken into account, would create pH inconsistencies of 0.028 over a 10-unit change in salinity. Spectrophotometric calibration can also be used to expeditiously determine the intercept potential (i.e., the potential corresponding to pH 0) of an electrode that has reliably demonstrated Nernstian behavior. Titrations to ascertain Nernstian behavior and salinity effects can be undertaken relatively infrequently (∼weekly to monthly). One-point determinations of intercept potential should be undertaken frequently (∼daily) to monitor for stable electrode behavior and ensure accurate potentiometric pH determinations. PMID:22463815

The effect of EDTA, cations, and various buffers on the morphology of erythrocyte membranes: an electron-microscopic study.

PubMed

Pinteric, L; Manery, J F; Chaudry, I H; Madapallimattam, G

1975-05-01

Membranes of human erythrocytes were prepared by stepwise osmotic hemolysis in Ca2+-free solutions. Examination with the electron microscope after negative staining showed some short, conelike protuberances on the surface of about 20 percent of the ghosts, while 80 percent were round, intact spheres. After Ca2+ treatment, all membranes were round and intact. After exposure to ethylenediaminetetraacetic acid (EDTA) (1.0 mM, pH 7.4), the entire ghost surface was covered with long, thin extrusions called stromalytic forms (about 460 per cell). Their sizes, shapes, and fine structure are described. Exposure to ionic calcium (1.4 times 10-minus 4M) abolished the EDTA-induced stromalytic forms. A second exposure to EDTA reversed this Ca2+ effect. ATP, like EDTA, produced stromalytic forms. EDTA-induced stromalytic forms were also abolished by Zn2+, La3+, and Nd3+ at concentrations of 1-5 times 10-minus 4 M. Mg2+ at 10-minus 2 M was ineffective. Ghosts were prepared by graded lysis in various buffers. Those prepared in phosphate were the most stable and provided consistent EDTA effects and Ca2+ reversal. Ghosts in Tris-HCl showed breakdown unless salt was added. Moderately satisfactory ghosts were also obtained in Hepes-NaOH buffer and salt.

Investigations of nanoscale variations in spin and charge transport in manganites and organic semiconductors using spin polarized scanning tunneling spectroscopy

NASA Astrophysics Data System (ADS)

Hughes, Cameron Richard

Analysis of DNA structure and behavior, up to and including full sequencing of a genome's bases, and of biological processes such as replication, transcription and translation, is essential for an understanding of genetic variation, heritable diseases and the effects of environmental factors. Recently, single-molecule techniques have been developed to study DNA properties in unprecedented detail. For a number of these techniques, controlled adsorption of linearly stretched DNA molecules on surfaces is necessary. In experiments where hybridization of adsorbed molecules to labeled probes is used to determine DNA structure, single-stranded DNA is needed. Conventionally, for long DNA's (up to Mbp), double-stranded DNA is deposited on a surface and denatured in-situ. While successful, this method has several disadvantages. This thesis reports efforts to directly adsorb long single-stranded DNA's out of solution as an alternative strategy. It consists of three parts: (1) Establishment of a simple method using Acridine Orange (AO) staining dye to determine whether DNA's are ss or ds on the surface. The method allows for the assessment of the degree of renaturation during deposition. Incubation of surface-adsorbed DNA in solutions of AO dye in the concentration range of 10--15uM were found to be effective for discriminating between ss DNA and ds DNA based on differences in the fluorescence emission spectra. (2) Deposition of ss DNA produced by heat denaturation on polymer-coated surfaces. Lambda DNA (48502bp) was adsorbed by drop evaporation or dipping/extraction of surface out of a buffered solution. The efficiency of deposition was optimized with respect to DNA concentration, buffer type and pH. (3) Separation of complementary single strands of Lambda, mono-cut digest and HindIII digest by gel electrophoresis. Using agarose gels in concentrations ranging from 0.4% to 1.4% (weight/volume), electric fields in the range 1--4V/cm in 1x Tris-Acetate-EDTA (TAE) buffer, good strand separation could be obtained. Both DC and pulsed electric fields were used and compared. Following separation, sense and anti-sense strands of lambda DNA were extracted from gels and deposited separately onto surfaces, and length distributions of the isolated molecules were measured by fluorescence microscopy.

Chemometrics-assisted spectrophotometric green method for correcting interferences in biowaiver studies: Application to assay and dissolution profiling study of donepezil hydrochloride tablets

NASA Astrophysics Data System (ADS)

Korany, Mohamed A.; Mahgoub, Hoda; Haggag, Rim S.; Ragab, Marwa A. A.; Elmallah, Osama A.

2018-06-01

A green, simple and cost effective chemometric UV-Vis spectrophotometric method has been developed and validated for correcting interferences that arise during conducting biowaiver studies. Chemometric manipulation has been done for enhancing the results of direct absorbance, resulting from very low concentrations (high incidence of background noise interference) of earlier points in the dissolution timing in case of dissolution profile using first and second derivative (D1 & D2) methods and their corresponding Fourier function convoluted methods (D1/FF& D2/FF). The method applied for biowaiver study of Donepezil Hydrochloride (DH) as a representative model was done by comparing two different dosage forms containing 5 mg DH per tablet as an application of a developed chemometric method for correcting interferences as well as for the assay and dissolution testing in its tablet dosage form. The results showed that first derivative technique can be used for enhancement of the data in case of low concentration range of DH (1-8 μg mL-1) in the three different pH dissolution media which were used to estimate the low drug concentrations dissolved at the early points in the biowaiver study. Furthermore, the results showed similarity in phosphate buffer pH 6.8 and dissimilarity in the other 2 pH media. The method was validated according to ICH guidelines and USP monograph for both assays (HCl of pH 1.2) and dissolution study in 3 pH media (HCl of pH 1.2, acetate buffer of pH 4.5 and phosphate buffer of pH 6.8). Finally, the assessment of the method greenness was done using two different assessment techniques: National Environmental Method Index label and Eco scale methods. Both techniques ascertained the greenness of the proposed method.

Chemometrics-assisted spectrophotometric green method for correcting interferences in biowaiver studies: Application to assay and dissolution profiling study of donepezil hydrochloride tablets.

PubMed

Korany, Mohamed A; Mahgoub, Hoda; Haggag, Rim S; Ragab, Marwa A A; Elmallah, Osama A

2018-06-15

A green, simple and cost effective chemometric UV-Vis spectrophotometric method has been developed and validated for correcting interferences that arise during conducting biowaiver studies. Chemometric manipulation has been done for enhancing the results of direct absorbance, resulting from very low concentrations (high incidence of background noise interference) of earlier points in the dissolution timing in case of dissolution profile using first and second derivative (D1 & D2) methods and their corresponding Fourier function convoluted methods (D1/FF& D2/FF). The method applied for biowaiver study of Donepezil Hydrochloride (DH) as a representative model was done by comparing two different dosage forms containing 5mg DH per tablet as an application of a developed chemometric method for correcting interferences as well as for the assay and dissolution testing in its tablet dosage form. The results showed that first derivative technique can be used for enhancement of the data in case of low concentration range of DH (1-8μgmL -1 ) in the three different pH dissolution media which were used to estimate the low drug concentrations dissolved at the early points in the biowaiver study. Furthermore, the results showed similarity in phosphate buffer pH6.8 and dissimilarity in the other 2pH media. The method was validated according to ICH guidelines and USP monograph for both assays (HCl of pH1.2) and dissolution study in 3pH media (HCl of pH1.2, acetate buffer of pH4.5 and phosphate buffer of pH6.8). Finally, the assessment of the method greenness was done using two different assessment techniques: National Environmental Method Index label and Eco scale methods. Both techniques ascertained the greenness of the proposed method. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a flow system for the determination of cadmium in fuel alcohol using vermicompost as biosorbent and flame atomic absorption spectrometry.

PubMed

Bianchin, Joyce Nunes; Martendal, Edmar; Mior, Renata; Alves, Vanessa Nunes; Araújo, Cleide Sandra Tavares; Coelho, Nívia Maria Melo; Carasek, Eduardo

2009-04-30

In this study a method for the determination of cadmium in fuel alcohol using solid-phase extraction with a flow injection analysis system and detection by flame atomic absorption spectrometry was developed. The sorbent material used was a vermicompost commonly used as a garden fertilizer. The chemical and flow variables of the on-line preconcentration system were optimized by means of a full factorial design. The selected factors were: sorbent mass, sample pH, buffer concentration and sample flow rate. The optimum extraction conditions were obtained using sample pH in the range of 7.3-8.3 buffered with tris(hydroxymethyl)aminomethane at 50 mmol L(-1), a sample flow rate of 4.5 mL min(-1) and 160 mg of sorbent mass. With the optimized conditions, the preconcentration factor, limit of detection and sample throughput were estimated as 32 (for preconcentration of 10 mL sample), 1.7 microg L(-1) and 20 samples per hour, respectively. The analytical curve was linear from 5 up to at least 50 microg L(-1), with a correlation coefficient of 0.998 and a relative standard deviation of 2.4% (35 microg L(-1), n=7). The developed method was successfully applied to spiked fuel alcohol, and accuracy was assessed through recovery tests, with recovery ranging from 94% to 100%.

Dissolution and time-dependent compaction of albite sand: experiments at 100°C and 160°C in pH-buffered organic acids and distilled water

NASA Astrophysics Data System (ADS)

Hajash, Andrew; Carpenter, Thomas D.; Dewers, Thomas A.

1998-09-01

Aqueous fluids are important in the diagenesis and deformation of crustal rocks. Both chemical and physical interactions are involved and often they are strongly coupled. For example, pore waters not only dissolve, transport, and precipitate chemical species, but they also substantially affect the mechanical behavior of the rocks that contain them. Stresses magnified at grain contacts by differences in pore-fluid pressure ( Pp) and confining pressure ( Pc) can, in turn, influence the rate and extent of chemical exchange. To begin investigation of these coupled systems, compaction experiments were conducted using albite sand (250-500 μm) and distilled water (pH 5.8), 0.07 M acetate (pH 4.7), and 0.07 M acetate + 0.005 M citrate (pH 4.4) solutions in a hydrothermal flow-through system at conditions that simulate diagenesis. Pore-fluid chemistry and pore-volume loss were monitored to quantify the effects of organic acids on time-dependent compaction rates. The effects of stress and fluid chemistry on the dissolution kinetics were also examined. Albite dissolution rates, monitored by steady-state fluid chemistry, increased when an effective pressure ( Pe= Pc- Pp) was applied, probably due to increases in total surface area caused by grain breakage at contacts. These effects were transient in distilled water, however, Si and Al concentrations remained elevated in the acetate pore fluid. The average Si-based release rates indicate ≈35% increase in reactive surface area by application of Pe=34.5 MPa. At 100°C with Pe=34.5 MPa, steady-state Si concentrations were ≈2.3 times higher in 0.07 M acetate and 5.8 times higher in 0.07 M acetate + 0.005 M citrate than in distilled water. Al increased by even larger factors (3× in the acetate buffer and 10× in the citrate solution). These changes in fluid chemistry are attributed to both pH and ligand-enhanced reactions. Albite dissolution appears to be controlled by surface complexation reactions at Al sites. Rapid dissolution of albite in the organic acid solutions is probably due to the ability of organic acid ligands to selectively complex with aluminum. Time-dependent compaction was observed at 100 and 160°C with Pe=34.5 MPa. Strain rates increased with temperature from ≈10 -9 s -l at 100°C to ≈10 -8 s -l at 160°C and decreased with strain in all pore fluids, especially at 100°C. Compaction rates in distilled water and in the acetate solution had similar magnitudes and strain dependencies; however, small amounts of citrate species apparently enhance compaction compared to the other fluids at similar strains. Textural data indicate that time-dependent compaction of the albite sand occurred primarily by brittle mechanisms at these temperatures. However, the deformation is clearly thermally activated and may be chemically assisted by the aqueous pore fluid.

Gold Nanocluster-DNase 1 Hybrid Materials for DNA Contamination Sensing

DTIC Science & Technology

2014-01-01

or 1 mM) was added to 2 mL of protein solution under vigorous stirring at 37 ̊ C. After 5 minutes 200 µL of NaOH (1 M) was added to raise the pH to...12 for the 1, 5, and 10 mM HAuCl4 samples whereas 400 µL of NaOH (1M) was required. The various protein/gold mixtures were then left to react for...1: AuNCs were carried out in a final volume of 20 µL buffer (100 mM sodium acetate, 6.25 mM magnesium sulfate pH 5.0), containing 2 µg of dsDNA

Stress state during fixation determines susceptibility to fatigue-linked biodegradation in bioprosthetic heart valve materials.

PubMed

Margueratt, Sean D; Lee, J Michael

2002-01-01

Mechanical loading contributes to the structural deterioration of bioprosthetic heart valves. The influence of stress state during fixation may play a substantial role in their failure, linking fatigue damage caused by buckling and tension and the enzymatic degradation of glutaraldehyde-crosslinked collagen. Bovine pericardia were obtained immediately postmortem and 100 mm x 15 mm samples were cut in the base-to-apex direction. Half the samples were subjected to a uniaxial tensile stress of 250 kPa and half remained unloaded during a crosslinking treatment in 0.5% glutaraldehyde. Tissue samples were rinsed and cut into 16 mm x 4 mm test strips. Half of these strips were exposed to cyclic compressive buckling and alternating tension at 30 Hz for 20 million cycles (approx. 7.5 days) using a custom-built multi-sample fatigue system. Fatigue-damaged and non-damaged samples were subsequently incubated at 37 C for 48 hrs in: (i) Type I bacterial collagenase (20 U/ml) buffered in 0.05 M Tris, 10 mM CaCl2 2H2O (pH 7.4) or (ii) 0.05 M Tris buffer (pH 7.4) only. In both cases, the samples were loaded sinusoidally between 40 and 80 g using a previously described microtensile culture system. Tissue removed from the bath was rinsed in 0.1 M EDTA solution and mounted in a servo-hydraulic mechanical testing system (MTS). Ultimate tensile strength (UTS), maximum tissue modulus, and fracture strain were determined. The percent collagen solubilized was assessed by a colourmetric hydroxyproline assay of the enzyme bath and tissue sample. All data were analyzed by analysis of variance (ANOVA). The results confirmed the synergy between fatigue damage and collagenase proteolysis in these materials; however, there were no significant differences in this effect between simple fixation and stress-fixation up to 20 million cycles. There were significant decreases in the mechanical properties and an increase in the amount of collagen solubilized with increased exposure to fatigue cycling.

Isolation of high quality RNA from cereal seeds containing high levels of starch.

PubMed

Wang, Guifeng; Wang, Gang; Zhang, Xiaowei; Wang, Fang; Song, Rentao

2012-01-01

Cereals are an important source of food, feed and fuel with a rapidly increasing global demand. However, cereal seeds contain high levels of starch and polysaccharides, making the isolation of high quality RNA extremely difficult. To develop a novel method for extracting high quality total RNA from various starch- and polysaccharides-rich cereal seeds, such as maize, rice, sorghum and wheat. We developed a modified sodium dodecyl sulphate (SDS)/TRIzol method. The combined use of a Tris buffer (pH 9.0) and SDS before TRIzol extraction effectively resolved the problem of seed homogenate solidification in such a buffer. A high concentration of SDS was used separately, not only to promote cell lysis but also to effectively dissolve seed sample containing high levels of starch. Moreover, acid phenol saturated with 0.1  M citrate buffer (pH 4.3) was used to separate RNA from DNAs, proteins and high levels of starch. This rapid protocol was compared with other RNA isolation methods preferentially used for plants rich in polysaccharides and secondary metabolites. Gel electrophoresis analysis indicated that the extracted total RNA had good integrity without apparent DNA contamination. Furthermore, an A₂₆₀/₂₈₀ ratio of approximately 2.0, an A₂₆₀/₂₃₀ ratio of more than 2.0 and RIN values of more than 8.6 indicated that the isolated RNA was of high purity. The isolated RNA was suitable for subsequent molecular manipulations, such as reverse-transcription polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE) and real-time PCR. The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds. Copyright © 2011 John Wiley & Sons, Ltd.

Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes.

PubMed

Ribeiro, Mariana Borsoi; Vijayalakshmi, Mookambesvaran; Todorova-Balvay, Daniele; Bueno, Sonia Maria Alves

2008-01-01

The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.

Extraction-spectrophotometric determination of tris(2-chloroethyl)amine using phthaleins.

PubMed

Rozsypal, Tomas; Halamek, Emil

2017-06-01

Procedures for the extraction-spectrophotometric determination of tris(2-chloroethyl)amine, an alkylating agent known as a drug as well as a chemical warfare agent (nitrogen mustard HN-3), with 7 acid-base indicators of a triphenylmethane lactone type, phthaleins, were developed. Representatives of phthaleins without an oxygen bridge (thymolphthalein, o-cresolphthalein, naphtholphthalein) and with an oxygen bridge (fluorescein, 2',7'-dichlorofluorescein, eosin B and eosin Y) were used. The methods were based on the formation of ion pair complexes. Chloroform was used as a non-polar solvent for an extraction. The conditions to determine were optimized for the optimal pH of the buffer and the concentration of a phthalein as a reagent. The dependence on the reaction time in a water phase and the stoichiometry of extraction products were studied. The detection limits and the limits of the determination of separate procedures and conditional extraction constants were determined. Comparison with the spectrophotometric method of the group determination of alkyl halides and acyl halides using alkaline ethanol-water solution of thymolphthalein, the so-called T-135 agent, was conducted. While studying the selectivity, the possible interference of bis(2-chloroethyl)sulphide and 3 nitrogen mustards in the proposed procedures were verified. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A rapid procedure for measurement of the free testosterone fraction in human plasma using the centria radioimmunoassay centrifugal analyzer.

PubMed

Schwarz, S; Boyd, J

1981-01-01

Following the incubation of plasma with a tracer amount of tritiated testosterone, the reaction mixture is separated into a sex hormone-binding globulin bound and an unbound fraction of radioligand using DEAE-cellulose columns placed in the incubator-separator module of the Centria radioimmunoassay centrifugal analyzer. Neural Tris-buffer elutes unbound steroid from the matrix, while acidic Tris-buffer can remove the protein-bound fraction in a subsequent step. Complementary and thus qualitatively equal results are obtained when counting either eluate. Comparison of this technique with an ammonium sulfate precipitation method showed high correlation. Free testosterone indices as determined by the Centria modification in a number of prepuberal children, normal men and women, as well as pregnant and hirsute women similar to those previously reported.

Separation of silver nanoparticles by hollow fiber flow field-flow fractionation: Addition of tannic acid into carrier liquid as a modifier.

PubMed

Saenmuangchin, Rattaporn; Mettakoonpitak, Jaruwan; Shiowatana, Juwadee; Siripinyanond, Atitaya

2015-10-09

A homemade hollow fiber flow-field fractionation (Hf-FlFFF) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was set-up for silver nanoparticles (AgNPs) separation by using polysulfone hollow fiber membrane (30,000 MW cutoff) as a separation channel. Tannic acid and citrate stabilized AgNPs were synthesized and introduced into Hf-FlFFF. The effects of carrier liquid and stabilizing agent on retention behavior of AgNPs were investigated. Different elution behaviors were observed as follows: with 0.02% (w/v) FL-70, all of AgNPs were eluted from Hf-FlFFF but differences in retention behaviors were observed for AgNPs with tannic acid and citrate stabilizing agents; and with 30mM TRIS buffer, only tannic acid stabilized AgNPs were eluted from Hf-FlFFF, whereas citrate stabilized AgNPs were not eluted. In this work, tannic acid addition into carrier liquid was proposed to modify the surface of AgNPs and the surface of the membrane, and thereby adjusting the retention behaviors of AgNPs. Various concentrations of tannic acid were added into FL-70 and TRIS buffer. With the use of 0.1mM tannic acid in 30mM TRIS buffer as the carrier liquid, retention behaviors of both tannic acid stabilized- and citrate stabilized-AgNPs were similar and with similar fractionation recovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Acidogenic fermentation of food waste for volatile fatty acid production with co-generation of biohydrogen.

PubMed

Dahiya, Shikha; Sarkar, Omprakash; Swamy, Y V; Venkata Mohan, S

2015-04-01

Fermentation experiments were designed to elucidate the functional role of the redox microenvironment on volatile fatty acid (VFA, short chain carboxylic acid) production and co-generation of biohydrogen (H2). Higher VFA productivity was observed at pH 10 operation (6.3g/l) followed by pH 9, pH 6, pH 5, pH 7, pH 8 and pH 11 (3.5 g/l). High degree of acidification, good system buffering capacity along with co-generation of higher H2 production from food waste was also noticed at alkaline condition. Experiments illustrated the role of initial pH on carboxylic acids synthesis. Alkaline redox conditions assist solubilization of carbohydrates, protein and fats and also suppress the growth of methanogens. Among the carboxylic acids, acetate fraction was higher at alkaline condition than corresponding neutral or acidic operations. Integrated process of VFA production from waste with co-generation of H2 can be considered as a green and sustainable platform for value-addition. Copyright © 2015 Elsevier Ltd. All rights reserved.

Technical Evaluation of Sample-Processing, Collection, and Preservation Methods

DTIC Science & Technology

2014-07-01

For the Gram-positive organism, B. atrophaeus var. globigii (Unified Culture Collection [ UCC ] designation: BACI051) was selected as a surrogate for...the well-known biothreat agent Bacillus anthracis. For the Gram-negative organism, Y. pestis CO92 (pgm–) ( UCC designation: YERS059) was selected...Diagnostics device) TAMRA tetramethylrhodamine TE buffer tris-ethylenediaminetetraacetic acid buffer UCC Unified Culture Collection USG U.S. Government

Enantioseparations of amino acids by capillary array electrophoresis with 532 nm laser induced fluorescence detection.

PubMed

Liu, Kaiying; Wang, Li

2013-06-21

Capillary array electrophoresis (CAE) is a promising technique for multiple enantiomeric separations. Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE), a rhodamine-core fluorescent probe, has rarely been applied as an original precolumn derivatization reagent for chiral amino acid (AA) analysis so far. For these purposes, high-throughput enantiomeric separations of 12 TAMRA SE-AAs by a home-made 532 nm CAE-LIF scanner are presented. The effect of cyclodextrins (CDs) and a variety of organic modifiers was quickly investigated. Baseline separations were achieved in 100 mM Tris-borate buffer (pH 10.0) containing 2 mM β-CD and 10 mM hexamethylenediamine (HDA). Multiple determination of the enantiomeric excess (ee) in non-racemic mixtures of alanine is successfully presented. Copyright © 2013 Elsevier B.V. All rights reserved.

The Ca(2+)-EDTA chelation as standard reaction to validate Isothermal Titration Calorimeter measurements (ITC).

PubMed

Ràfols, Clara; Bosch, Elisabeth; Barbas, Rafael; Prohens, Rafel

2016-07-01

A study about the suitability of the chelation reaction of Ca(2+)with ethylenediaminetetraacetic acid (EDTA) as a validation standard for Isothermal Titration Calorimeter measurements has been performed exploring the common experimental variables (buffer, pH, ionic strength and temperature). Results obtained in a variety of experimental conditions have been amended according to the side reactions involved in the main process and to the experimental ionic strength and, finally, validated by contrast with the potentiometric reference values. It is demonstrated that the chelation reaction performed in acetate buffer 0.1M and 25°C shows accurate and precise results and it is robust enough to be adopted as a standard calibration process. Copyright © 2016 Elsevier B.V. All rights reserved.

The influence of chicken eggshell powder as a buffer on biohydrogen production from rotten orange (Citrus nobilis var. microcarpa) with immobilized mixed culture

NASA Astrophysics Data System (ADS)

Damayanti, Astrilia; Sarto, Syamsiah, Siti; Sediawan, Wahyudi B.

2017-06-01

This research observed the influence of chicken eggshell on hydrogen production from anaerobic fermentation of rotten orange (Citrus nobilis var. microcarpa) using batch method at 36 °C and pH 7. Fermentation material were varied in several types, the first type was meat and peel of oranges with VS of 59.152 g.L-1 in A, B, C, and D compositions. The second type was orange meat added with peel (OMP) with VS of 36.852 g.L-1. The immobilized ingredients used in the experiment consisted of 2 % (w/v) alginate and active carbon with the ratio of 1:1. 3.2 g chicken eggshell powder was added to the first type of material (substrates A, B, C, and D). Results showed that pH during fermentation process using chicken eggshell as a buffer was constant at 5.5; however, without the use of chicken eggshell, the pH decreased to 3.8 and increased slightly before it stayed stable at 4.0. The total amount of gas produced in sample using the chicken eggshell was 46,35 mL.mg VS-1 and in sample produced without the eggshell, it was 3,4 mL.mg VS-1. The production of hydrogen in substrate that used chicken eggshell was 1,276 mL.gVS-1 in average on the first day. Meanwhile, for the substrate with no addition of chicken eggshell, the average production of hydrogen was 0,163 mL.gVS-1. The reduction of volatile solid (VS) in sample that used chicken eggshell was 24 %, while in sample produced without addition of chicken eggshell, the reduction was 12 %. The liquid compounds (VFA) produced in the fermentation using chicken eggshell were acetic acid and butyric acid. Meanwhile, without addition of chicken eggshell, the products were acetic acid, butyric acid, and propionic acid. This study shows that addition of chicken eggshell as a buffer effectively contributed to hydrogen production during fermentation of rotten oranges.

Photocurrent enhancement of n-type Cu2O electrodes achieved by controlling dendritic branching growth.

PubMed

McShane, Colleen M; Choi, Kyoung-Shin

2009-02-25

Cu(2)O electrodes composed of dendritic crystals were produced electrochemically using a slightly acidic medium (pH 4.9) containing acetate buffer. The buffer played a key role for stabilizing dendritic branching growth as a pH drop during the synthesis prevents formation of morphologically unstable branches and promotes faceted growth. Dendritic branching growth enabled facile coverage of the substrate with Cu(2)O while avoiding growth of a thicker Cu(2)O layer and increasing surface areas. The resulting electrodes showed n-type behavior by generating anodic photocurrent without applying an external bias (zero-bias photocurrent under short-circuit condition) in an Ar-purged 0.02 M K(2)SO(4) solution. The zero-bias photocurrent of crystalline dendritic electrodes was significantly higher than that of the electrodes containing micrometer-size faceted crystals deposited without buffer. In order to enhance photocurrent further a strategy of improving charge-transport properties by increasing dendritic crystal domain size was investigated. Systematic changes in nucleation density and size of the dendritic Cu(2)O crystals were achieved by altering the deposition potential, Cu(2+) concentration, and acetate concentration. Increasing dendritic crystal size consistently resulted in the improvement of photocurrent regardless of the method used to regulate crystal size. The electrode composed of dendritic crystals with the lateral dimension of ca. 12000 microm(2) showed more than 20 times higher zero-bias photocurrent than that composed of dendritic crystals with the lateral dimension of ca. 100 microm(2). The n-type nature of the Cu(2)O electrodes prepared by this study were confirmed by linear sweep voltammetry with chopped light and capacitance measurements (i.e., Mott-Schottky plots). The flatband potential in a 0.2 M K(2)SO(4) solution (pH 6) was estimated to be -0.78 vs Ag/AgCl reference electrode. The IPCE measured without applying an external bias was approximately 1% for the visible region. With appropriate doping studies and surface treatment to improve charge transport and interfacial kinetics more efficient n-type Cu(2)O electrodes will be prepared for use in various photoelectrochemical and photovoltaic devices.

Fluorescence behaviour of 5,10-methenyltetrahydrofolate, 10-formyltetrahydrofolate, 10-formyldihydrofolate, and 10-formylfolate in aqueous solution at pH 8

NASA Astrophysics Data System (ADS)

Tyagi, A.; Penzkofer, A.; Batschauer, A.; Wolf, E.

2009-06-01

The fluorescence spectroscopic behaviour of (6R,S)-5,10-methenyltetrahydrofolate (MTHF), (6R,S)-10-formyltetrahydrofolate (10-HCO-H4folate), 10-formyldihydrofolate (10-HCO-H2folate), and 10-formylfolate (10-HCO-folate) in aqueous Tris-HCl buffer at pH 8 is studied. MTHF and 10-HCO-folate were commercially available. 10-HCO-H4folate was prepared from MTHF by hydrolysis at room temperature under anaerobic conditions. 10-HCO-H2folate was prepared by oxidation of 10-HCO-H4folate under aerobic conditions. Fluorescence quantum distributions at room temperature and fluorescence signal decays at room temperature and liquid nitrogen temperature were measured. The fluorescence lifetimes determined at room temperature (liquid nitrogen temperature) are 10 ps (2.9 ns) for MTHF, 38 ps (3.7 ns) for 10-HCO-H4folate, 80 ps (10.5 ns) for 10-HCO-H2folate, and 7.1 ns (20 ns) for 10-HCO-folate. The results are discussed in terms of dyadic (pterin-benzoyl-glutamate) photo-induced electron transfer and dyadic fluorescent dynamics.

Comparison of Four Nuclear Isolation Buffers for Plant DNA Flow Cytometry

PubMed Central

LOUREIRO, JOÃO; RODRIGUEZ, ELEAZAR; DOLEŽEL, JAROSLAV; SANTOS, CONCEIÇÃO

2006-01-01

• Background and Aims DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue. • Methods Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1·30–26·90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days. • Key Results Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a ‘fingerprint’ for each buffer. The variation between days was more significant than the variation between operators. • Conclusions Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining. PMID:16820407

Gravimetric method for the determination of diclofenac in pharmaceutical preparations.

PubMed

Tubino, Matthieu; De Souza, Rafael L

2005-01-01

A gravimetric method for the determination of diclofenac in pharmaceutical preparations was developed. Diclofenac is precipitated from aqueous solution with copper(II) acetate in pH 5.3 (acetic acid/acetate buffer). Sample aliquots had approximately the same quantity of the drug content in tablets (50 mg) or in ampules (75 mg). The observed standard deviation was about +/- 2 mg; therefore, the relative standard deviation (RSD) was approximately 4% for tablet and 3% for ampule preparations. The results were compared with those obtained with the liquid chromatography method recommended in the United States Pharmacopoeia using the statistical Student's t-test. Complete agreement was observed. It is possible to obtain more precise results using higher aliquots, for example 200 mg, in which case the RSD falls to 1%. This gravimetric method, contrary to what is expected for this kind of procedure, is relatively fast and simple to perform. The main advantage is the absolute character of the gravimetric analysis.

Electroneutral, HCO3(-)-independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea.

PubMed Central

Vasseur, M; Caüzac, M; Alvarado, F

1989-01-01

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2597129

Studies on the intracellular Ca2+ concentration of thawed dog spermatozoa: influence of Equex from different sources, two thawing diluents and post-thaw incubation in capacitating conditions.

PubMed

Peña, A I; López-Lugilde, L; Barrio, M; Becerra, J J; Quintela, L A; Herradón, P G

2003-02-01

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris-egg yolk extender was demonstrated to improve the longevity of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C. The aim of the first experiment was to compare the effects of two SDS-containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris-egg yolk based extender, on the post-thaw longevity of dog spermatozoa, as well as on the intracellular Ca2+ concentration of spermatozoa, during post-thaw incubation at 38 degrees C. The post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca2+ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post-thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca2+ concentration of cryopreserved dog spermatozoa during post-thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 microg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca2+ content. However, after 8-10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2-4-h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris-egg yolk based extender significantly improved the post-thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post-thaw sperm longevity; (c) the addition of 5 microg/ml of heparin to CCM had no significant capacitating effects on frozen-thawed dog spermatozoa.

Nonaqueous capillary electrophoresis with indirect electrochemical detection.

PubMed

Matysik, Frank-Michael; Marggraf, Daniela; Gläser, Petra; Broekaert, José A C

2002-11-01

Nonaqueous capillary electrophoresis (NACE) which makes use of organic solvents in place of conventional aqueous electrophoresis buffers is gaining increasing importance among modern separation techniques. Recently, it has been shown that amperometric detection in conjunction with acetonitrile-based NACE offers an extended accessible potential range and an enhanced long-term stability of the amperometric responses generated at solid electrodes. The present contribution takes advantage of the latter aspect to develop reliable systems for NACE with indirect electrochemical detection (IED). In this context, several compounds such as (ferrocenylmethyl)trimethylammonium perchlorate, tris(1,10-phenanthroline)cobalt(III) perchlorate and bis(1,4,7-triazacyclononane)nickel(II) perchlorate were studied regarding their suitability to act as electroactive buffer additives for IED in NACE. The performance characteristics for the respective buffer systems were evaluated. Tetraalkylammonium perchlorates served as model compounds for the optimization of the NACE-IED system. Target analytes choline and acetylcholine could easily be separated and determined by means of NACE-IED. In the case of a buffer system containing 10(-4) M tris(1,10-phenanthroline)cobalt(III) perchlorate the limits of detection were 2.5 x 10(-7) M and 4.6 x 10(-7) M for choline and acetylcholine, respectively. With the elaborated analytical procedure choline could be determined in pharmaceutical preparations.

Influence of dissolution media pH and USP1 basket speed on erosion and disintegration characteristics of immediate release metformin hydrochloride tablets.

PubMed

Desai, Divyakant; Wong, Benjamin; Huang, Yande; Tang, Dan; Hemenway, Jeffrey; Paruchuri, Srinivasa; Guo, Hang; Hsieh, Daniel; Timmins, Peter

2015-01-01

To investigate the influence of the pH of the dissolution medium on immediate release 850 mg metformin hydrochloride tablets. A traditional wet granulation method was used to manufacture metformin hydrochloride tablets with or without a disintegrant. Tablet dissolution was conducted using the USP apparatus I at 100 rpm. In spite of its pH-independent high solubility, metformin hydrochloride tablets dissolved significantly slower in 0.1 N HCl (pH 1.2) and 50 mM pH 4.5 acetate buffer compared with 50 mM pH 6.8 phosphate buffer, the dissolution medium in the USP. Metformin hydrochloride API compressed into a round 1200 mg disk showed a similar trend. When basket rotation speed was increased from 100 to 250 rpm, the dissolution of metformin hydrochloride tablets was similar in all three media. Incorporation of 2% w/w crospovidone in the tablet formulation improved the dissolution although the pH-dependent trend was still evident, but incorporation of 2% w/w croscarmellose sodium resulted in rapid pH-independent tablet dissolution. In absence of a disintegrant in the tablet formulation, the dissolution was governed by the erosion-diffusion process. Even for a highly soluble drug, a super-disintegrant was needed in the formulation to overcome the diffusion layer limitation and change the dissolution mechanism from erosion-diffusion to disintegration.

Fluorescence lifetime imaging of oxygen in dental biofilm

NASA Astrophysics Data System (ADS)

Gerritsen, Hans C.; de Grauw, Cees J.

2000-12-01

Dental biofilm consists of micro-colonies of bacteria embedded in a matrix of polysaccharides and salivary proteins. pH and oxygen concentration are of great importance in dental biofilm. Both can be measured using fluorescence techniques. The imaging of dental biofilm is complicated by the thickness of the biofilms that can be up to several hundred micrometers thick. Here, we employed a combination of two-photon excitation microscopy with fluorescence lifetime imaging to quantify the oxygen concentration in dental biofilm. Collisional quenching of fluorescent probes by molecular oxygen leads to a reduction of the fluorescence lifetime of the probe. We employed this mechanism to measure the oxygen concentration distribution in dental biofilm by means of fluorescence lifetime imaging. Here, TRIS Ruthenium chloride hydrate was used as an oxygen probe. A calibration procedure on buffers was use to measure the lifetime response of this Ruthenium probe. The results are in agreement with the Stern-Volmer equation. A linear relation was found between the ratio of the unquenched and the quenched lifetime and the oxygen concentration. The biofilm fluorescence lifetime imaging results show a strong oxygen gradient at the buffer - biofilm interface and the average oxygen concentration in the biofilm amounted to 50 μM.

The influence of calcium and pH on growth in primary roots of Zea mays

NASA Technical Reports Server (NTRS)

Hasenstein, K. H.; Evans, M. L.

1988-01-01

We investigated the interaction of Ca2+ and pH on root elongation in Zea mays L. cv. B73 x Missouri 17 and cv. Merit. Seedlings were raised to contain high levels of Ca2+ (HC, imbibed and raised in 10 mM CaCl2) or low levels of Ca2+ (LC, imbibed and raised in distilled water). In HC roots, lowering the pH (5 mM MES/Tris) from 6.5 to 4.5 resulted in strong, long-lasting growth promotion. Surprisingly, increasing the pH from 6.5 to 8.5 also resulted in strong growth promotion. In LC roots acidification of the medium (pH 6.5 to 4.5) resulted in transient growth stimulation followed by a gradual decline in the growth rate toward zero. Exposure of LC roots to high pH (pH shift from 6.5 to 8.5) also promoted growth. Addition of EGTA resulted in strong growth promotion in both LC and HC roots. The ability of EGTA to stimulate growth appeared not to be related to H+ release from EGTA upon Ca2+ chelation since, 1) LC roots showed a strong and prolonged response to EGTA, but only a transient response to acid pH, and 2) promotion of growth by EGTA was observed in strongly buffered solutions. We also examined the pH dependence of the release of 45Ca2+ from roots of 3-day-old seedlings grown from grains imbibed in 45Ca2+. Release of 45Ca2+ from the root into agar blocks placed on the root surface was greater the more acidic the pH of the blocks. The results indicate that Ca2+ may be necessary for the acid growth response in roots.

The Antemortem Detection and Conformational Switches of Prion Proteins

DTIC Science & Technology

2006-07-01

distribution of di-, mono-, and unglycosylated PrPC molecules (Fig. 7B, lane 2). Subsequent elution of the column with N-acetyl glucosamine yielded...pellet was washed in 50 mM Tris, pH 8.0, 150 mM NaCl, and then solubilized in 6 M guanidinium hydrochloride , 50 mM Tris pH 8.0, 150 mM NaCl. The...and 150mM sodium chloride (TCl) containing 6M guanidinium hydrochloride . The supernatant was centrifuged for 30min at 18,000g. The protein was

Polydopamine and Polydopamine-Silane Hybrid Surface Treatments in Structural Adhesive Applications.

PubMed

Tran, Ngon T; Flanagan, David P; Orlicki, Joshua A; Lenhart, Joseph L; Proctor, Kenneth L; Knorr, Daniel B

2018-01-30

Numerous studies have focused on the remarkable adhesive properties of polydopamine, which can bind to substrates with a wide range of surface energies, even under aqueous conditions. This behavior suggests that polydopamine may be an attractive option as a surface treatment in structural bonding applications, where good bond durability is required. Here, we assessed polydopamine as a surface treatment for bonding aluminum plates with an epoxy resin. A model epoxy adhesive consisting of diglycidyl ether of bisphenol A (DGEBA) and Jeffamine D230 polyetheramine was employed, and lap shear measurements (ASTM D1002 10) were made (i) under dry conditions to examine initial bond strength and (ii) after exposure to hot/wet (63 °C in water for 14 days) conditions to assess bond durability. Surprisingly, our results showed that polydopamine alone as a surface treatment provided no benefit beyond that obtained by exposing the substrates to an alkaline solution of tris buffer used for the deposition of polydopamine. This implies that polydopamine has a potential Achilles' heel, namely, the formation of a weak boundary layer that was identified using X-ray photoelectron spectroscopy (XPS) of the fractured surfaces. In fact, for longer deposition times (2.5 and 18 h), the tris buffer-treated surface outperformed the polydopamine surface treatments, suggesting that tris buffer plays a unique role in improving adhesive performance even in the absence of polydopamine. We further showed that the use of polydopamine-3-aminopropyltriethoxysilane (APTES) hybrid surface treatments provided significant improvements in bond durability at extended deposition times relative to both polydopamine and an untreated control.

Efficacy of a novel mucolytic agent on pseudomyxoma peritonei mucin, with potential for treatment through peritoneal catheters

PubMed Central

Akhter, Javed; Pillai, Krishna; Chua, Terence C; Alzarin, Naeef; Morris, David Lawson

2014-01-01

Compared to current treatment for pseudomyxoma peritonei (PMP), the extraction of solubilised mucin through peritoneal catheter can be minimally invasive. However, mucin has variable appearance that may influence mucolysis. Hence, we investigated the mucolysis of 36 mucin samples with a novel agent. Using visual inspection and hardness index, PMP mucin was classified into three grades. The mucin pathological category was identified from patient record. Subsequently, the dissolution of the samples was tested. For in vitro, 1 g of mucin was treated to the mucolytic agent in 10 ml TRIS buffer at 37 deg. Celsius for 3 hours, with weighing of residual mucin. Control treatment was similar but received TRIS buffer. For in vivo, 2 g of implanted intra-peritoneal mucin in nude rats was treated to mucolytic (2 X 500 ul/24 hr, over 48 hours, plus another treatment before sacrifice at 56 hours, with weighing of residual mucin. Controls were treated but only with TRIS buffer. Six animals were used for each mucin grade (3 mucolytic treated & and 3 controls). Grades of mucin were soft mucin (62%), semi hard (20%) and hard mucin (18%). Diffuse peritoneal adenomucinosis had 50% of soft mucin and peritoneal mucinous carcinoma had 11% (P = 0.0382). In vitro and in vivo absolute disintegration was 100% for soft, 57.38% and 48.67% for semi hard, 50% and 28.67% for hard mucin. Majority of mucin were soft with complete disintegration, the rest showed variable disintegration, suggesting that the mucolytic has potential for treating PMP. PMID:25232491

Efficient purification of Apolipoprotein A1 (ApoA1) from plasma by HEA HyperCel™: An alternative approach.

PubMed

G, Arun Govind; Kamalanathan, Agamudi Shivasankaran; Vijayalakshmi, Mookambeswaran Arunachalam; Venkataraman, Krishnan

2018-01-15

HDL-ApoA1 plays a pivotal role in the prevention of atherosclerosis and cardiovascular diseases. ApoA1 purification from blood plasma has always remained tedious, involving multiple steps, large volumes of plasma and substantial loss in the final yield of pure ApoA1. In this study, a two-step method has been developed and optimized for the purification of ApoA1 from plasma. Plasma was first subjected to 60% ammonium sulphate (NH 4 ) 2 SO 4 precipitation and subsequently, ApoA1 was recovered using mixed mode chromatographic sorbent, HEA HyperCel™. ApoA1 was found to be enriched in 60% (NH 4 ) 2 SO 4 supernatant that was dialyzed and injected onto HEA sorbent with 50 mM phosphate buffer pH 7.4. The bound proteins were eluted by decreasing the pH in step-gradient from pH 7.4 to pH 4.0 and subsequently to pH 3.5 using 50 mM sodium acetate buffer. Gel electrophoresis showed elution of homogeneous apoA1 at pH 3.5, with purity and yield of 63%. An interesting feature of this approach is that the purified ApoA1 was monomeric with a mass of 28,079.30 Da as confirmed by MS analysis. This simple and efficient method of purification of apoA1 serves as an alternative method which can be combined with traditional approaches and has a great potential for biochemical and clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Anodic voltammetric behavior and determination of cefixime in pharmaceutical dosage forms and biological fluids.

PubMed

Golcu, Ayşegul; Dogan, Burcu; Ozkan, Sibel A

2005-10-15

The voltammetric behavior of cefixime was studied using cyclic, linear sweep, differential pulse and square wave voltammetric techniques. The oxidation of cefixime was irreversible and exhibited diffusion controlled process depending on pH. The oxidation mechanism was proposed and discussed. Different parameters were tested to optimize the conditions for the determination of cefixime. The dependence of current intensities and potentials on pH, concentration, scan rate, nature of the buffer was investigated. According to the linear relationship between the peak current and the concentration, differential pulse (DPV) and square wave (SWV) voltammetric methods for cefixime assay in pharmaceutical dosage forms and biological fluids were developed. For the determination of cefixime were proposed in acetate buffer at pH 4.5, which allows quantitation over the 6 x 10(-6)-2 x 10(-4)M range in supporting electrolyte and spiked serum sample; 8 x 10(-6)-2 x 10(-4)M range in urine sample; 6 x 10(-6)-1 x 10(-4)M range in breast milk samples for both techniques. The repeatability, reproducibility, precision and accuracy of the methods in all media were investigated. No electroactive interferences from the excipients and endogenous substances were found in the pharmaceutical dosage forms and in the biological samples, respectively.

A phosphorus-free anolyte to enhance coulombic efficiency of microbial fuel cells

NASA Astrophysics Data System (ADS)

Tang, Xinhua; Li, Haoran; Du, Zhuwei; Ng, How Yong

2014-12-01

In this study, a phosphorus-free anolyte is prepared by using bicarbonate to replace phosphate buffer for application in two chamber microbial fuel cells (MFCs). Optical density test and Bradford protein assay shows that this phosphorus-free anolyte effectively inhibits the growth and reproduction of microorganisms suspended in the solution and greatly reduces the suspended cell mass. As a result, it considerably enhances the coulombic efficiency (CE) of MFCs. When the acetate concentration is 11 mM, the CE of the MFC using the pH 7 phosphate-containing anolyte is 9.7% and the CE with the pH 8.3 phosphate-containing anolyte is 9.1%, while the CE of the MFC using the phosphorus-free anolyte (pH 8.3) achieves 26.6%. This study demonstrates that this phosphorus-free anolyte holds the potential to enhance the feasibility for practical applications of MFCs.

Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries

PubMed Central

Fan, Yuwei; Wen, Zezhang T; Liao, Sumei; Lallier, Thomas; Hagan, Joseph L; Twomley, Jefferson T; Zhang, Jian-Feng; Sun, Zhi; Xu, Xiaoming

2013-01-01

Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels. PMID:23338820

Simultaneous determination of triacetin, acetic ether, butyl acetate and amorolfine hydrochloride in amorolfine liniment by HPLC.

PubMed

Gao, Yuan; Li, Li; Zhang, Jianjun; Shu, Wenjuan; Gao, Liqiong

2012-04-01

A simple, rapid, specific and precise reversed-phase high-performance liquid chromatographic method was developed for simultaneous estimation of triacetin, acetic ether, butyl acetate and amorolfine in marketed pharmaceutical liniment. Chromatographic separation was performed on a Shimadzu VP-ODS C(18) column using the mixture of citric acid-hydrochloric acid-sodium hydrate buffer (pH 3.0), acetonitrile and methanol (32:30:38) as the mobile phase at a flow rate of 1.0 mL/min with UV-detection at 215 nm. The method separated the four components simultaneously in less than 10 min. The validation of the method was performed with respect to specificity, linearity, accuracy, and precision. The calibration curves were linear in the range of 35.1-81.9 μ/mL for triacetin, 431.1-1005.9 μ/mL for acetic ether, 167.0-389.7 μ/mL for butyl acetate and 151.0-352.3 μ/mL for amorolfine. The mean 100% spiked recovery for triacetin, acetic ether, butyl acetate and amorolfine is 99.43 ± 0.42, 101.5 ± 1.09, 101.4 ± 1.02 and 100.8 ± 0.69, respectively. The intra-day and inter-day relative standard deviation values were <2.0%. The limits of detection of these compounds ranged from 0.08 to 5.88 ng. The utility of the procedure was verified by its application to the commercial liniment.

Structure and optical properties of ZnO produced from microwave hydrothermal hydrolysis of tris(ethylenediamine)zinc nitrate complex

NASA Astrophysics Data System (ADS)

Mostafa, Nasser Y.; Heiba, Zein K.; Ibrahim, Mohamed M.

2015-01-01

ZnO powders were synthesized using a solution microwave hydrothermal hydrolysis process and tris(ethylenediamine)zinc nitrate {[Zn(en)3](NO3)2} (en = ethylenediamine) as a precursor. Hydrolysis of the precursor complex at different pH produced zinc oxide with a diversity of well-defined morphologies. The effect of hydrolysis pH values on the structural and optical properties has been explored using XRD, SEM, and UV-visible diffuse reflectance spectroscopy (DRS). At pH = 7.0, randomly dispersed rods were formed. Whereas flower-like morphologies were obtained by treating the complex precursor in water at pH = 10.0 and 12.0. The ZnO4 tetrahedrons are greatly affected by the pH value. The band gap decreased sharply with increasing the pH value from 7.0 to 10.0, then slightly decreased with further increasing the pH to 12.0. The relationship between band gap and both structure and surface defects of the samples is also discussed.

Sarcolemmal mechanisms for pHi recovery from alkalosis in the guinea-pig ventricular myocyte

PubMed Central

Leem, Chae-Hun; Vaughan-Jones, Richard D

1998-01-01

The mechanism of pHi recovery from an intracellular alkali load (induced by acetate prepulse or by reduction/removal of ambient PCO2) was investigated using intracellular SNARF fluorescence in the guinea-pig ventricular myocyte. In Hepes buffer (pHo 7.40), pHi recovery was inhibited by removal of extracellular Cl−, but not by removal of Na+o or elevation of K+o. Recovery was unaffected by the stilbene drug DIDS (4,4-diisothiocyanatostilbene-disulphonic acid), but was slowed dose dependently by the stilbene drug DBDS (dibenzamidostilbene-disulphonic acid). In 5 % CO2/HCO3− buffer (pHo 7.40), pHi recovery was faster than in Hepes buffer. It consisted of an initial rapid recovery phase followed by a slow phase. Much of the rapid phase has been attributed to CO2-dependent buffering. The slow phase was inhibited completely by Cl− removal but not by Na+o removal or K+o elevation. At a test pHi of 7.30 in CO2/HCO3− buffer, the slow phase was inhibited 70 % by DIDS. The mean DIDS-inhibitable acid influx was equivalent in magnitude to the HCO3−-stimulated acid influx. Similarly, the DIDS-insensitive influx was equivalent to that estimated in Hepes buffer. We conclude that two independent sarcolemmal acid-loading carriers are stimulated by a rise of pHi and account for the slow phase of recovery from an alkali load. The results are consistent with activation of a DIDS-sensitive Cl−-HCO3− anion exchanger (AE) to produce HCO3− efflux, and a DIDS-insensitive Cl−-OH− exchanger (CHE) to produce OH− efflux. H+-Cl− co-influx as the alternative configuration for CHE is not, however, excluded. The dual acid-loading system (AE plus CHE), previously shown to be activated by a fall of extracellular pH, is thus activated by a rise of intracellular pH. Activity of the dual-loading system is therefore controlled by pH on both sides of the cardiac sarcolemma. PMID:9575297

A biocompatible synthesis of gold nanoparticles by Tris(hydroxymethyl)aminomethane

NASA Astrophysics Data System (ADS)

Chen, Feng; Wang, Yanwei; Ma, Jun; Yang, Guangcan

2014-05-01

Gold nanoparticles' novel properties are widely realized in catalysis, plasmonics, electronics, and biomedical applications. For biomedical application, one challenge is to find a non-toxic chemical and/or physical method of functionalizing gold nanoparticles with biomolecular compounds that can promote efficient binding, clearance, and biocompatibility and to assess their safety to other biological systems and their long-term effects on human health and reproduction. In the present study, we describe a new method by using Tris(hydroxymethyl)aminomethane (Tris), a widely used buffer solvent of nucleic acid and proteins, as the reducing agent for synthesizing gold nanoparticles by one step. It is found that Tris carries out the reduction reactions in relatively mild conditions for biomacromolecules. Particularly, it can be used to modify the DNA during the process of preparation of gold nanoparticles. The morphology and size distribution of gold nanoparticles are consistent and were confirmed by many different approaches including dynamic light scattering (DLS), UV-visible (UV-vis) spectrophotometry, atomic force microscopy (AFM), and transmission electron microscopy (TEM).

Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin.

PubMed

Perry, Jennifer L; Goldsmith, Michael R; Williams, T Richard; Radack, Kyle P; Christensen, Trine; Gorham, Justin; Pasquinelli, Melissa A; Toone, Eric J; Beratan, David N; Simon, John D

2006-01-01

Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W-) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 x 10(5) M(-1). ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W- is accompanied by uptake of 0.30+/-0.02 protons from the solvent. This measurement suggests that the binding of W- is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W-.

A macrocyclic polyamine as an anion receptor in the capillary electrochromatographic separation of carbohydrates.

PubMed

Liu, Chuen-Ying; Chen, Tse-Hsien; Misra, Tarun Kumar

2007-06-22

An analytical approach of the 32-membered macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N8) was described for the capillary electrochromatographic (CEC) separation of derivatized mono- and disaccharides. The column displayed reversal electroosmotic flow (EOF) at pH below 7.0, while a cathodic EOF was shown at pH above 7.0. The reductive amination of saccharides was carried out with p-aminobenzoic acid. Some parameters that affect the CEC separations were investigated. Several competitive ligands, such as Tris, EDTA and phosphate were also examined for the effect on the performance. We achieved a complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side). The relative standard deviation of the retention time measured for each sample was less than 4% in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. With the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves.

PubMed

Oztürk, Lokman; Bülbül, Metin; Elmastas, Mahfuz; Ciftçi, Mehmet

2007-01-01

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.

Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata.

PubMed

Ishisaki, Kana; Arai, Sachiko; Hamada, Tatsuro; Honda, Yuji

2012-11-01

A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)(n) (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)(6). The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)(6) hydrolytic reaction was 3.9 at 37 °C. The optimal temperature for activity was 65 °C in 50 mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid. Copyright © 2012 Elsevier Ltd. All rights reserved.

Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli

PubMed Central

Giri, Janhavi; Tang, John M.; Wirth, Christophe; Peneff, Caroline M.

2012-01-01

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (Vreversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer. PMID:22246445

The dynamics of the metabolism of acetate and bicarbonate associated with use of hemodialysates in the ABChD trial: a phase IV, prospective, single center, single blind, randomized, cross-over, two week investigation.

PubMed

Smith, William B; Gibson, Sandy; Newman, George E; Hendon, Kendra S; Askelson, Margarita; Zhao, James; Hantash, Jamil; Flanagan, Brigid; Larkin, John W; Usvyat, Len A; Thadhani, Ravi I; Maddux, Franklin W

2017-08-29

In the United States, hemodialysis (HD) is generally performed via a bicarbonate dialysate. It is not known if small amounts of acid used in dialysate to buffer the bicarbonate can meaningfully contribute to overall buffering administered during HD. We aimed to investigate the metabolism of acetate with use of two different acid buffer concentrates and determine if it effects blood bicarbonate concentrations in HD patients. The Acid-Base Composition with use of hemoDialysates (ABChD) trial was a Phase IV, prospective, single blind, randomized, cross-over, 2 week investigation of peridialytic dynamics of acetate and bicarbonate associated with use of acid buffer concentrates. Eleven prevalent HD patients participated from November 2014 to February 2015. Patients received two HD treatments, with NaturaLyte® and GranuFlo® acid concentrates containing 4 and 8 mEq/L of acetate, respectively. Dialysate order was chosen in a random fashion. The endpoint was to characterize the dynamics of acetate received and metabolized during hemodialysis, and how it effects overall bicarbonate concentrations in the blood and dialysate. Acetate and bicarbonate concentrations were assessed before, at 8 time points during, and 6 time points after the completion of HD. Data from 20 HD treatments for 11 patients (10 NaturaLyte® and 10 GranuFlo®) was analyzed. Cumulative trajectories of arterialized acetate were unique between NaturaLyte® and GranuFlo® (p = 0.003), yet individual time points demonstrated overlap without remarkable differences. Arterialized and venous blood bicarbonate concentrations were similar at HD initiation, but by 240 min into dialysis, mean arterialized bicarbonate concentrations were 30.2 (SD ± 4.16) mEq/L in GranuFlo® and 28.8 (SD ± 4.26) mEq/L in NaturaLyte®. Regardless of acid buffer concentrate, arterial blood bicarbonate was primarily dictated by the prescribed bicarbonate level. Subjects tolerated HD with both acid buffer concentrates without experiencing any related adverse events. A small fraction of acetate was delivered to HD patients with use of NaturaLyte® and GranuFlo® acid buffers; the majority of acetate received was observed to be rapidly metabolized and cleared from the circulation. Blood bicarbonate concentrations appear to be determined mainly by the prescribed concentration of bicarbonate. This trial was registered on ClinicalTrials.gov on 11 Dec 2014 ( NCT02334267 ).

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm.

PubMed

Mohammadi, M R; Vadamalai, G; Joseph, H

2010-01-01

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.

Determination of josamycin residues in porcine tissues using high-performance liquid chromatography with pre-column derivatization and spectrofluorimetric detection.

PubMed

Leroy, P; Decolin, D; Nicolas, A; Archimbault, P

1994-12-01

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of josamycin residues in four porcine tissues (i.e., muscle, liver, kidney and fat). The sample preparation consisted of a homogenization step in an acetonitrile-10 mmol l-1 phosphate buffer mixture, pH 6.0 (35 + 65), centrifugation and a liquid-liquid extractive clean-up of the resulting supernatant with isooctane. Pre-column derivatization of josamycin was performed using cyclohexa-1,3-dione in ammonium acetate buffer, pH 5.0 (90 degrees C for 2 h). The derivative was chromatographed in an isocratic reversed-phase HPLC system. A LiChrospher RP 18 end-capped (5 microns) column was eluted with an acetonitrile-methanol-10 mmol l-1 phosphate buffer mixture, pH 6.0 (45 + 5 + 50). The capacity factor of the josamycin derivative was 17.5. Detection was achieved using spectrofluorimetry (lambda ex = 375 nm; lambda em = 450 nm). The structure of the derivative was assessed by using mass spectrometry. Full selectivity was obtained in the HPLC system versus other macrolide antibiotics (tylosin, spiramycin and erythromycin), aldehydes (formaldehyde, acetaldehyde and benzaldehyde) and endogenous compounds. Linearity and repeatability were tested. Correlation coefficients, for calibration curves in the range of 0.1-3.2 micrograms g-1, were greater than 0.999 for all tissues and the relative standard deviation (S(r)) was 4.9% (1.6 micrograms g-1; n = 6); recovery was higher than 88%.

A mathematical model of the influence of salivary urea on the pH of fasted dental plaque and on the changes occurring during a cariogenic challenge.

PubMed

Dibdin, G H; Dawes, C

1998-01-01

Urea diffusing from saliva into dental plaque is converted to ammonia and carbon dioxide by bacterial ureases. The influence of normal salivary urea levels on the pH of fasted plaque and on the depth and duration of a Stephan curve is uncertain. A numerical model which simulates a cariogenic challenge (a 10% sucrose rinse alone or one followed by use of chewing-gum with or without sugar) was modified to include salivary urea levels from 0 to 30 mmol/l. It incorporated: site-dependent exchange between bulk saliva and plaque surfaces via a salivary film; sugar and urea diffusion into plaque; pH-dependent rates of acid formation and urea breakdown; diffusion and dissociation of end-products and other buffers (acetate, lactate, phosphate, ammonia and carbonate); diffusion of protons and other ions; equilibration with fixed and mobile buffers; and charge-coupling between ionic flows. The Km (2.12 mmol/l) and Vmax (0.11 micromol urea/min/mg dry weight) values for urease activity and the pH dependence of Vmax were taken from the literature. From the results, it is predicted that urea concentrations normally present in saliva (3-5 mmol/l) will increase the pH at the base of a 0.5-mm-thick fasted plaque by up to 1 pH unit, and raise the pH minimum after a sucrose rinse or sugar-containing chewing-gum by at least half a pH unit. The results suggest that plaque cariogenicity may be inversely related to salivary urea concentrations, not only when the latter are elevated because of disease, but even when they are in the normal range.

Assessment of Mexican Arnica (Heterotheca inuloides Cass) and Rosemary (Rosmarinus officinalis) Extracts on Dopamine and Selected Biomarkers of Oxidative Stress in Stomach and Brain of Salmonella typhimurium Infected rats.

PubMed

Guzmàn, David Calderón; Herrera, Maribel Ortiz; Brizuela, Norma Osnaya; Mejía, Gerardo Barragàn; García, Ernestina Hernàndez; Olguín, Hugo Juàrez; Peraza, Armando Valenzuela; Ruíz, Norma Labra; Del Angel, Daniel Santamaría

2017-01-01

The effects of some natural products on dopamine (DA) and 5-hydroxyindole acetic acid (5-HIAA) in brain of infected models are still unclear. The purpose of this study was to measure the effect of Mexican arnica/rosemary (MAR) water extract and oseltamivir on both biogenic amines and some oxidative biomarkers in the brain and stomach of young rats under infection condition. Female Wistar rats (weight 80 g) in the presence of MAR or absence (no-MAR) were treated as follows: group 1, buffer solution (controls); oseltamivir (100 mg/kg), group 2; culture of Salmonella typhimurium ( S.Typh ) (1 × 10 6 colony-forming units/rat) group 3; oseltamivir (100 mg/kg) + S.Typh (same dose) group 4. Drug and extracts were administered intraperitoneally every 24 h for 5 days, and S.Typh was given orally on days 1 and 3. On the fifth day, blood was collected to measure glucose and hemoglobin. The brains and stomachs were obtained to measure levels of DA, 5-HIAA, glutathione (GSH), TBARS, H 2 O 2 , and total ATPase activity using validated methods. DA levels increased in MAR group treated with oseltamivir alone but decreased in no-MAR group treated with oseltamivir plus S.Typh . 5-HIAA, GSH, and H 2 O 2 decreased in this last group, and ATPase activity increased in MAR group treated with oseltamivir plus S.Typh . TBARS (lipid peroxidation) increased in MAR group that received oseltamivir alone. Most of the biomarkers were not altered significantly in the stomach. MAR extract alters DA and metabolism of 5-HIAA in the brain of young animals infected. Antioxidant capacity may be involved in these effects. The purpose of this study was to measure the effect of Mexican arnica/rosemary water extract and oseltamivir on both biogenic amines and some oxidative biomarkers in the brain and stomach of young rats under infection condition. Results: Mexican arnica and rosemary extract alter dopamine and metabolism of 5-HIAA in the brain of young animals infected. Antioxidant capacity may be involved in these effects. Abbreviations used: AS: Automated system, ATP: Adenosine triphosphate, CNS: Central nervous system, CFU: Colony-forming unit, DA: Dopamine EDTA: Ethylenediaminetetraacetic acid, 5-HIAA: Äcido 5-hidroxindolacético (serotonina), GABA: γ-aminobutyric acid, GSH: Glutathione, H2O2: Hidrogen peroxide, HCLO4: Perchloric acid, iNOS: Inducible nitric oxide synthase, LPS: Lipopolysaccharides, MAR: Arnica/Rosemary, NaCl: Sodium Chloride, NOGSH: nitrosoglutathione, NOS: Nitric oxide, OPT: Ortho-phtaldialdehyde, Pbs: Phosphate buffered saline, pH: potential of Hydrogen, Pi: Inorganic phosphate, ROS: Reactive oxygen species, RNSs: Reactive nitrogen species Tba: Thiobarbaturic acid, TBARS: Thiobarbituric aid reactive, Tca: Trichloroacetic, Tris-HCL: Tris hydrochloride, TSA: Trypticasein Soya Agar.

Assessment of Mexican Arnica (Heterotheca inuloides Cass) and Rosemary (Rosmarinus officinalis) Extracts on Dopamine and Selected Biomarkers of Oxidative Stress in Stomach and Brain of Salmonella typhimurium Infected rats

PubMed Central

Guzmàn, David Calderón; Herrera, Maribel Ortiz; Brizuela, Norma Osnaya; Mejía, Gerardo Barragàn; García, Ernestina Hernàndez; Olguín, Hugo Juàrez; Peraza, Armando Valenzuela; Ruíz, Norma Labra; Del Angel, Daniel Santamaría

2017-01-01

Background: The effects of some natural products on dopamine (DA) and 5-hydroxyindole acetic acid (5-HIAA) in brain of infected models are still unclear. Objective: The purpose of this study was to measure the effect of Mexican arnica/rosemary (MAR) water extract and oseltamivir on both biogenic amines and some oxidative biomarkers in the brain and stomach of young rats under infection condition. Methods: Female Wistar rats (weight 80 g) in the presence of MAR or absence (no-MAR) were treated as follows: group 1, buffer solution (controls); oseltamivir (100 mg/kg), group 2; culture of Salmonella typhimurium (S.Typh) (1 × 106 colony-forming units/rat) group 3; oseltamivir (100 mg/kg) + S.Typh (same dose) group 4. Drug and extracts were administered intraperitoneally every 24 h for 5 days, and S.Typh was given orally on days 1 and 3. On the fifth day, blood was collected to measure glucose and hemoglobin. The brains and stomachs were obtained to measure levels of DA, 5-HIAA, glutathione (GSH), TBARS, H2O2, and total ATPase activity using validated methods. Results: DA levels increased in MAR group treated with oseltamivir alone but decreased in no-MAR group treated with oseltamivir plus S.Typh. 5-HIAA, GSH, and H2O2 decreased in this last group, and ATPase activity increased in MAR group treated with oseltamivir plus S.Typh. TBARS (lipid peroxidation) increased in MAR group that received oseltamivir alone. Most of the biomarkers were not altered significantly in the stomach. Conclusion: MAR extract alters DA and metabolism of 5-HIAA in the brain of young animals infected. Antioxidant capacity may be involved in these effects. SUMMARY The purpose of this study was to measure the effect of Mexican arnica/rosemary water extract and oseltamivir on both biogenic amines and some oxidative biomarkers in the brain and stomach of young rats under infection condition. Results: Mexican arnica and rosemary extract alter dopamine and metabolism of 5-HIAA in the brain of young animals infected. Antioxidant capacity may be involved in these effects. Abbreviations used: AS: Automated system, ATP: Adenosine triphosphate, CNS: Central nervous system, CFU: Colony-forming unit, DA: Dopamine EDTA: Ethylenediaminetetraacetic acid, 5-HIAA: Äcido 5-hidroxindolacético (serotonina), GABA: γ-aminobutyric acid, GSH: Glutathione, H2O2: Hidrogen peroxide, HCLO4: Perchloric acid, iNOS: Inducible nitric oxide synthase, LPS: Lipopolysaccharides, MAR: Arnica/Rosemary, NaCl: Sodium Chloride, NOGSH: nitrosoglutathione, NOS: Nitric oxide, OPT: Ortho-phtaldialdehyde, Pbs: Phosphate buffered saline, pH: potential of Hydrogen, Pi: Inorganic phosphate, ROS: Reactive oxygen species, RNSs: Reactive nitrogen species Tba: Thiobarbaturic acid, TBARS: Thiobarbituric aid reactive, Tca: Trichloroacetic, Tris-HCL: Tris hydrochloride, TSA: Trypticasein Soya Agar PMID:28539708

Heterotrophic Nature of the Cell-Free Protein-Synthesizing System from the Strict Chemolithotroph, Thiobacillus thiooxidans

PubMed Central

Amemiya, K.; Umbreit, W. W.

1974-01-01

A cell-free protein-synthesizing system prepared from the strict chemolithotroph, Thiobacillus thiooxidans, was similar to that of heterotrophs. The poly-U directed system had a temperature optimum of 37 C, but in the presence of spermidine (3 mM) the optimum shifted to 45 C. Although growth of the chemolithotroph occurs only in acid conditions, the pH optimum for the cell-free system was pH 7.2. The endogenous-directed activity in the presence or absence of spermidine was maximal at pH 7.8. Spermidine had a stimulatory effect; however, this effect was dependent on the magnesium and tris(hydroxymethyl)aminomethane (Tris) concentrations. At low Tris concentrations (10 mM), spermidine (3 to 5 mM) could completely replace magnesium. When the Tris concentration was increased (50 mM), spermidine could not replace magnesium. Supernatant and ribosomal fractions from T. thiooxidans were exchanged with those of Bacillus thuringiensis and Escherichia coli, and the ribosomal fraction from the chemolithotroph gave good to moderate stimulation when exchanged with the supernatant from the heterotrophs. On the other hand, the supernatant from T. thiooxidans gave good stimulation when mixed with ribosomes from B. thuringiensis but poor activity with ribosomes from E. coli. Both supernatant and ribosomal fractions prepared from stationary phase extracts of T. thiooxidans were inactive in the cell-free system. PMID:4590488

Enantioselective determination of (R)-zopiclone and (S)-zopiclone (eszopiclone) in human hair by micropulverized extraction and chiral liquid chromatography/high resolution mass spectrometry.

PubMed

Miyaguchi, Hajime; Kuwayama, Kenji

2017-10-13

Zopiclone and its (S)-enantiomer (eszopiclone) are commonly prescribed for insomnia. Despite the high demand for enantioselective differentiation, the chiral analysis of zopiclone in hair has not been reported. In this study, a method for the enantioselective quantification of zopiclone in human hair was developed. The extraction medium and duration were optimized using real eszopiclone-positive hair samples. Specifically, micropulverized extraction with 3.0M ammonium phosphate buffer (pH 8.4) involving salting-out assisted liquid-liquid extraction with acetonitrile was utilized to minimize the degradation of zopiclone and for rapid and facile operation. On the other hand, recovery of the conventional solid-liquid extraction involved overnight soaking in 3.0M ammonium phosphate buffer (pH 8.4) was only 0.58±0.12% of the maximum recovery achieved by the present method due to the decomposition in the phosphate buffer. An excellent chiral separation (Rs=5.0) was achieved using a chiral stationary phase comprising cellulose tris(3,5-dichlorophenylcarbamate) and a volatile mobile phase of 10mM ammonium carbonate (pH 8.0)-acetonitrile (25:75, v/v). Detection was carried out using liquid chromatography/high resolution mass spectrometry (LC/HRMS) with electrospray ionization. A Q Exactive mass spectrometer equipped with a quadrupole-Orbitrap analyzer was used for detection. The concentration of 0.50pg/mg was defined as the lowest limit of quantification using 5mg of hair sample. Using the developed approach, the concentration of eszopiclone in hair after a single 2-mg dose was found to be 441pg/mg, which was higher than all the reported values regarding a single administration of zopiclone. After daily administration of racemic zopiclone (3.75mg/day), the concentrations of (R)-enantiomer and (S)-enantiomer in the black hair were 5.30-8.31ng/mg and 7.96-12.8ng/mg, respectively, and the concentration of the (S)-enantiomer was always higher than that of the (R)-enantiomer due to the enantioselective difference in the pharmacokinetics. Copyright © 2017 Elsevier B.V. All rights reserved.

Control of Hepatic Glucose Metabolism by the Oral Hypoglycemic Sulfonylureas

DTIC Science & Technology

1984-05-11

another In vitro study, Vignerl, et al. (1982) were unable to detect any effect on Insulin binding to MCF-7 human breast cancer cells, IM-9 human cultured...according to the method of Huljlng (1970). Aspirgillus niger amyloglucosidase (1,4 a- glucan glucohy- drolase; E.C. 3.2.1.3), (0.5 U) and 0.38 U of porcine...pancreatic a-amylase (1,6 a- glucan glucohydrolase; E.C. 3.2.1.1), in 1.0 ml of 100 mM sodium acetate buffer (pH 4.8) were added to the glycogen 34

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns.

PubMed

Rocco, Anna; Maruška, Audrius; Fanali, Salvatore

2012-03-01

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R(s) = 1.80 for naproxen to R(s) = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R(s) value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R(s) = 0.89).

Dual-Analyte Fluorescent Sensor Based on [5]Helicene Derivative with Super Large Stokes Shift for the Selective Determinations of Cu2+ or Zn2+ in Buffer Solutions and Its Application in a Living Cell.

PubMed

Sakunkaewkasem, Siwakorn; Petdum, Anuwut; Panchan, Waraporn; Sirirak, Jitnapa; Charoenpanich, Adisri; Sooksimuang, Thanasat; Wanichacheva, Nantanit

2018-05-10

A new fluorescent sensor, M201-DPA, based on [5]helicene derivative was utilized as dual-analyte sensor for determination of Cu 2+ or Zn 2+ in different media and different emission wavelengths. The sensor could provide selective and bifunctional determination of Cu 2+ in HEPES buffer containing Triton-X100 and Zn 2+ in Tris buffer/methanol without interference from each other and other ions. In HEPES buffer, M201-DPA demonstrated the selective ON-OFF fluorescence quenching at 524 nm toward Cu 2+ . On the other hand, in Tris buffer/methanol, M201-DPA showed the selective OFF-ON fluorescence enhancement upon the addition of Zn 2+ , which was specified by the hypsochromic shift at 448 nm. Additionally, M201-DPA showed extremely large Stokes shifts up to ∼150 nm. By controlling the concentration of Zn 2+ and Cu 2+ in a living cell, the imaging of a HepG2 cellular system was performed, in which the fluorescence of M201-DPA in the blue channel was decreased upon addition of Cu 2+ and was enhanced in UV channel upon addition of Zn 2+ . The detection limits of M201-DPA for Cu 2+ and Zn 2+ in buffer solutions were 5.6 and 3.8 ppb, respectively. Importantly, the Cu 2+ and Zn 2+ detection limits of the developed sensors were significantly lower than permitted Cu 2+ and Zn 2+ concentrations in drinking water as established by the U.S. EPA and WHO.

A simple spectrophotometric method for the determination of arsenic in industrial and environmental samples using 2,4-Dihydroxy benzophenone-2-amino thiophenol.

PubMed

Deepa, K; Lingappa, Y

2014-04-24

2,4-Dihydroxy benzophenone-2-amino thiophenol (BPBT) has been proposed as new analytical reagent for the direct non-extractive spectrophotometric determination of arsenic. The reagent reacts with arsenic in acidic medium (pH=6.0, sodium acetate-acetic acid buffer) to form light greenish yellow colored 1:1 (M:L) complex. Maximum absorbance was obtained at 343 nm and remains constant for over 24 h. The molar absorptivity and Sandell's sensitivity of BPBT are found to be 6.01×10(4) L mol(-1)cm(-1) and 0.0016 μg cm(-2) respectively. The system obeys Beer's law in the range of 0.125-2.637 μg/ml of As (III). Since BPBT method is more sensitive, it was applied for the determination of arsenic in some environmental water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Syringe-cartridge solid-phase extraction method for patulin in apple juice.

PubMed

Eisele, Thomas A; Gibson, Midori Z

2003-01-01

A syringe-cartridge solid-phase extraction (SPE) method was developed for determination of patulin in apple juice. A 2.5 mL portion of test sample was passed through a conditioned macroporous SPE cartridge and washed with 2 mL 1% sodium bicarbonate followed by 2 mL 1% acetic acid. Patulin was eluted with 1 mL 10% ethyl acetate in ethyl ether and determined by reversed-phase liquid chromatography using a mobile phase consisting of 81% acetonitrile, 9% water, and 10% 0.05M potassium phosphate buffer, pH 2.4. Recoveries averaged 92% and the relative standard deviation was 8.0% in test samples spiked with 50 ng/mL patulin. The method appears to be applicable for monitoring apple juice samples to meet the U.S. Food and Drug Administration compliance action level of 50 microg/kg in an industrial quality assurance laboratory environment.

Determination of gallium at trace levels using a spectrofluorimetric method in synthetic U-Ga and Ga-As solutions.

PubMed

Kara, Derya; Fisher, Andrew; Foulkes, Mike; Hill, Steve J

2010-01-01

A simple, easy to use and selective spectrofluorimetric method for the determination of trace levels of gallium has been developed. A new Schiff base, N-o-vanillidine-2-amino-p-cresol (OVAC) was synthesized and its fluorescence activity with gallium investigated. Based on this chelation reaction, a spectrofluorimetric method has been developed for the determination of gallium in synthetically prepared Ga-U and Ga-As samples buffered at pH 4.0 using acetic acid-sodium acetate. The chelation reaction between Ga(III) and N-o-vanillidine-2-amino-p-cresol was very fast, requiring only 30min at room temperature to complex completely. The limit of detection (LOD) (3sigma) for Ga(III) was 7.17 nM (0.50 microgL(-1)), determined from the analysis of 11 different solutions of 20 microg L(-1) Ga(III). Copyright 2009 Elsevier B.V. All rights reserved.

Speciation of chromium (VI) and total chromium determination in welding dust samples by flow-injection analysis coupled to atomic absorption spectrometry.

PubMed

Girard, L; Hubert, J

1996-11-01

We have studied the speciation of chromium (VI) in stainless-steel welding dusts. The approach used for the analysis of Cr(VI) and total Cr relies on a flow-injection analyzer (FIA) equipped with two different sequential detectors. The system measures Cr(VI). by colorimetry (with 1,5-diphenyl carbohydrazide) and total chromium content by flame atomic absorption spectroscopy (AAS). The extraction of the samples of welding-fume dusts is achieved in a buffer solution (acetic acid and sodium acetate at pH 4). This extraction procedure gives a 96% recovery of chromium (VI). The FIA-AAS system that has been described is also more sensitive, has a lower detection limit (0.005 mug ml(-1)) and gives a better precision (< 1%) than other equivalent systems that have been previously described.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaramurthi, Prakash; Suryanarayanan, Raj

To effectively inhibit succinate buffer crystallization and the consequent pH changes in frozen solutions. Using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), the crystallization behavior of succinate buffer in the presence of either (i) a crystallizing (glycine, mannitol, trehalose) or (ii) a non-crystallizing cosolute (sucrose) was evaluated. Aqueous succinate buffer solutions, 50 or 200 mM, at pH values 4.0 or 6.0 were cooled from room temperature to -25 C at 0.5 C/min. The pH of the solution was measured as a function of temperature using a probe designed to function at low temperatures. The final lyophiles prepared from thesemore » solutions were characterized using synchrotron radiation. When the succinic acid solution buffered to pH 4.0, in the absence of a cosolute, was cooled, there was a pronounced shift in the freeze-concentrate pH. Glycine and mannitol, which have a tendency to crystallize in frozen solutions, remained amorphous when the initial pH was 6.0. Under this condition, they also inhibited buffer crystallization and prevented pH change. At pH 4.0 (50 mM initial concentration), glycine and mannitol crystallized and did not prevent pH change in frozen solutions. While sucrose, a non-crystallizing cosolute, did not completely prevent buffer crystallization, the extent of crystallization was reduced. Sucrose decomposition, based on XRD peaks attributable to {beta}-D-glucose, was observed in frozen buffer solutions with an initial pH of 4.0. Trehalose completely inhibited crystallization of the buffer components when the initial pH was 6.0 but not at pH 4.0. At the lower pH, the crystallization of both trehalose dihydrate and buffer components was evident. When retained amorphous, sucrose and trehalose effectively inhibited succinate buffer component crystallization and the consequent pH shift. However, when trehalose crystallized or sucrose degraded to yield a crystalline decomposition product, crystallization of buffer was observed. Similarly, glycine and mannitol, two widely used bulking agents, inhibited buffer component crystallization only when retained amorphous. In addition to stabilizing the active pharmaceutical ingredient, lyoprotectants may prevent solution pH shift by inhibiting buffer crystallization.« less

[Chiral separation of five beta-blockers using di-n-hexyl L-tartrate-boric acid complex as mobile phase additive by reversed-phase liquid chromatography].

PubMed

Yang, Juan; Wang, Lijuan; Guo, Qiaoling; Yang, Gengliang

2012-03-01

A reversed-phase high performance liquid chromatographic (HPLC) method using the di-n-hexyl L-tartrate-boric acid complex as a chiral mobile phase additive was developed for the enantioseparation of five beta-blockers including propranolol, esmolol, metoprolol, bisoprolol and sotalol. In order to obtain a better enantioseparation, the influences of concentrations of di-n-butyl L-tartrate and boric acid, the type, concentration and pH of the buffer, methanol content as well as the molecular structure of analytes were extensively investigated. The separation of the analytes was performed on a Venusil MP-C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase was 15 mmol/L ammonium acetate-methanol containing 60 mmol/L boric acid, 70 mmol/L di-n-hexyl L-tartrate (pH 6.00). The volume ratios of 15 mmol/L ammonium acetate to methanol were 20: 80 for propranolol, esmolol, metoprolol, bisoprolol and 30: 70 for sotalol. The flow rate was 0.5 mL/min and the detection wavelength was set at 214 nm. Under the optimized conditions, baseline enantioseparation was obtained separately for the five pairs of analytes.

Novel RuCoSe as non-platinum catalysts for oxygen reduction reaction in microbial fuel cells

NASA Astrophysics Data System (ADS)

Rozenfeld, Shmuel; Schechter, Michal; Teller, Hanan; Cahan, Rivka; Schechter, Alex

2017-09-01

Microbial electrochemical cells (MECs) are explored for the conversion of acetate directly to electrical energy. This device utilizes a Geobacter sulfurreducens anode and a novel RuCoSe air cathode. RuCoSe synthesized in selected compositions by a borohydride reduction method produces amorphous structures of powdered agglomerates. Oxygen reduction reaction (ORR) was measured in a phosphate buffer solution pH 7 using a rotating disc electrode (RDE), from which the kinetic current (ik) was measured as a function of potential and composition. The results show that ik of RuxCoySe catalysts increases in the range of XRu = 0.25 > x > 0.7 and y < 0.15 for all tested potentials. A poisoning study of RuCoSe and Pt catalysts in a high concentration acetate solution shows improved tolerance of RuCoSe to this fuel at acetate concentration ≥500 mM. MEC discharge plots under physiological conditions show that ∼ RuCo2Se (sample S3) has a peak power density of 750 mW cm-2 which is comparable with Pt 900 mW cm-2.

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study.

PubMed

Lee, Heon-Woo; Seo, Ji-Hyung; Choi, Seung-Ki; Lee, Kyung-Tae

2007-01-30

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5>166.1 for itopride and m/z 342.3>111.6 for IS, respectively. Analytes were chromatographed on an YMC C18 reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r2=0.9999) over the studied range (0.5-1000 ng mL(-1)) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

Pyclen Tri-n-butylphosphonate Ester as Potential Chelator for Targeted Radiotherapy: From Yttrium(III) Complexation to (90)Y Radiolabeling.

PubMed

Le Fur, Mariane; Beyler, Maryline; Lepareur, Nicolas; Fougère, Olivier; Platas-Iglesias, Carlos; Rousseaux, Olivier; Tripier, Raphaël

2016-08-15

The Y(3+) complex of PCTMB, the tri-n-butyl phosphonate ester of pyclen (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene), was synthesized as well as its Ho(3+) and Lu(3+) analogues. X-ray diffraction analyses revealed isomorphous dimeric M2(PCTMB)2·9H2O (M = Y, Ho, Lu) structures that crystallize in the centrosymmetric P1̅ triclinic space group. (1)H NMR and UV studies in aqueous solutions indicated that Y(3+) complexation is fast, being quantitative in 167 min at pH 3.8 and in 13 min at pH 5.5 (25 °C, acetate buffer, I = 0.150 M, [Y(3+)] = [PCTMB] = 0.2 mM). (1)H NMR DOSY and photon correlation spectroscopy experiments evidenced the formation of aggregates in chloroform with a bimodal distribution that changes slightly with concentration (11-24 and 240-258 nm). The behavior of the acid-assisted dissociation of the complex of Y(3+) with PCTMB was studied under pseudo-first-order conditions, and the half-life of the [Y(PCTMB)] complex in 0.5 M HCl at 25 °C was found to be 37 min, a value that decreases to 2.6 min in 5 M HCl. The Y(3+) complex of PCTMB is thermodynamically very stable, with a stability constant of log KY-PCTMB = 19.49 and pY = 16.7 measured by potentiometry. (90)Y complexation studies revealed fast radiolabeling kinetics; optimal radiolabeling conditions were obtained for (90)Y in acetate medium, PCTMB at 10(-4) to 10(-2) M in acetate buffer pH = 4.75, 15 min at 45-60 °C. In vitro stability studies in human serum showed that [(90)Y(PCTMB)] is quite stable, with about 90% of the activity still in the form of the radiotracer at 24 h and 80% from 48 h to 72 h. A comparison with other ligands such as PCTA, DOTA, and DTPA already used for in vivo application shows that [(90)Y(PCTMB)] is an interesting lipophilic and neutral analogue of these reference chelates for therapeutic applications in aqueous and nonaqueous media.

Glass corrosion in natural environments

NASA Technical Reports Server (NTRS)

Thorpe, Arthur N.; Barkatt, Aaron

1992-01-01

Experiments carried out during the progress period are summarized. Experiments carried out involving glass samples exposed to solutions of Tris have shown the appearance of 'spikes' upon monitoring glass dissolution as a function of time. The periodic 'spikes' observed in Tris-based media were interpreted in terms of cracking due to excessive stress in the surface region of the glass. Studies of the interactions of silicate glasses with metal ions in buffered media were extended to systems containing Al. Caps buffer was used to establish the pH. The procedures used are described and the results are given. Preliminary studies were initiated as to the feasibility of adding a slowly dissolving solid compound of the additive to the glass-water system to maintain a supply of dissolved additive. It appears that several magnesium compounds have a suitable combination of solubility and affinity towards silicate glass surfaces to have a pronounced retarding effect on the extraction of uranium from the glass. These preliminary findings raise the possibility that introducing a magnesium source into geologic repositories for nuclear waste glass in the form of a sparingly soluble Mg-based backfill material may cause a substantial reduction in the extent of long-term glass corrosion. The studies described also provide mechanistic understanding of the roles of various metal solutes in the leachant. Such understanding forms the basis for developing long-term predictions of nuclear waste glass durability under repository conditions. From what is known about natural highly reduced glasses such as tektites, it is clear that iron is dissolved as ferrous iron with little or no ferric iron. The reducing conditions were high enough to cause metallic iron to exsolve out of the glass in the form of submicroscopic spherules. As the nuclear waste glass is much less reduced, a study was initiated on other natural glasses in addition to the nuclear waste glass. Extensive measurements were carried out on these glasses in order to characterize their magnetic properties. Results of these studies are described.

Relative contribution of ruminal buffering systems to pH regulation in feedlot cattle fed either low- or high-forage diets.

PubMed

Chibisa, G E; Beauchemin, K A; Penner, G B

2016-07-01

The relative contribution of ruminal short-chain fatty acid (SCFA) absorption and salivary buffering to pH regulation could potentially change under different dietary conditions. Therefore, the objective of this study was to investigate the effects of altering the ruminal supply of rapidly fermentable carbohydrate (CHO) on absorptive function and salivation in beef cattle. Eight heifers (mean BW±SD=410±14 kg) were randomly allocated to two treatments in a crossover design with 37-day periods. Dietary treatments were barley silage at 30% low forage (LF) or 70% high forage (HF) of dietary dry matter (DM), with the remainder of the diet consisting of barley grain (65% or 25% on a DM basis) and a constant level (5%) of supplement. The LF and HF diets contained 45.3% and 30.9% starch, and 4.1% and 14.0% physically effective fiber (DM basis), respectively. Ruminal pH was continuously measured from day 17 to day 23, whereas ruminal fluid was collected on day 23 to determine SCFA concentration. Ruminal liquid passage rate was determined on day 23 using Cr-ethylenediaminetetraacetic acid. Eating or resting salivation was measured by collecting masticate (days 28 and 29) or saliva samples (days 30 and 31) at the cardia, respectively. On days 30 and 31, the temporarily isolated and washed reticulo-rumen technique was used to measure total, and Cl--competitive (an indirect measure of protein-mediated transport) absorption of acetate, propionate and butyrate. As a result of the higher dietary starch content and DM intake, the ruminal supply of rapidly fermentable CHO, total ruminal SCFA concentration (118 v. 95 mM; P<0.001) and osmolality (330 v. 306 mOsm/kg; P=0.018) were greater in cattle fed LF compared with HF. In addition, feeding LF resulted in a longer duration (2.50 v. 0.09 h/day; P=0.02) and a larger area (0.44 v. 0.01 (pH×h)/day; P=0.050) that pH was below 5.5. There was no diet effect on total and Cl--competitive absorption (mmol/h and %/h) of acetate, propionate, butyrate and total SCFA (acetate+propionate+butyrate), but eating salivation was less (131 v. 152 ml/min; P=0.02), and resting salivation tended to be less (87 v. 104 ml/min; P=0.10) in cattle fed an LF diet. In summary, lower ruminal pH in cattle with greater rapidly fermentable CHO intake was attributed to an increase in SCFA production and decrease in salivation, which were not compensated for by an increase in epithelial permeability.

Preparation of polydopamine nanocapsules in a miscible tetrahydrofuran-buffer mixture.

PubMed

Ni, Yun-Zhou; Jiang, Wen-Feng; Tong, Gang-Sheng; Chen, Jian-Xin; Wang, Jie; Li, Hui-Mei; Yu, Chun-Yang; Huang, Xiao-hua; Zhou, Yong-Feng

2015-01-21

A miscible tetrahydrofuran-tris buffer mixture has been used to fabricate polydopamine hollow capsules with a size of 200 nm and with a shell thickness of 40 nm. An unusual non-emulsion soft template mechanism has been disclosed to explain the formation of capsules. The results indicate that the capsule structure is highly dependent on the volume fraction of tetrahydrofuran as well as the solvent, and the shell thickness of capsules can be controlled by adjusting the reaction time and dopamine concentration.

Purification and characterization of a novel thermostable luciferase from Benthosema pterotum.

PubMed

Homaei, Ahmad Abolpour; Mymandi, Asma Bahari; Sariri, Reyhaneh; Kamrani, Ehsan; Stevanato, Roberto; Etezad, Seyed-Masoud; Khajeh, Khosro

2013-08-05

A novel luciferase from Benthosema pterotum, collected from Port of Jask, close to Persian Gulf, was purified for the first time, using Q-Sepharose anion exchange chromatography. The molecular mass of the novel enzyme, measured by SDS-PAGE technique, was about 27 kDa and its Km value is 0.4 μM; both values are similar to those of other coelenterazine luciferases. B. pterotum (BP) luciferase showed maximum intensity of emitted light at 40°C, in 20mM Tris buffer, pH 9 and 20 mM magnesium concentration. Experimental measurements indicated that BP luciferase is a relatively thermostable enzyme; furthermore it shows a high residual activity at extreme pH values. Its biological activity is strongly inhibited by 1 mM Cu(2+), Zn(2+) and Ni(2+), while calcium and mainly magnesium ions strongly increase BP luciferase activity. The B. pterotum luciferase generated blue light with a maximum emission wavelength at 475 nm and showed some similarity with other luciferases, while other parameters appeared quite different, in this way, confirming that a novel protein has been purified. Copyright © 2013 Elsevier B.V. All rights reserved.

Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.

PubMed

Jiang, Ting-Fu; Lv, Zhi-Hua; Wang, Yuan-Hong; Yue, Mei-E

2006-06-01

A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of buffer pH, the concentration of HDB and the voltage on the resolution were studied systematically. By this method, the contents of plant hormone in biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery.

Synthesis and characterization of polylactide/doxorubicin/magnetic nanoparticles composites for drug delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mhlanga, Nikiwe; Ray, Suprakas Sinha; DST/CSIR National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, Pretoria, 0001

Magnetic iron oxide nanoparticles have potential to transform conventional therapeutics, through targeted delivery by external magnetic field modulation. Conventional drug delivery lacks specificity; both normal and infected cells are exposed to toxic drugs. Consequently, the toxicity towards healthy cells leads to detrimental side effects which are formidable. However, iron oxide research in biomedicine has been hindered by their lack of stability. This study reports on the stabilization of iron oxide by polylactide (PLA). Besides affording stable iron oxide, PLA is also good for sustained delivery of the drug. PLA/doxorubicin/magnetic nanoparticles (PLA/DOX/MNPs) spheres were synthesized by solvent evaporation method and DOXmore » anticancer drug was encapsulated. The spheres were characterized using scanning electron microscope, Fourier transform infrared microscope, thermogravimetric analyzer and UV-visible spectroscopy, which ascertained formation of the anticipated spheres and incorporation of DOX. In vitro drug release studies were carried out in both phosphate buffer (pH 7.4) and acetate buffer (pH 4.6) and they showed the same trend in both mediums. Drug release kinetics followed Higuchi model, which proved drug release by diffusion via a diffusion gradient.« less

In-vitro and in-vivo study of amorphous spironolactone prepared by adsorption method using supercritical CO2.

PubMed

Jiang, Qikun; Li, Yuanyuan; Fu, Qiang; Geng, Yajie; Zhao, Juanhang; Ma, Panqin; Zhang, Tianhong

2015-02-01

The aim of this study was to improve the oral bioavailability of spironolactone (SP). SP was adsorbed on the fumed silica using supercritical CO2 (scCO2) technology and further compressed into tablets. The morphology was observed by scanning electron microscopy (SEM), and the crystalline form was investigated by differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). The dissolution test was performed in water, 0.1 M HCl solution, pH 4.5 acetate buffers and pH 6.8 phosphate buffers using the paddle method. The pharmacokinetics was undertaken in six dogs in a crossover fashion. SP was successfully prepared into tablets and presented in amorphous state. SP-silica scCO2 tablets displayed higher dissolution profiles than SP-silica physical mixtures tablets in different media. The AUC0-t and Cmax of SP-silica supercritical CO2 was 1.61- and 1.52-fold greater than those of SP-silica physical mixtures (p < 0.05), respectively. It is a promising method in improving dissolution and bioavailability by adsorbing SP, a poorly soluble drug, on the fumed silica using rapid expansion of supercritical solutions.

Hydrophilic interaction liquid chromatography in analysis of granisetron HCl and its related substances. Retention mechanisms and method development.

PubMed

Maksić, Jelena; Tumpa, Anja; Stajić, Ana; Jovanović, Marko; Rakić, Tijana; Jančić-Stojanović, Biljana

2016-05-10

In this paper separation of granisetron and its two related substances in HILIC mode is presented. Separation was done on silica column derivatized with sulfoalkylbetaine groups (ZIC-HILIC). Firstly, retention mechanisms were assessed whereby retention factors of substances were followed in wide range of acetonitrile content (80-97%), at constant concentration of aqueous buffer (10mM) as well as at constant pH value of 3.0. Further, in order to developed optimal HILIC method, Design of Experiments (DoE) methodology was applied. For optimization full factorial design 3(2) was employed. Influence of acetonitrile content and ammonium acetate concentration were investigated while pH of the water phase was kept at 3.3. Adequacy of obtained mathematical models was confirmed by ANOVA. Optimization goals (α>1.15 and minimal run time) were accomplished with 94.7% of acetonitrile in mobile phase and 70 mM of ammonium acetate in water phase. Optimal point was in the middle of defined Design Space. In the next phase, robustness was experimetally tested by Rechtschaffen design. The investigated factors and their levels were: acetonitrile content (±1%), ammonium acetate molarity in water phase (±2 mM), pH value of water phase (±0.2) and column temperature (±4 °C). The validation scope included selectivity, linearity, accuracy and precision as well as determination of limit of detection (LOD) and limit of quantification (LOQ) for the related substances. Additionally, the validation acceptance criteria were met in all cases. Finally, the proposed method could be successfully utilized for estimation of granisetron HCl and its related substances in tablets and parenteral dosage forms, as well as for monitoring degradation under various stress conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Indirect photometric detection of boron cluster anions electrophoretically separated in methanol.

PubMed

Vítová, Lada; Fojt, Lukáš; Vespalec, Radim

2014-04-18

3,5-Dinitrobenzoate and picrate are light absorbing anions pertinent to indirect photometric detection of boron cluster anions in buffered methanolic background electrolytes (BGEs). Tris(hydroxymethyl)aminomethane and morpholine have been used as buffering bases, which eliminated baseline steps, and minimized the baseline noise. In methanolic BGEs, mobilities of boron cluster anions depend on both ionic constituents of the BGE buffer. This dependence can be explained by ion pair interaction of detected anions with BGE cations, which are not bonded into ion pairs with the BGE anions. The former ion pair interaction decreases sensitivity of the indirect photometric detection. Copyright © 2014 Elsevier B.V. All rights reserved.

High-performance liquid chromatographic determination of pantoprazole and its main impurities in pharmaceuticals.

PubMed

Letica, Jelena; Marković, Slavko; Zirojević, Jelena; Nikolić, Katarina; Agbaba, Danica

2010-01-01

An RP-HPLC method for simultaneous separation and quantification of pantoprazole and its five main impurities in pharmaceutical formulations was developed and validated. The separation was accomplished on a Zorbax Eclipse XDB C18 column (5 microm particle size, 150 x 4.6 mm id) using a gradient with mobile phase A [buffer-acetonitrile (70 + 30, v/v)], and mobile phase B [buffer-acetonitrile (30 + 70, v/v)]. The buffer was 0.01 M ammonium acetate solution with addition of 1 mL triethylamine/L of the solution, adjusted to pH 4.5 with orthophosphoric acid. The eluent flow rate was 1 mL/min, the temperature of the column was 30 degrees C, and the eluate was monitored at 290 nm. Linearity (r = 0.999), recovery (97.6-105.8%), RSD (0.55-1.90%), and LOQ (0.099-1.48 microg/mL) were evaluated and found to be satisfactory. The proposed method can be used for simultaneous identification and quantification of the analyzed compounds in pharmaceutical formulations.

Adsorption and Reduction of Hexavalent Chromium on the Surface of Vivianite at Acidic Environment

NASA Astrophysics Data System (ADS)

HA, S.; Hyun, S. P.; Lee, W.

2016-12-01

Due to the rapid increase of chemical use in industrial activities, acid spills have frequently occurred in Korea. The acid spill causes soil and water acidification and additional problems such as heavy metal leaching from the soil. Hexavalent chromium (Cr(VI)) is relatively mobile in the environment and toxic and mutagenic. Monoclinic octa-hydrated ferrous phosphate, vivianite, is one of commonly found iron-bearing soil minerals occurring in phosphorous-enriched reducing environments. We have investigated reductive sorption of Cr(VI) on the vivianite surfaces using batch experimental tests under diverse groundwater conditions. Cr(VI) (5 mg/L) was added in 6.5 g/L vivianite suspension buffered at pH 5, 7, and 9, using 0.05 M HEPES or tris buffer solution, to check the effect of pH on the reductive sorption of Cr(VI) on the vivianite surface. The aqueous Cr(VI) removal was fastest at pH 5, followed by pH 7, and pH 9. The effect of ionic strength on the removal kinetics of Cr(VI) was negligible. It could be subsequently removed via sorption and reduction on the surface of vivianite of which reactive chemical species could be aqueous Fe(II), iron oxides, and metavivianite. Adsorption test was conducted using the same amount of Cr(III) to check the selectivity of chromium species on the vivianite surface for the reductive adsorption. Through Cr extraction test, amount of strong-bound Cr to vivianite is similar for Cr(III) and Cr(VI) injection but amount of weak-bound Cr is bigger for Cr(VI) injection. Reaction mechanism for the sorption and reductive transformation of Cr(VI) to Cr(III) species at reactive sites of vivianite surface are discussed based on surface complexation modeling and K-edge Fe X-ray absorption near edge structure (XANES) results. Since vivianite is reacted with Cr(VI), two smooth peaks of absorption edge changed to one sharp peak. Pre-edge that contains 1s-3d transition information tends to show high peak when reaction time is increased and pH is low. This fact indicated that the Fe(II) is oxidized to Fe(III) at the surface of viviante and this phenomena is optimized at pH 5 and longer elapsed time.

Development and Validation of a Stability-Indicating HPLC Method for Imidapril and Its Degradation Products Using a Design of Experiment (DoE) Approach.

PubMed

Arumugam, Abiramasundari; Joshi, Amita; Vasu, Kamala K

2017-11-01

The present work focused on the application of design of experiment (DoE) principles to the development and optimization of a stability-indicating method (SIM) for the drug imidapril hydrochloride and its degradation products (DPs). The resolution of peaks for the DPs and their drug in a SIM can be influenced by many factors. The factors studied here were pH, gradient time, organic modifier, flow rate, molar concentration of the buffer, and wavelength, with the aid of a Plackett-Burman design. Results from the Plackett-Burman study conspicuously showed influence of two factors, pH and gradient time, on the analyzed response, particularly, the resolution of the closely eluting DPs (DP-5 and DP-6) and the retention time of the last peak. Optimization of the multiresponse processes was achieved through Derringer's desirability function with the assistance of a full factorial design. Separation was achieved using a C18 Phenomenex Luna column (250 × 4.6 mm id, 5 µm particle size) at a flow rate of 0.8 mL/min at 210 nm. The optimized mobile phase composition was ammonium-acetate buffer (pH 5) in pump A and acetonitrile-methanol (in equal ratio) in pump B with a run time of 40 min using a gradient method.

Molecular-level insights of early-stage prion protein aggregation on mica and gold surface determined by AFM imaging and molecular simulation.

PubMed

Lou, Zhichao; Wang, Bin; Guo, Cunlan; Wang, Kun; Zhang, Haiqian; Xu, Bingqian

2015-11-01

By in situ time-lapse AFM, we investigated early-stage aggregates of PrP formed at low concentration (100 ng/mL) on mica and Au(111) surfaces in acetate buffer (pH 4.5). Remarkably different PrP assemblies were observed. Oligomeric structures of PrP aggregates were observed on mica surface, which was in sharp contrast to the multi-layer PrP aggregates yielding parallel linear patterns observed Au(111) surface. Combining molecular dynamics and docking simulations, PrP monomers, dimers and trimers were revealed as the basic units of the observed aggregates. Besides, the mechanisms of the observed PrP aggregations and the corresponding molecular-substrate and intermolecular interactions were suggested. These interactions involved gold-sulfur interaction, electrostatic interaction, hydrophobic interaction, and hydrogen binding interaction. In contrast, the PrP aggregates observed in pH 7.2 PBS buffer demonstrated similar large ball-like structures on both mica and Au(111) surfaces. The results indicate that the pH of a solution and the surface of the system can have strong effects on supramolecular assemblies of prion proteins. This study provides in-depth understanding on the structural and mechanistic nature of PrP aggregation, and can be used to study the aggregation mechanisms of other proteins with similar misfolding properties. Copyright © 2015 Elsevier B.V. All rights reserved.

Anodic voltammetric behavior and determination of rosiglitazone in pharmaceutical dosage forms and biological fluids on solid electrode.

PubMed

Dogan-Topal, Burcu; Tuncel, Secil; Ozkan, Sibel A

2010-09-01

The anodic voltammetric behavior and electroanalytical determination of rosiglitazone was studied using cyclic, linear sweep, differential pulse and square wave voltammetric techniques on glassy carbon electrode. The oxidation of rosiglitazone was irreversible and exhibited diffusion controlled process depending on pH. Different parameters were tested to optimize the conditions for the determination of the oxidation mechanism of rosiglitazone. The dependence of current intensities and potentials on pH, concentration, scan rate, nature of the buffer was also investigated. According to the linear relationship between the peak current and the concentration, differential pulse and square wave voltammetric methods for rosiglitazone assay in pharmaceutical dosage forms and biological fluids were developed. A linear response was obtained within the range of 1x10-6M - 6x10-5M in 0.1 M H2SO4 and acetate buffer at pH 5.70 for both voltammetric methods in human serum samples. The practical analytical value of the method is demonstrated by quantitative determination of rosiglitazon in pharmaceutical formulation and human serum, without the need for separation or complex sample preparation, since there was no interference from the excipients and endogenous substances. The methods were fully validated and successfully applied to the high throughput determination of the drug in tablets and human serum with good recoveries.

In-vitro evaluation of enteric coated insulin tablets containing absorption enhancer and enzyme inhibitor.

PubMed

Wong, Chun Y; Martinez, Jorge; Carnagarin, Revathy; Dass, Crispin R

2017-03-01

The aim of this study was to develop an enteric coated insulin tablet formulation using polymers, absorption enhancer and enzyme inhibitor, which protect the tablets in acidic pH and enhance systemic bioavailability. In this study, the influence of coating by cellulose acetate hydrogen phthalate solution and chosen excipients on Glut-4 transporter translocation in C2C12 skeletal muscle cells was examined. Following the determination of optimum number of coating layers, two dissolution buffers such as 0.01 m hydrochloric acid, pH 2, and 50 mm phosphate, pH 7.4, were employed to determine the in-vitro release of insulin. Insulin was protected by the coating during the dissolution process. Five (5-CL) coating layers and eight (8-CL) coating layers had minimal insulin release in hydrochloric acid, but not three (3-CL) coating layers. Glut-4 translocation in C2C12 cells was promoted by the chosen excipients. No detrimental metabolic effects were observed in these cells. To date, limited studies combine the overall effectiveness of multiple excipients. Our study showed that the coated tablets have an immediate release effect in phosphate buffer. In Glut-4 translocation assay, insulin was still functional after releasing from the tablet. Such tablet formulation can be potentially beneficial to type 1 diabetes patients. © 2017 Royal Pharmaceutical Society.

In silico and in vitro prediction of gastrointestinal absorption from potential drug eremantholide C.

PubMed

Caldeira, Tamires G; Saúde-Guimarães, Dênia A; Dezani, André B; Serra, Cristina Helena Dos Reis; de Souza, Jacqueline

2017-11-01

Analysis of the biopharmaceutical properties of eremantholide C, sesquiterpene lactone with proven pharmacological activity and low toxicity, is required to evaluate its potential to become a drug. Preliminary analysis of the physicochemical characteristics of eremantholide C was performed in silico. Equilibrium solubility was evaluated using the shake-flask method, at 37.0 °C, 100 rpm during 72 h in biorelevant media. The permeability was analysed using parallel artificial membrane permeability assay, at 37.0 °C, 50 rpm for 5 h. The donor compartment was composed of an eremantholide C solution in intestinal fluid simulated without enzymes, while the acceptor compartment consisted of phosphate buffer. Physicochemical characteristics predicted in silico indicated that eremantholide C has a low solubility and high permeability. In-vitro data of eremantholide C showed low solubility, with values for the dose/solubility ratio (ml): 9448.82, 10 389.61 e 15 000.00 for buffers acetate (pH 4.5), intestinal fluid simulated without enzymes (pH 6.8) and phosphate (pH 7.4), respectively. Also, it showed high permeability, with effective permeability of 30.4 × 10 -6 cm/s, a higher result compared with propranolol hydrochloride (9.23 × 10 -6 cm/s). The high permeability combined with its solubility, pharmacological activity and low toxicity demonstrate the importance of eremantholide C as a potential drug candidate. © 2017 Royal Pharmaceutical Society.

Miniaturized and direct spectrophotometric multi-sample analysis of trace metals in natural waters.

PubMed

Albendín, Gemma; López-López, José A; Pinto, Juan J

2016-03-15

Trends in the analysis of trace metals in natural waters are mainly based on the development of sample treatment methods to isolate and pre-concentrate the metal from the matrix in a simpler extract for further instrumental analysis. However, direct analysis is often possible using more accessible techniques such as spectrophotometry. In this case a proper ligand is required to form a complex that absorbs radiation in the ultraviolet-visible (UV-Vis) spectrum. In this sense, the hydrazone derivative, di-2-pyridylketone benzoylhydrazone (dPKBH), forms complexes with copper (Cu) and vanadium (V) that absorb light at 370 and 395 nm, respectively. Although spectrophotometric methods are considered as time- and reagent-consuming, this work focused on its miniaturization by reducing the volume of sample as well as time and cost of analysis. In both methods, a micro-amount of sample is placed into a microplate reader with a capacity for 96 samples, which can be analyzed in times ranging from 5 to 10 min. The proposed methods have been optimized using a Box-Behnken design of experiments. For Cu determination, concentration of phosphate buffer solution at pH 8.33, masking agents (ammonium fluoride and sodium citrate), and dPKBH were optimized. For V analysis, sample (pH 4.5) was obtained using acetic acid/sodium acetate buffer, and masking agents were ammonium fluoride and 1,2-cyclohexanediaminetetraacetic acid. Under optimal conditions, both methods were applied to the analysis of certified reference materials TMDA-62 (lake water), LGC-6016 (estuarine water), and LGC-6019 (river water). In all cases, results proved the accuracy of the method. Copyright © 2015 Elsevier Inc. All rights reserved.

A diazonium ion cascade from the nitrosation of tolazoline, an imidazoline-containing drug.

PubMed

Loeppky, Richard N; Shi, Jianzheng; Barnes, Charles L; Geddam, Sailaja

2008-02-01

Tolazoline (1-benzylimidazoline), a representative imidazoline-containing drug, reacts readily with nitrite in acetic acid to produce a complex product mixture. Fourteen compounds have been identified as products of this transformation when an 8-fold excess of HNO2 is used. The products, which include N-nitrosoamides, esters, alcohols, and phenylacetic acid, are rationalized as arising from a cascade of reactive diazonium ions. N-Nitrosotolazoline can be isolated from the nitrosation reaction in good yield when the mixture is extracted with CH2Cl2 as the transformation progresses. It nitrosates much more rapidly (50x) than tolazoline to give, among other products, the oxime [1-( N-nitroso-2-imidazolinyl)benzylidene]hydroxylamine, which can also be produced in good yield from the reaction of tolazoline with isopropyl nitrite. At low substrate and nitrite concentrations, the main reaction products are N-nitrosotolazoline, its decomposition product N-2-hydroxyethylphenylacetamide, the above-mentioned oxime, phenyl acetic acid, and 2-hydroxyethyl phenylacetate. The tolazoline nitrosation rate in three buffer systems has been determined at pH 3.4 and 37 degrees C ( kobs = 6.25 x 10 (-5) s (-1) in 0.5 M acetate buffer with a 10 * [NO2(-)] = 250 mM). Because N-nitrosotolazoline exhibits the chemical properties of a direct-acting mutagen and carcinogen, we have used the rate data to estimate its level of formation at nitrite concentrations <3 mM. Cursory examination of the nitrosation chemistry of oxymetazoline, a related drug, is primarily focused at its electron-rich aromatic ring.

Nature and origin of the calcium asymmetry-arising during gravitropic response in etiolated pea epicotyls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Migliaccio, F.; Galston, A.W.

1987-10-01

Seven day old etiolated pea epicotyls were loaded symmetrically with /sup 3/H-indole 3-acetic acid (IAA) or /sup 45/Ca/sup 2 +/, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca/sup 2 +/ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca/sup 2 +/ asymmetries, but substances known to interfere with normal Ca/sup 2 +/ transport do not significantly alter either IAA or Ca/sup 2 +/ asymmetries. These substances,more » however, are active in modifying both Ca/sup 2 +/ uptake and efflux through oat and pea leaf protoplast membranes. The authors conclude that the /sup 45/Ca/sup 2 +/ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca/sup 2 +/ movement secondary in gravitropism. They hypothesize that apoplastic Ca/sup 2 +/ changes during the graviresponse because it is displaced by H/sup +/ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increased calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H/sup +/ efflux, increase Ca/sup 2 +/ release from pea epicotyl segments, whereas cycloheximide, which inhibits H/sup +/ efflux, has the reverse effect.« less

Study to optimize gellant polymer-water systems for the control of hypergolic spills and fires

NASA Technical Reports Server (NTRS)

Jennings, R. R.; Macwilliams, D. C.; Foshee, W. C.; Katzer, M. F.

1973-01-01

A system of buffered gelled water was developed to prevent and control fires from small spills of nitrogen tetroxide-(N2O4)-Aerozine 50-hypergolic fuel. Laboratory studies on various alkalis, buffers, and seavengers for the fuel components are described. Chilling and sodium acetate-acetic acid buffer was found to be the best additives to the gelled water. Field tests and a delivery system (airborne) for the extinguishant are described. A short movie showing the field testing is available upon request.

Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

PubMed

Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

2016-05-01

Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermophysical properties of carboxylic and amino acid buffers at subzero temperatures: relevance to frozen state stabilization.

PubMed

Sundaramurthi, Prakash; Suryanarayanan, Raj

2011-06-02

Macromolecules and other thermolabile biologicals are often buffered and stored in frozen or dried (freeze-dried) state. Crystallization of buffer components in frozen aqueous solutions and the consequent pH shifts were studied in carboxylic (succinic, malic, citric, tartaric acid) and amino acid (glycine, histidine) buffers. Aqueous buffer solutions were cooled from room temperature (RT) to -25 °C and the pH of the solution was measured as a function of temperature. The thermal behavior of frozen solutions was investigated by differential scanning calorimetry (DSC), and the crystallized phases were identified by X-ray diffractometry (XRD). Based on the solubility of the neutral species of each buffer system over a range of temperatures, it was possible to estimate its degree of supersaturation at the subambient temperature of interest. This enabled us to predict its crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartrate systems, at initial pH < pK(a)(2), only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. In glycine buffer solutions, when the initial pH was ∼3 units < isoelectric pH (pI = 5.9), β-glycine crystallization caused a small decrease in pH, while a similar effect but in the opposite direction was observed when the initial pH was ∼3 units > pI. In the histidine buffer system, depending on the initial pH, either histidine or histidine HCl crystallized.

Characterization of a recombinant flocculent Saccharomyces cerevisiae strain that co-ferments glucose and xylose: II. influence of pH and acetic acid on ethanol production.

PubMed

Matsushika, Akinori; Sawayama, Shigeki

2012-12-01

The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0-6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150-200 mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.

Iron as a catalyst of human low-density lipoprotein oxidation: Critical factors involved in its oxidant properties.

PubMed

Lapenna, Domenico; Ciofani, Giuliano; Obletter, Gabriele

2017-05-01

Iron-induced human LDL oxidation, which is relevant to atherosclerosis, has not yet been properly investigated. We addressed such issue using iron(II) and (III) basically in the presence of phosphates, which are present in vivo and influence iron oxidative properties, at pH 4.5 and 7.4, representative, respectively, of the lysosomal and plasma environment. In 10mM phosphate buffered saline (PBS), iron(II) induces substantial LDL oxidation at pH 4.5 at low micromolar concentrations, while at pH 7.4 has low oxidative effects; iron(III) promotes small LDL oxidation only at pH 4.5. In 10mM sodium acetate/NaCl buffer, pH 4.5, iron-induced LDL oxidation is far higher than in PBS, highlighting the relevance of phosphates in the inhibitory modulation of iron-induced LDL oxidation. LDL oxidation is related to iron binding to the protein and lipid moiety of LDL, and requires the presence of iron(II) bound to LDL together with iron(III). Chemical modification of LDL carboxyl groups, which could bind iron especially at pH 4.5, decreases significantly iron binding to LDL and iron-induced LDL oxidation. Hydroxyl radical scavengers are ineffective on iron-induced LDL oxidation, which is inhibited by metal chelation, scavengers of alkoxyl/peroxyl radicals, or removal of LDL lipid hydroperoxides (LOOH). Overall, substantial human LDL oxidation is induced LOOH-dependently by iron(II) at pH 4.5 even in the presence of phosphates, suggesting the occurrence of iron(II)-induced LDL oxidation in vivo within lysosomes, where pH is about 4.5, iron(II) and phosphates coexist, plasma with its antioxidants is absent, and glutathione peroxidase is poorly expressed resulting in LOOH accumulation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Effect of royal jelly on experimental colitis induced by acetic acid and alteration of mast cell distribution in the colon of rats

PubMed Central

Karaca, T.; Bayiroglu, F.; Yoruk, M.; Kaya, M.S.; Uslu, S.; Comba, B.; Mis, L.

2010-01-01

This study investigated the effects of royal jelly (RJ) on acetic acid-induced colitis in rats. Twenty adult female Wistar albino rats were divided into four treatment groups of 5 animals each, including a control group (Group I); Group II was treated orally with RJ (150 mg kg−1 body weight); Group III had acetic acid-induced colitis; and Group IV had acetic acid-induced colitis treated orally with RJ (150 mg kg−1 body weight) for 4 weeks. Colitis was induced by intracolonic instillation of 4% acetic acid; the control group received physiological saline (10 mL kg−1). Colon samples were obtained under deep anaesthesia from animals in all groups. Tissues were fixed in 10% formalin neutral buffer solution for 24 h and embedded in paraffin. Six-micrometre-thick sections were stained with Mallory’s triple stain and toluidine blue in 1% aqueous solution at pH 1.0 for 5 min (for Mast Cells). RJ was shown to protect the colonic mucosa against the injurious effect of acetic acid. Colitis (colonic damage) was confirmed histomorphometrically as significant increases in the number of mast cells (MC) and colonic erosions in rats with acetic acid-induced colitis. The RJ treatment significantly decreased the number of MC and reduced the area of colonic erosion in the colon of RJ-treated rats compared with rats with untreated colitis. The results suggest that oral treatment with RJ could be used to treat colitis. PMID:21263740

Self-adaption of methane-producing communities to pH disturbance at different acetate concentrations by shifting pathways and population interaction.

PubMed

Hao, Liping; Lü, Fan; Li, Lei; Wu, Qing; Shao, Liming; He, Pinjing

2013-07-01

To investigate the competition among acetate-utilizing microorganisms at different acetate levels, bioconversion processes of 50, 100, 150 and 200 mM acetate in the presence and absence of methanogenic inhibitor CH3F were monitored in thermophilic methanogenic system. The successive response of methane-producing community during the deteriorative and recovery phases caused by pH disturbance was analyzed. High acetate concentration (>50mM) inhibited the activity of acetoclastic methanogenesis (AM). The increasing pH (>7.5) enhanced this inhibition. The syntrophic acetate oxidizing (SAO) bacteria and hydrogenotrophic methanogens including Methanomicrobiales and Methanobacteirales were more tolerant to the stress from high acetate concentration and high pH. Resumption from alkali condition to normal pH stimulated the growth of acetate oxidizing syntrophs. The reaction rate of SAO-HM was lower than that of AM. These results point to the possibility to regenerate the deteriorated anaerobic digesters by addition of acclimatized inocula rich in acetate-oxidizing syntrophs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Buffer Therapy for Cancer

PubMed Central

Ribeiro, Maria de Lourdes C; Silva, Ariosto S.; Bailey, Kate M.; Kumar, Nagi B.; Sellers, Thomas A.; Gatenby, Robert A.; Ibrahim-Hashim, Arig; Gillies, Robert J.

2013-01-01

Oral administration of pH buffers can reduce the development of spontaneous and experimental metastases in mice, and has been proposed in clinical trials. Effectiveness of buffer therapy is likely to be affected by diet, which could contribute or interfere with the therapeutic alkalinizing effect. Little data on food pH buffering capacity was available. This study evaluated the pH and buffering capacity of different foods to guide prospective trials and test the effect of the same buffer (lysine) at two different ionization states. Food groups were derived from the Harvard Food Frequency Questionnaire. Foods were blended and pH titrated with acid from initial pH values until 4.0 to determine “buffering score”, in mmol H+/pH unit. A “buffering score” was derived as the mEq H+ consumed per serving size to lower from initial to a pH 4.0, the postprandial pH of the distal duodenum. To differentiate buffering effect from any metabolic byproduct effects, we compared the effects of oral lysine buffers prepared at either pH 10.0 or 8.4, which contain 2 and 1 free base amines, respectively. The effect of these on experimental metastases formation in mice following tail vein injection of PC-3M prostate cancer cells were monitored with in vivo bioluminescence. Carbohydrates and dairy products’ buffering score varied between 0.5 and 19. Fruits and vegetables showed a low to zero buffering score. The score of meats varied between 6 and 22. Wine and juices had negative scores. Among supplements, sodium bicarbonate and Tums® had the highest buffering capacities, with scores of 11 and 20 per serving size, respectively. The “de-buffered” lysine had a less pronounced effect of prevention of metastases compared to lysine at pH 10. This study has demonstrated the anti-cancer effects of buffer therapy and suggests foods that can contribute to or compete with this approach to manage cancer. PMID:24371544

Gel Electrophoresis--The Easy Way for Students

ERIC Educational Resources Information Center

VanRooy, Wilhelmina; Sultana, Khalida

2010-01-01

This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis.

PubMed

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-03-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.

Quantitative thin layer chromatographic multi-sulfonamide screening procedure.

PubMed

Thomas, M H; Soroka, K E; Thomas, S H

1983-07-01

In-situ optical scanning of fluorescamine derivatives on thin layer silica gel plates provides a rapid method for the determination of multiple sulfonamides at levels below 0.1 ppm. Sample preparation is minimal. Homogenized liver or muscle is extracted with ethyl acetate and then back-extracted into 0.2M glycine buffer. After pH adjustment, the extract is washed with hexane and extracted with methylene chloride. The organic phase is evaporated to dryness and reconstituted in methanol. Pre-adsorbent layer silica gel plates are used for chromatography. The method has been applied to residues of sulfamethazine, sulfadimethoxine, sulfathiazole, sulfaquinoxaline, and sulfabromomethazine in cattle, swine, turkey, and duck tissues.

Preparation of hemoglobin-modified boron-doped diamond for acrylamide biosensors

NASA Astrophysics Data System (ADS)

Umam, K.; Saepudin, E.; Ivandini, T. A.

2017-04-01

Boron-doped diamond (BDD) electrode was modified with haemoglobin to develop electrochemical biosensors of acrylamide. Prior to modify with haemoglobin, the BDD was modified by gold nanoparticles to increase the affinity of BDD against haemoglobin. The electrochemical behaviour of the electrode in the presence of acrylamide was studied in comparison to haemoglobin-modified gold electrodes. Cyclic voltammetry indicated the optimum responses in 0.1 M sodium acetate buffer at pH 5. The responses were linear to the acrylamide concentration range of 5-50 μM with an estimated detection limit of 5.14 μM, suggesting that the electrode was promising for acrylamide biosensors.

A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

PubMed

Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

2016-01-01

The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.

Determination of ultra-trace formaldehyde in air using ammonium sulfate as derivatization reagent and capillary electrophoresis coupled with on-line electrochemiluminescence detection.

PubMed

Deng, Biyang; Liu, Yang; Yin, Huihui; Ning, Xi; Lu, Hua; Ye, Li; Xu, Quanxiu

2012-03-15

The reaction between formaldehyde and ammonium ion to produce hexamethylenetetramine is well known. The reaction conditions are very easily controlled in situ and the experiment operation is very simple. However, such derivatization reaction for trace formaldehyde determination using capillary electrophoresis (CE) electrochemiluminescence (ECL) has not been reported before. In this study, the application of ammoniun sulfate as derivatization reagent to in-situ determination of formaldehyde in air was reported. Based on ECL enhancement of tris(2,2'-bipyridyl)ruthenium(II) with hexamethylenetetramine, a novel approach for the determination of ultra-trace formaldehyde in air using CE coupled with on-line ECL of tris(2,2'-bipyridyl)ruthenium(II) has been developed. The parameters affecting separation and detection such as detection potential, concentration and pH of phosphate buffer, and electrokinetic voltage, were investigated. Under the optimal conditions, the linear concentration range of formaldehyde in air was from 0.48 μg/m(3) to 96 mg/m(3) (linear range covering 5 orders of magnitude). The limit of detection (3σ) was 0.15 μg/m(3). The relative standard deviations of peak height and migration time for six consecutive injection of 1 ng/mL formaldehyde derivative were 0.9% and 0.8%, respectively. The recoveries of formaldehyde in air were between 99.3% and 101%. Copyright © 2012 Elsevier B.V. All rights reserved.

Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations

DOE PAGES

Chavez, Brittany K.; Agarabi, Cyrus D.; Read, Erik K.; ...

2016-01-01

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potentialmore » storage buffer due to significant visible precipitate formation. An additional 2 4full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Therefore, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules.« less

Spectrophotometric method development and validation for determination of chlorpheniramine maleate in bulk and controlled release tablets.

PubMed

Ashfaq, Maria; Sial, Ali Akber; Bushra, Rabia; Rehman, Atta-Ur; Baig, Mirza Tasawur; Huma, Ambreen; Ahmed, Maryam

2018-01-01

Spectrophotometric technique is considered to be the simplest and operator friendly among other available analytical methods for pharmaceutical analysis. The objective of the study was to develop a precise, accurate and rapid UV-spectrophotometric method for the estimation of chlorpheniramine maleate (CPM) in pure and solid pharmaceutical formulation. Drug absorption was measured in various solvent systems including 0.1N HCl (pH 1.2), acetate buffer (pH 4.5), phosphate buffer (pH 6.8) and distil water (pH 7.0). Method validation was performed as per official guidelines of ICH, 2005. High drug absorption was observed in 0.1N HCl medium with λ max of 261nm. The drug showed the good linearity from 20 to 60μg/mL solution concentration with the correlation coefficient linear regression equation Y= 0.1853 X + 0.1098 presenting R 2 value of 0.9998. The method accuracy was evaluated by the percent drug recovery, presents more than 99% drug recovery at three different levels assessed. The % RSD value <1 was computed for inter and intraday analysis indicating the high accuracy and precision of the developed technique. The developed method is robust because it shows no any significant variation in with minute changes. The LOD and LOQ values were assessed to be 2.2μg/mL and 6.6μg/mL respectively. The investigated method proved its sensitivity, precision and accuracy hence could be successfully used to estimate the CPM content in bulk and pharmaceutical matrix tablets.

Coaxial atomic force microscope probes for dielectrophoresis of DNA under different buffer conditions

NASA Astrophysics Data System (ADS)

Tao, Yinglei; Kumar Wickramasinghe, H.

2017-02-01

We demonstrate a coaxial AFM nanoprobe device for dielectrophoretic (DEP) trapping of DNA molecules in Tris-EDTA (TE) and phosphate-buffered saline (PBS) buffers. The DEP properties of 20 nm polystyrene beads were studied with coaxial probes in media with different conductivities. Due to the special geometry of our DEP probe device, sufficiently high electric fields were generated at the probe end to focus DNA molecules with positive DEP. DEP trapping for both polystyrene beads and DNA molecules was quantitatively analyzed over the frequency range from 100 kHz to 50 MHz and compared with the Clausius-Mossotti theory. Finally, we discussed the negative effect of medium salinity during DEP trapping.

Pseudotetrahedral manganese complexes supported by the anionic tris(phosphino)borate ligand [PhBPiPr3

PubMed Central

Lu, Connie C.; Peters, Jonas C.

2008-01-01

This paper presents aspects of the coordination chemistry of mono- and divalent manganese complexes supported by the anionic tris(phosphino)borate ligand, [PhBPiPr3] (where [PhBPiPr3] = [PhB(CH2PiPr2)3]−). The Mn(II) halide complexes, [PhBPiPr3]MnCl (1 and [PhBPiPr3]MnI (2), have been characterized by X-ray diffraction, SQUID magnetometry, and EPR spectroscopy. Compound 2 serves as a precursor to a series of Mn azide, alkyl, and amide species: [PhBPiPr3]Mn(N3) (3), [PhBPiPr3]Mn(CH2Ph) (4), [PhBPiPr3]Mn(Me) (5), [PhBPiPr3]Mn(NH(2,6-iPr2-C6H3)) (6), [PhBPiPr3]Mn(dbabh) (7), and [PhBPiPr3]Mn(1-Ph(isoindolate)) (8). The complexes 2 – 8 feature a divalent-metal center and are pseudotetrahedral. They collectively represent an uncommon structural motif for low-coordinate, polyphosphine-supported Mn complexes. Two Mn(I) species have also been prepared. These include the Tl-Mn adduct, [PhBPiPr3]Tl-MnBr(CO)4 (9), and the octahedral complex [PhBPiPr3]Mn(CNtBu)3 (10). Some of our initial synthetic efforts to generate [PhBPiPr3]Mn≡Nx species are briefly described, as are DFT studies that probe the electronic viability of these types of multiply bonded target structures. PMID:17029370

Total enzymatic synthesis of cholecystokinin CCK-5.

PubMed

Xiang, H; Xiang, G Y; Lu, Z M; Guo, L; Eckstein, H

2004-08-01

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Reference measurement procedure for total glycerides by isotope dilution GC-MS.

PubMed

Edwards, Selvin H; Stribling, Shelton L; Pyatt, Susan D; Kimberly, Mary M

2012-04-01

The CDC's Lipid Standardization Program established the chromotropic acid (CA) reference measurement procedure (RMP) as the accuracy base for standardization and metrological traceability for triglyceride testing. The CA RMP has several disadvantages, including lack of ruggedness. It uses obsolete instrumentation and hazardous reagents. To overcome these problems the CDC developed an isotope dilution GC-MS (ID-GC-MS) RMP for total glycerides in serum. We diluted serum samples with Tris-HCl buffer solution and spiked 200-μL aliquots with [(13)C(3)]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b). The calibration response was linear for the range of calibrator concentrations examined (0-1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123-0.7225) and 0.3122 (95% CI, 0.3096-0.3140), respectively. The limit of detection was 14.8 μmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%. This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.

Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue.

PubMed

Shepherd, Lara D; McLay, Todd G B

2011-03-01

The high polysaccharide content of some plant species hinders the successful isolation of their DNA. As an alternative to the macro-extraction methods previously published for polysaccharide-rich plants, we present two techniques (STE/CTAB and HEPES/CTAB), which are performed in microcentrifuge tubes. These protocols are suitable for small amounts of silica gel-preserved plant tissue such as are commonly available from endangered plants. The critical step to remove polysaccharides was performing initial washes in either STE (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA) or HEPES (2% β-mercaptoethanol, 0.2% PVP, 0.1 M HEPES, pH 8.0) buffer. Precipitating the DNA at room temperature with isopropanol also aided in decreasing polysaccharide co-precipitation. Of the two protocols we present the STE/CTAB method has the advantages of being more cost-effective and avoiding the use of the hazardous chemical β-mercaptoethanol.

Encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-in-water emulsion and ionic gelation processes.

PubMed

Yoksan, Rangrong; Jirawutthiwongchai, Jatesuda; Arpo, Kridsada

2010-03-01

The encapsulation of ascorbyl palmitate (AP) in chitosan particles was carried out by droplet formation via an oil-in-water emulsion, followed by droplet solidification via ionic gelation using sodium triphosphate pentabasic (TPP) as a cross-linking agent. The success of AP encapsulation was confirmed by FT-IR, UV-vis spectrophotometry, TGA, and XRD techniques. The obtained AP-loaded chitosan particles were spherical in shape with an average diameter of 30-100nm as observed by SEM and TEM. Loading capacity (LC) and encapsulation efficiency (EE) of AP in the nanoparticles were about 8-20% and 39-77%, respectively, when the initial AP concentration was in the range of 25-150% (w/w) of chitosan. Augmentation of the initial AP concentration led to an increase of LC and a reduction of EE. The amount of AP released from the nanoparticles in ethanol and tris buffer (pH approximately 8.0) increased with increasing LC and decreasing TPP concentration.

Detection of vitamin b1 (thiamine) using modified carbon paste electrodes with polypyrrole

NASA Astrophysics Data System (ADS)

Muppariqoh, N. M.; Wahyuni, W. T.; Putra, B. R.

2017-03-01

Vitamin B1 (thiamine) is oxidized in alkaline medium and can be detected by cyclic voltammetry technique using carbon paste electrode (CPE) as a working electrode. polypyrrole-modified CPE were used in this study to increase sensitivity and selectivity measurement of thiamine. Molecularly imprinted polymers (MIP) of the modified CPE was prepared through electrodeposition of pyrrole. Measurement of thiamine performed in KCl 0.05 M (pH 10, tris buffer) using CPE and the modified CPE gave an optimum condition anodic current of thiamine at 0.3 V, potential range (-1.6_1 V), and scan rate of 100 mV/s. Measurement of thiamine using polypyrrole modified CPE (CPE-MIPpy) showed better result than CPE itself with detection limit of 6.9×10-5 M and quantitation limit 2.1×10-4 M. CPE-MIPpy is selective to vita min B1. In conclusion, CPE-MIPpy as a working electrode showed better performance of thiamine measurement than that of CPE.

Enantioselective hydrolysis of racemic styrene oxide and its substituted derivatives using newly-isolated Sphingopyxis sp. exhibiting a novel epoxide hydrolase activity.

PubMed

Woo, Jung-Hee; Lee, Eun Yeol

2014-02-01

(S)-Styrene oxide, (S)-2-chlorostyrene oxide (CSO), (S)-3-CSO and (S)-4-CSO with 99.9 %ee were obtained with a yield of 20.6, 39.3, 28.7 and 26.8 % from 4 mM corresponding racemic substrates using 10 mg cells of a newly-isolated Sphingopyxis sp. at pH 8.0 and 25 °C in 1 ml 100 mM Tris/HCl buffer after 420, 100, 120 and 55 min, respectively. For racemic 2CSO, well-known for one of the racemates that is difficult to obtained in enantiomerically pure form, (S)-2-CSO with 99.9 %ee, 39.3 % yield (theoretical yield 50 %) and enantiomeric ratio of 42.1 was obtained. The newly-isolated strain can thus be used as whole-cell biocatalyst in the production of various (S)-CSO with a chlorine group at different positions.

Mechanism of Decarboxylation of Pyruvic Acid in the Presence of Hydrogen Peroxide

PubMed Central

Lopalco, Antonio; Dalwadi, Gautam; Niu, Sida; Schowen, Richard L.; Douglas, Justin; Stella, Valentino J.

2015-01-01

The purpose of this work was to probe the rate and mechanism of rapid decarboxylation of pyruvic acid in the presence of hydrogen peroxide (H2O2) to acetic acid and carbon dioxide over the pH range 2 – 9 at 25°C, utilizing UV spectrophotometry, high performance liquid chromatography (HPLC), and proton and carbon nuclear magnetic resonance spectrometry (1H, 13C-NMR). Changes in UV absorbance at 220 nm were used to determine the kinetics since the reaction was too fast to follow by HPLC or NMR in much of the pH range. The rate constants for the reaction were determined in the presence of molar excess of H2O2 resulting in pseudo first order kinetics. No buffer catalysis was observed. The calculated second order rate constants for the reaction followed a sigmoidal shape with pH independent regions below pH 3 and above pH 7 but increased between pH 4 and 6. Between pH 4 and 9, the results were in agreement with a change from rate determining nucleophilic attack of the deprotonated peroxide species, HOO−, on the α-carbonyl group followed by rapid decarboxylation at pH values below 6 to rate-determining decarboxylation above pH 7. The addition of H2O2 to ethyl pyruvate was also characterized. PMID:26422524

Efficient Enzyme-Free Biomimetic Sensors for Natural Phenol Detection.

PubMed

Ferreira Garcia, Luane; Ribeiro Souza, Aparecido; Sanz Lobón, Germán; Dos Santos, Wallans Torres Pio; Alecrim, Morgana Fernandes; Fontes Santiago, Mariângela; de Sotomayor, Rafael Luque Álvarez; de Souza Gil, Eric

2016-08-13

The development of sensors and biosensors based on copper enzymes and/or copper oxides for phenol sensing is disclosed in this work. The electrochemical properties were studied by cyclic and differential pulse voltammetry using standard solutions of potassium ferrocyanide, phosphate/acetate buffers and representative natural phenols in a wide pH range (3.0 to 9.0). Among the natural phenols herein investigated, the highest sensitivity was observed for rutin, a powerful antioxidant widespread in functional foods and ubiquitous in the plant kingdom. The calibration curve for rutin performed at optimum pH (7.0) was linear in a broad concentration range, 1 to 120 µM (r = 0.99), showing detection limits of 0.4 µM. The optimized biomimetic sensor was also applied in total phenol determination in natural samples, exhibiting higher stability and sensitivity as well as distinct selectivity for antioxidant compounds.

[Construction and Characterization of B850-Only LH2 Energy Transfer System in Purple Bacteria].

PubMed

Li, Kai; Zhao, Chun-gui; Yue, Hui-ying; Yang, Su-ping; Qu, Yin-bo; Jiao, Nian-zhi

2015-04-01

To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis, the conditions for construction of B850-only peripheral light-harvesting complex (LH2) and their properties were investigated using absorption, fluorescence spectroscopy, molecular sieve chromatography, ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the purple bacteria. The results indicated that bacteriochlorophylls (BChl) of B800 incubated in 10 mmo · L(-1) Tris-HCl (pH 8.0) buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans (A-LH2) by 0.08% (W/V) SDS. B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration. B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature (RT). While BChl of B800 incubated in pH 1.9 buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris (P-LH2). The authors acquired two components using molecular sieve chromatography. Free BChl of one component was not removed and self-assembled to P-LH2. The other removed free BChl and B850-only P-LH2 was constructed. B850 unchanged after P-LH2 was incubated. P-LH2 α and β subunits have different molecular weights, but those of A-LH2 are in the contrary. It is concluded that B850-only P-LH2 is more stable than A-LH2. The enigmatic split of the B800 absorption band was not observed in these LH2, but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris. The authors' results may provide a new light to separate homogeneous Apoprotein LH2.

Reaction of 2-acetylaminofluorene-N-sulfate with RNA and glutathione: evidence for the generation of two reactive intermediates with different reactivities towards RNA and glutathione.

PubMed

van den Goorbergh, J A; Meerman, J H; de Wit, H; Mulder, G J

1985-11-01

The sulfate ester of N-hydroxy-2-acetylaminofluorene (AAF-N-sulfate) is one of the reactive intermediates of this carcinogen. This ester breaks down spontaneously to a very reactive nitrenium ion, which reacts with nucleophilic groups in protein, DNA, RNA and glutathione (GSH). Reactions involving the nitrenium ion with several nucleophiles under various conditions were studied. The adduct formation to RNA was much higher in Tris-HCI buffer than in phosphate buffer (at pH 7.4), while adduct formation to deoxy-guanosine monomers was the same in both buffers. The presence of 150 mM KCI had the same decreasing effect in both cases. Ionic strength effects may be involved in these phenomena. GSH decreased RNA adduct formation by 20-45%, while other thiols were much more effective. On the other hand, RNA did not decrease the formation of GSH conjugates from AAF-N-sulfate. The decrease in RNA adduct formation by thiols corresponded with an increase in the formation of 2-acetylaminofluorene (AAF) from AAF-N-sulfate, while no N-hydroxy-AAF was formed. These results suggest that two independent reactive intermediates are formed from AAF-N-sulfate, with different reactivities towards RNA and glutathione. Possibly these intermediates are the 'hard' triplet state nitrenium ion and the 'soft' singlet state nitrenium ion. Cysteine, cysteamine and penicillamine were most effective in the inhibition of RNA adduct formation; the extent of inhibition correlated with the extent of AAF formation. The mechanisms involved are discussed.

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient.

PubMed

Fritze, Andreas; Hens, Felicitas; Kimpfler, Andrea; Schubert, Rolf; Peschka-Süss, Regine

2006-10-01

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependent drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 degrees C or 30% at 37 degrees C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.

Supramolecular gel electrophoresis of large DNA fragments.

PubMed

Tazawa, Shohei; Kobayashi, Kazuhiro; Oyoshi, Takanori; Yamanaka, Masamichi

2017-10-01

Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C 3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous injection effective mixing flow analysis of urinary albumin using dye-binding reaction.

PubMed

Ratanawimarnwong, Nuanlaor; Ponhong, Kraingkrai; Teshima, Norio; Nacapricha, Duangjai; Grudpan, Kate; Sakai, Tadao; Motomizu, Shoji

2012-07-15

A new four-channel simultaneous injection effective mixing flow analysis (SIEMA) system has been assembled for the determination of urinary albumin. The SIEMA system consisted of a syringe pump, two 5-way cross connectors, four holding coils, five 3-way solenoid valves, a 50-cm long mixing coil and a spectrophotometer. Tetrabromophenol blue anion (TBPB) in Triton X-100 micelle reacted with albumin at pH 3.2 to form a blue ion complex with a λ(max) 625nm. TBPB, Triton X-100, acetate buffer and albumin standard solutions were aspirated into four individual holding coils by a syringe pump and then the aspirated zones were simultaneously pushed in the reverse direction to the detector flow cell. Baseline drift, due to adsorption of TBPB-albumin complex on the wall of the hydrophobic PTFE tubing, was minimized by aspiration of Triton X-100 and acetate buffer solutions between samples. The calibration graph was linear in the range of 10-50μg/mL and the detection limit for albumin (3σ) was 0.53μg/mL. The RSD (n=11) at 30μg/mL was 1.35%. The sample throughput was 37/h. With a 10-fold dilution, interference from urine matrix was removed. The proposed method has advantages in terms of simple automation operation and short analysis time. Copyright © 2012 Elsevier B.V. All rights reserved.

Time-related Changes in pH, Buffering Capacity and Phosphate and Urea Concentration of Stimulated Saliva.

PubMed

Vuletic, Lea; Peros, Kristina; Spalj, Stjepan; Rogic, Dunja; Alajbeg, Ivan

2014-01-01

To quantify changes in pH, buffering capacity and hydrogen carbonate, phosphate, protein and urea concentrations of stimulated saliva which occur during a 30-min measurement delay after saliva collection. The correlation between time-related chemical changes and changes of salivary pH and buffering capacity was assessed in order to explain the observed changes in salivary pH and buffering capacity. Stimulated saliva samples were collected from 30 volunteers after inducing salivation by chewing a piece of parafilm. Measurements of salivary variables were made immediately after saliva collection and again 30 min later, during which time the specimens were exposed to the atmosphere in collection cups at room temperature. Postponement of measurements resulted in a significant increase in pH and a significant decrease of buffering capacity, phosphate and urea concentration. The results suggest that the time-related pH increase could primarily be attributed to loss of dissolved carbon dioxide from saliva, and confirm the importance of hydrogen carbonate in the neutralisation of hydrogen ions, but they do not support the principle of catalysed phase-buffering for the hydrogen carbonate buffer system in saliva. A decrease in phosphate and urea concentration affects salivary buffering capacity. This study emphasises the importance of the standardisation of measurement time when measuring salivary pH, buffering capacity, phosphate and urea concentrations following the collection of saliva in order to obtain comparable results. It also provides a partial explanation of the mechanisms underlying the observed changes of pH and buffering capacity over time.

Dissolution patterns of biocompatible glasses in 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffer.

PubMed

Fagerlund, S; Hupa, L; Hupa, M

2013-02-01

A continuous flow measurement system with sensitive on-line ion analysis has been applied to study the initial dissolution behaviour of biocompatible glasses in Tris. Altogether 16 glasses with widely varying compositions were studied. The measurement system allowed for quantitative determination of the time-dependent rates of dissolution of sodium, potassium, calcium, magnesium, silicon and phosphorus during the first 10-15 min in contact with Tris solution. The dissolution rates of the different ions showed significant glass to glass variations, but all glasses studied showed one of four distinct dissolution patterns. The ion dissolution rates after an exposure of 1000 s, expressed as the normalized surface-specific mass loss rates, were compared with the in vitro and in vivo reactivity of the glasses as predicted by models in the literature. The results showed a clear correlation between the dissolution rates of the glasses in Tris and their reactivity as measured by other different methods. Consequently, the measured short-term dissolution patterns could be used to determine which glasses are suitable as bioactive, biodegradable, or inert biomaterials for medical devices. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparison of different extenders on the recovery and longevity of epididymal sperm from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831).

PubMed

da Silva, Andréia Maria; Sousa, Patrícia Cunha; Campos, Lívia Batista; Bezerra, José Artur Brilhante; de Araújo Lago, Arthur Emannuel; de Oliveira, Moacir Franco; Silva, Alexandre Rodrigues

2017-04-01

The aim of this study was to evaluate the performance of cavy (Galea spixii) epididymal sperm following addition to TES or TRIS extenders and using a thermal resistance test (TRT), as well as fluorescence analysis as a complementary method to predict the viability of these gametes. Nine testicle-epididymis complexes were used for sperm collection using a flotation method. Epididymis tails were sliced and one was immersed in 3 ml of TRIS buffer, and the other in 3 ml of TES, for 5 min. After sperm recovery, the samples were subjected to a TRT which involved incubation in a water bath at 37°C for 3 h. During incubation, sample parameters were assessed at 0, 15, 30, 60, 90, 120, 150 or 180 min intervals. Results indicated that the TRIS diluent was more efficient than TES (P < 0.05) for the maintenance of sperm parameters in Spix's yellow-toothed cavies over the whole TRT, maintaining sperm longevity for an extended time. In conclusion, we indicate the use of TRIS diluent for recovery and maintenance of longevity of epididymal sperm from cavies (G. spixii).

Mapping Soil pH Buffering Capacity of Selected Fields

NASA Technical Reports Server (NTRS)

Weaver, A. R.; Kissel, D. E.; Chen, F.; West, L. T.; Adkins, W.; Rickman, D.; Luvall, J. C.

2003-01-01

Soil pH buffering capacity, since it varies spatially within crop production fields, may be used to define sampling zones to assess lime requirement, or for modeling changes in soil pH when acid forming fertilizers or manures are added to a field. Our objective was to develop a procedure to map this soil property. One hundred thirty six soil samples (0 to 15 cm depth) from three Georgia Coastal Plain fields were titrated with calcium hydroxide to characterize differences in pH buffering capacity of the soils. Since the relationship between soil pH and added calcium hydroxide was approximately linear for all samples up to pH 6.5, the slope values of these linear relationships for all soils were regressed on the organic C and clay contents of the 136 soil samples using multiple linear regression. The equation that fit the data best was b (slope of pH vs. lime added) = 0.00029 - 0.00003 * % clay + 0.00135 * % O/C, r(exp 2) = 0.68. This equation was applied within geographic information system (GIS) software to create maps of soil pH buffering capacity for the three fields. When the mapped values of the pH buffering capacity were compared with measured values for a total of 18 locations in the three fields, there was good general agreement. A regression of directly measured pH buffering capacities on mapped pH buffering capacities at the field locations for these samples gave an r(exp 2) of 0.88 with a slope of 1.04 for a group of soils that varied approximately tenfold in their pH buffering capacities.

Improved biocompatibility of bicarbonate/lactate-buffered PDF is not related to pH.

PubMed

Zareie, Mohammad; Keuning, Eelco D; ter Wee, Piet M; Schalkwijk, Casper G; Beelen, Robert H J; van den Born, Jacob

2006-01-01

Chronic exposure to conventional peritoneal dialysis fluid (PDF) is associated with functional and structural alterations of the peritoneal membrane. The bioincompatibility of conventional PDF can be due to hypertonicity, high glucose concentration, lactate buffering system, presence of glucose degradation products (GDPs) and/or acidic pH. Although various investigators have studied the sole effects of hyperosmolarity, high glucose, GDPs and lactate buffer in experimental PD, less attention has been paid to the chronic impact of low pH in vivo. Rats received daily 10 ml of either conventional lactate-buffered PDF (pH 5.2; n=7), a standard bicarbonate/lactate-buffered PDF with physiological pH (n=8), bicarbonate/lactate-buffered PDF with acidic pH (adjusted to pH 5.2 with 1 N hydrochloride, n=5), or bicarbonate/lactate buffer, without glucose, pH 7.4 (n=7). Fluids were instilled via peritoneal catheters connected to implanted subcutaneous mini vascular access ports for 8 weeks. Control animals with or without peritoneal catheters served as control groups (n=8/group). Various functional (2 h PET) and morphological/cellular parameters were analyzed. Compared with control groups and the buffer group, conventional lactate-buffered PDF induced a number of morphological/cellular changes, including angiogenesis and fibrosis in various peritoneal tissues (all parameters P<0.05), accompanied by increased glucose absorption and reduced ultrafiltration capacity. Daily exposure to standard or acidified bicarbonate/lactate-buffered PDF improved the performance of the peritoneal membrane, evidenced by reduced new vessel formation in omentum (P<0.02) and parietal peritoneum (P<0.008), reduced fibrosis (P<0.02) and improved ultrafiltration capacity. No significant differences were found between standard and acidified bicarbonate/lactate-buffered PDF. During PET, acidic PDF was neutralized within 15 to 20 min. The bicarbonate/lactate-buffered PDF, acidity per se did not contribute substantially to peritoneal worsening in our in vivo model for PD, which might be explained by the buffering capacity of the peritoneum.

In vitro effects of sodium bicarbonate buffer on rumen fermentation, levels of lipopolysaccharide and biogenic amine, and composition of rumen microbiota.

PubMed

Mao, Shengyong; Huo, Wenjie; Liu, Junhua; Zhang, Ruiyang; Zhu, Weiyun

2017-03-01

Diets containing high levels of carbohydrates provoke a rapid decrease of rumen pH and high levels of biogenic amines and lipopolysaccharides (LPS), which severely impair the health and performance of ruminants. The goal of this study was to evaluate the effects of sodium bicarbonate (BC) buffer on rumen fermentation, levels of LPS and biogenic amine, and composition of rumen microbiota using in vitro rumen cultures. Sodium bicarbonate supplementation increased (P < 0.05) the final pH levels and concentrations of total volatile fatty acids and LPS, as well as the proportions of acetate, propionate, isobutyrate, isovalerate and valerate, and it decreased (P < 0.05) the proportion of butyrate and the levels of lactic acid, methylamine, tryptamine, tyramine, histamine and putrescine compared with the control. Pyrosequencing of the 16S rRNA gene showed that BC inclusion increased (P < 0.05) the bacterial diversity index compared with the control. Adding BC also decreased (P < 0.05) the relative abundance of Streptococcus and Butyrivibrio and increased (P < 0.05) the proportions of Ruminococcus, Succinivibrio and Prevotella. Sodium bicarbonate supplementation has beneficial effects in the reduction of bioamine levels and the increase in ruminal pH, and in modifying the microbial ecology of the rumen; however, it results in an accumulation of LPS under high-grain diet conditions. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Preparation, characterization and in vitro release study of carvacrol-loaded chitosan nanoparticles.

PubMed

Keawchaoon, Lalita; Yoksan, Rangrong

2011-05-01

The fabrication of carvacrol-loaded chitosan nanoparticles was achieved by a two-step method, i.e., oil-in-water emulsion and ionic gelation of chitosan with pentasodium tripolyphosphate. The obtained particles possessed encapsulation efficiency (EE) and loading capacity (LC) in the ranges of 14-31% and 3-21%, respectively, when the initial carvacrol content was 0.25-1.25 g/g of chitosan. The individual particles exhibited a spherical shape with an average diameter of 40-80 nm, and a positively charged surface with a zeta potential value of 25-29 mV. The increment of initial carvacrol content caused a reduction of surface charge. Carvacrol-loaded chitosan nanoparticles showed antimicrobial activity against Staphylococcus aureus, Bacillus cereus and Escherichia coli with an MIC of 0.257 mg/mL. The release of carvacrol from chitosan nanoparticles reached plateau level on day 30, with release amounts of 53% in acetate buffer solution with pH of 3, and 23% and 33% in phosphate buffer solutions with pH of 7 and 11, respectively. The release mechanism followed a Fickian behavior. The release rate was superior in an acidic medium to either alkaline or neutral media, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, Chiho; Quantum Beam Science Directorate, Japan Atomic Energy Agency; Taura, Futoshi

Polyketide synthase-1 from C. sativa has been crystallized. The crystal diffracted to 1.55 Å resolution with sufficient quality for further structure determination. Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa (Mexican strain). PKS-1 was overproduced in Escherichia coli, purified and finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w/v) polyethylene glycol 3350 diffracted to 1.65 Å resolution and belonged to space group P1, with unit-cell parameters a = 54.3, b =more » 59.3, c = 62.6 Å, α = 69, β = 81, γ = 80°. Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w/v) polyethylene glycol 3350 diffracted to 1.55 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.3, b = 110, c = 130 Å. These data will enable us to determine the crystal structure of PKS-1.« less

Eu(III)-Sensitized Luminescence Probe for Determination of Tolnaftate in Pharmaceuticals and Biological Fluids.

PubMed

Alarfaj, Nawal A; El-Tohamy, Maha F

2016-01-01

A highly selective, sensitive, accurate, and reproducible luminescence procedure for determination of antifungal drug tolnaftate was developed. The introduced method was based on the formation of Europa Universalis III (Eu(III))-tolnaftate complex using sodium sulfite as a deoxygenated agent in the presence of acetate buffer (pH = 6) and micellar solution of anionic surfactant sodium dodecyl sulfate. The optimum conditions (effect of pH, buffer, surfactant, Eu(III), and sodium sulfite concentrations) for the luminescence signal were investigated and optimized. The luminescence signals were recorded at λex = 270 nm and λem = 460 nm. The method has a good linear response (0.2-130 μg/mL(-1)) between the luminescence intensity and the concentrations of the drug (r = 0.999), with a LOD 0.07 μg/mL(-1) and LOQ 0.2 μg/mL(-1). The luminescence signals of Eu (III)-tolnaftate-sodium dodecyl sulfate were found to be 200-fold more sensitive without the presence of micelle solution. The interferences of some additives, metals, amino acids, sugars, and other related pharmacological action drugs were examined and no interference was recorded. The proposed method was used for quick and simple determination of tolnaftate in its pharmaceuticals and biological fluids.

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis.

PubMed

Masarik, Michal; Gumulec, Jaromir; Sztalmachova, Marketa; Hlavna, Marian; Babula, Petr; Krizkova, Sona; Ryvolova, Marketa; Jurajda, Michal; Sochor, Jiri; Adam, Vojtech; Kizek, Rene

2011-12-01

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Separation of catechins and methylxanthines in tea samples by capillary electrochromatography.

PubMed

Uysal, Ulku Dilek; Aturki, Zeineb; Raggi, Maria Augusta; Fanali, Salvatore

2009-04-01

In this paper, the simultaneous separation of several polyphenols such as (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, theophylline, caffeine in green and black teas by capillary electrochromatography (CEC) was developed. Several experimental parameters such as stationary phase type, mobile phase composition, buffer and pH, inner diameter of the columns, sample injection, were evaluated to obtain the complete separation of the analysed compounds. Baseline resolution of the studied polyphenols was achieved within 30 min by using a capillary column (id 100 microm) packed with bidentate C(18) particles for 24.5 cm and a mobile phase composed of 5 mM ammonium acetate buffer pH 4 with H(2)O/ACN (80:20, v/v). The applied voltage and the temperature were set at 30 kV and 20 degrees C. Precision, detection and quantification limits, linearity, and accuracy were investigated. A good linearity (R(2) > 0.9992) was achieved over a concentration working range of 2-100 microg/mL for all the analytes. LOD and LOQ were 1 and 2 microg/mL, respectively, for all studied compounds. The CEC method was applied to the analysis of those polyphenols in green and black tea samples after an extraction procedure. Good recovery data from accuracy studies ranged between 90% and 112% for all analytes.

Modelling anaerobic digestion acclimatisation to a biodegradable toxicant: application to cyanide.

PubMed

Zaher, U; Moussa, M S; Widyatmika, I N; van Der Steen, P; Gijzen, H J; Vanrolleghem, P A

2006-01-01

The observed acclimatisation to biodegradable toxicants in anaerobic cassava wastewater treatment is explained by modelling anaerobic cyanide degradation. A complete degradation pathway is proposed for cyanide. Cyanide degradation is modelled as enzymatic hydrolysis to formate and ammonia. Ammonia is added to the inorganic nitrogen content of the digester while formate is degraded by the hydrogenotrophic methanogens. Cyanide irreversible enzyme inhibition is modelled as an inhibition factor to acetate uptake processes. Cyanide irreversible toxicity is modelled as a decay factor to the acetate degraders. Cyanide as well as added phosphorus buffer solution were considered in the chemical equilibrium calculations of pH. The observed reversible effect after acclimatisation of sludge is modelled by a population shift between two aceticlastic methanogens that have different tolerance to cyanide toxicity. The proposed pathway is added to the IWA Anaerobic Digestion Model no.1 (ADM1). The ADM1 model with the designed extension is validated by an experiment using three lab-scale upflow anaerobic sludge bed reactors which were exposed to different cyanide loadings.

Simultaneous Determination of Potassium Sorbate, Sodium Benzoate, Quinoline Yellow and Sunset Yellow in Lemonades and Lemon Sauces by HPLC Using Experimental Design.

PubMed

Dinç Zor, Şule; Aşçı, Bürge; Aksu Dönmez, Özlem; Yıldırım Küçükkaraca, Dilek

2016-07-01

In this study, development and validation of a HPLC method was described for simultaneous determination of potassium sorbate, sodium benzoate, quinoline yellow and sunset yellow. A Box-Behnken design using three variables at three levels was employed to determine the optimum conditions of chromatographic separation: pH of mobile phase, 6.0-7.0; flow rate, 0.8-1.2 mL min(-1) and the ratio of mobile phase composed of a 0.025 M sodium acetate/acetic acid buffer, 80-90%. Resolution was chosen as a response. The optimized method was validated for linearity, the limits of detection and quantification, accuracy, precision and stability. All the validation parameters were within the acceptance range. The applicability of the developed method to the determination of these food additives in commercial lemonade and lemon sauce samples was successfully demonstrated. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

PubMed Central

Chu, S; Montrose, M H

1995-01-01

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments. Images Fig. 1 Fig. 3 PMID:7724557

Matrix Effect Evaluation and Method Validation of Azoxystrobin and Difenoconazole Residues in Red Flesh Dragon Fruit (Hylocereus polyrhizus) Matrices Using QuEChERS Sample Preparation Methods Followed by LC-MS/MS Determination.

PubMed

Noegrohati, Sri; Hernadi, Elan; Asviastuti, Syanti

2018-06-01

Production of red flesh dragon fruit (Hylocereus polyrhizus) was hampered by Colletotrichum sp. Pre-harvest application of azoxystrobin and difenoconazole mixture is recommended, therefore, a selective and sensitive multi residues analytical method is required in monitoring and evaluating the commodity's safety. LC-MS/MS is a well-established analytical technique for qualitative and quantitative determination in complex matrices. However, this method is hurdled by co-eluted coextractives interferences. This work evaluated the pH effect of acetate buffered and citrate buffered QuEChERS sample preparation in their effectiveness of matrix effect reduction. Citrate buffered QuEChERS proved to produce clean final extract with relative matrix effect 0.4%-0.7%. Method validation of the selected sample preparation followed by LC-MS/MS for whole dragon fruit, flesh and peel matrices fortified at 0.005, 0.01, 0.1 and 1 g/g showed recoveries 75%-119%, intermediate repeatability 2%-14%. The expanded uncertainties were 7%-48%. Based on the international acceptance criteria, this method is valid.

Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

PubMed Central

Morgner, Frank; Stufler, Stefan; Geißler, Daniel; Medintz, Igor L.; Algar, W. Russ; Susumu, Kimihiro; Stewart, Michael H.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Hildebrandt, Niko

2011-01-01

Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma. PMID:22163719

Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems.

PubMed

Pikal-Cleland, Katherine A; Cleland, Jeffrey L; Anchordoquy, Thomas J; Carpenter, John F

2002-09-01

Previous studies have established that the selective precipitation of a less soluble buffer component during freezing can induce a significant pH shift in the freeze concentrate. During freezing of sodium phosphate solutions, crystallization of the disodium salt can produce a pH decrease as great as 3 pH units which can dramatically affect protein stability. The objective of our study was to determine how the presence of glycine (0-500 mM), a commonly used bulking agent in pharmaceutical protein formulations, affects the pH changes normally observed during freezing in sodium phosphate buffer solutions and to determine whether these pH changes contribute to instability of model proteins in glycine/phosphate formulations. During freezing in sodium phosphate buffers, the presence of glycine significantly influenced the pH. Glycine at the lower concentrations (< or = 50 mM) suppressed the pH decrease normally observed during freezing in 10 and 100 mM sodium phosphate buffer, possibly by reducing the nucleation rate of salt and thereby decreasing the extent of buffer salt crystallization. The presence of glycine at higher concentration (> 100 mM) in the sodium phosphate buffer resulted in a more complete crystallization of the disodium salt as indicated by the frozen pH values closer to the equilibrium value (pH 3.6). Although high concentrations of glycine can facilitate more buffer salt crystallization and these pH shifts may prove to be potentially damaging to the protein, glycine, in its amorphous state, can also act to stabilize a protein via the preferential exclusion mechanism. Copyright 2002 Wiley-Liss Inc.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

PubMed Central

2002-01-01

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column. PMID:11972900

Enantioseparation of dopa and related compounds by cyclodextrin-modified microemulsion electrokinetic chromatography.

PubMed

Borst, Claudia; Holzgrabe, Ulrike

2008-09-19

A chiral microemulsion electrokinetic chromatography method has been developed for the enantiomeric separation of 3,4-dihydroxyphenylalanine (dopa), its precursors phenylalanine and tyrosine, and the structurally related substance methyldopa. The separations were achieved using an oil-in-water microemulsion, which consisted of the oil-compound ethyl acetate, the surfactant sodium dodecylsulfate (SDS), the co-surfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 or 2.0 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The resolution of each racemate was optimized by varying the concentration of the buffer and all components of the microemulsion. Enantioseparation could be achieved for dl-dopa, dl-phenylalanine and dl-tyrosine within 13 min with a resolution of 4.3, 3.1 and 3.3, respectively, and for methyldopa in 17 min (Rs: 1.4). The established methods allowed the detection of dopa, phenylalanine, tyrosine and methyldopa with a limit at 0.5, 1.0, 0.2 and 2.0 microg/ml.

Ensifer meliloti overexpressing Escherichia coli phytase gene ( appA) improves phosphorus (P) acquisition in maize plants

NASA Astrophysics Data System (ADS)

Sharma, Vikas; Kumar, Ajit; Archana, G.; Kumar, G. Naresh

2016-10-01

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 ( lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti { E. m (pVA1)} increased its phosphatase and phytase activity by ˜9- and ˜50-fold, respectively, compared to the transformants containing empty plasmid as control { E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein { E. m (pVA1)} and { E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both { E. m (pVA1)} and { E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with { E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

PubMed

Sharma, Vikas; Kumar, Ajit; Archana, G; Kumar, G Naresh

2016-10-01

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

PubMed

Ni, He; Guo, Peng-Cheng; Jiang, Wei-Ling; Fan, Xiao-Min; Luo, Xiang-Yu; Li, Hai-Hang

2016-08-10

Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolism of Cytokinin 1

PubMed Central

Chen, Chong-Maw; Kristopeit, Susan M.

1981-01-01

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N6-(Δ2-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The Km values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N6-(Δ2-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells. PMID:16661701

[The effects of electromagnetic radiation of extremely high frequency and low intensity on the growth rate of bacteria Escherichia coli and the role of medium pH].

PubMed

Tadevosian, A; Kalantarian, V; Trchunian, A

2007-01-01

It has been shown that coherent electromagnetic irradiation (EMI) of extremely high frequency (45-53 GHz) or millimeter waves (wavelength 5.6-6.7 mm) of low intensity (flux capacity 0.06 mW/cm2) of Escherichia coli K12, grown under anaerobic conditions during the fermentation of sugar (glucose) for 30 min or 1 h, caused a decrease in their growth rate, the maximum inhibitory effect being achieved at a frequency of 51.8 or 53 GHz. This effect depended on medium pH when the maximal action was determined at pH 7.5. In addition, separate 30-min of 1-h irradiation (frequency 51.8 or 53 GHz) of doubly distilled water or some inorganic ions contained in Tris-phosphate buffer where the cells were transferred induced oppositely directed changes in further growth of these bacteria under anaerobic conditions; irradiation of water caused a decrease in the growth rate of bacteria. A significant change in pH of water (0.5-1.5 unit) was induced by a 30-irradiation at a frequency of 49, 50.3, 51.8, or 53 GHz, when the initial pH value was 6.0 or 8.0, but not 7.5. These results indicate the changes in the properties of water and its role in the effects of EMI of extremely high frequency. The marked effect of EMI on bacteria disappeared upon repeated irradiation for 1 h at a frequency of 51.8 or 53 GHz with an interval of 2 hours. This result indicates some compensatory mechanisms in bacteria.

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid.

PubMed

Anumula, K R; Dhume, S T

1998-07-01

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.

Solubility of ammonium acid urate nephroliths from bottlenose dolphins (Tursiops truncatus).

PubMed

Argade, Sulabha; Smith, Cynthia R; Shaw, Timothy; Zupkas, Paul; Schmitt, Todd L; Venn-Watson, Stephanie; Sur, Roger L

2013-12-01

Nephrolithiasis has been identified in managed populations of bottlenose dolphins (Tursiops truncatus); most of these nephroliths are composed of 100% ammonium acid urate (AAU). Several therapies are being investigated to treat and prevent nephrolithiasis in dolphins including the alkalization of urine for dissolution of nephroliths. This study evaluates the solubility of AAU nephroliths in a phosphate buffer, pH range 6.0-8.0, and in a carbonate-bicarbonate buffer, pH range 9.0-10.8. AAU nephroliths were obtained from six dolphins and solubility studies were conducted using reverse-phase high performance liquid chromatography with ultraviolet detection at 290 nm. AAU nephroliths were much more soluble in a carbonate-bicarbonate buffer, pH range 9.0-10.8 compared to phosphate buffer pH range 6.0-8.0. In the pH range 6.0-8.0, the solubility was 45% lower in potassium phosphate buffer compared to sodium phosphate buffer. When citrate was used along with phosphate in the same pH range, the solubility was improved by 13%. At pH 7 and pH 8, 150 mM ionic strength buffer was optimum for dissolution. In summary, adjustment of urinary pH alone does not appear to be a useful way to treat AAU stones in bottlenose dolphins. Better understanding of the pathophysiology of AAU nephrolithiasis in dolphins is needed to optimize kidney stone prevention and treatment.

Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application.

PubMed

Kröner, Frieder; Hubbuch, Jürgen

2013-04-12

pH gradient protein separations are widely used techniques in the field of protein analytics, of which isoelectric focusing is the most well known application. The chromatographic variant, based on the formation of pH gradients in ion exchange columns is only rarely applied due to the difficulties to form controllable, linear pH gradients over a broad pH range. This work describes a method for the systematic generation of buffer compositions with linear titration curves, resulting in well controllable pH gradients. To generate buffer compositions with linear titration curves an in silico method was successfully developed. With this tool, buffer compositions for pH gradient ion exchange chromatography with pH ranges spanning up to 7.5 pH units were established and successfully validated. Subsequently, the buffer systems were used to characterize the elution behavior of 22 different model proteins in cation and anion exchange pH gradient chromatography. The results of both chromatographic modes as well as isoelectric focusing were compared to describe differences in between the methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Local conformation and intermolecular interaction of rigid ring polymers are not always the same as the linear analogue: cyclic amylose tris(phenylcarbamate) in Θ solvents.

PubMed

Asano, Natsuki; Kitamura, Shinichi; Terao, Ken

2013-08-15

Small-angle X-ray scattering and static and dynamic light scattering measurements were made for cyclic amylose tris(phenylcarbamate) (cATPC) of which weight-average molar mass M(w) ranges from 1.3 × 10(4) to 1.5 × 10(5) to determine their z-average mean square radius of gyration z, particle scattering function P(q), hydrodynamic radius R(H), and second virial coefficient A2 in methyl acetate (MEA), ethyl acetate (EA), and 4-methyl-2-pentanone (MIBK). The obtained z, P(q), and R(H) data were analyzed in terms of the wormlike ring to estimate the helix pitch per residue h and the Kuhn segment length λ(-1) (the stiffness parameter, twice the persistence length). Both h and λ(-1) for cATPC in MEA, EA, and MIBK are smaller than those for linear amylose tris(phenylcarbamate) (ATPC) in the corresponding solvent and the discrepancy becomes more significant with increasing the molar volume of the solvent. This indicates that not every rigid ring has the same local helical structure and chain stiffness as that for the linear polymer in the M(w) range investigated while infinitely long ring chains should have the same local conformation. This conformational difference also affects A2. In actuality, negative A2 was observed for cATPC in MIBK at the Θ temperature of linear ATPC whereas intermolecular topological interaction of ring polymers increases A2.

Reduction-Triggered Self-Assembly of Nanoscale Molybdenum Oxide Molecular Clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Panchao; Wu, Bin; Li, Tao

A 2.9 nm molybdenum oxide cluster {Mo 132} (Formula: [Mo VI 72Mo V 60O 372(CH 3COO) 30(H 2O) 72] 42-) can be obtained by reducing ammonium molybdate with hydrazine sulfate in weakly acidic CH 3COOH/CH 3COO- buffers. This reaction has been monitored by time-resolved UV-Vis, 1H-NMR, small angle X-ray/neutron scattering, and X-ray absorption near edge structure spectroscopy. The growth of {Mo 132} cluster shows a typical sigmoid curve, suggesting a multi-step assembly mechanism for this reaction. The reaction starts with a lag phase period when partial MoVI centers of molybdate precursors are reduced to form {MoV2(acetate)} structures under the coordinationmore » effect of the acetate groups. Once the concentration of {Mo V 2(acetate)} reaches a critical value, it triggers the assembly of Mo V and Mo VI species into {Mo 132} clusters. Parameters such as the type and amount of reducing agent, the pH, the type of cation, and the type of organic ligand in the reaction buffer, have been studied for the roles they play in the formation of the target clusters.Understanding the formation mechanism of giant molecular clusters is essential for rational design and synthesis of cluster-based nanomaterials with required morphologies and functionalities. Here, typical synthetic reactions of a 2.9 nm spherical molybdenum oxide cluster, {Mo 132} (formula: [Mo VI 72Mo V 60O 372(CH 3COO) 30(H 2O) 72] 42), with systematically varied reaction parameters have been fully explored to determine the morphologies and concentration of products, reduction of metal centers, and chemical environments of the organic ligands. The growth of these clusters shows a typical sigmoid curve, suggesting a general multistep self-assembly mechanism for the formation of giant molecular clusters. The reaction starts with a lag phase period when partial MoVI centers of molybdate precursors are reduced to form {Mo V 2(acetate)} structures under the coordination effect of the acetate groups. Once the concentration of {MoV2(acetate)} reaches a critical value, it triggers the co-assembly of Mo V and Mo VI species into the giant clusters.« less

Reduction-Triggered Self-Assembly of Nanoscale Molybdenum Oxide Molecular Clusters

DOE PAGES

Yin, Panchao; Wu, Bin; Li, Tao; ...

2016-07-26

A 2.9 nm molybdenum oxide cluster {Mo 132} (Formula: [Mo VI 72Mo V 60O 372(CH 3COO) 30(H 2O) 72] 42-) can be obtained by reducing ammonium molybdate with hydrazine sulfate in weakly acidic CH 3COOH/CH 3COO- buffers. This reaction has been monitored by time-resolved UV-Vis, 1H-NMR, small angle X-ray/neutron scattering, and X-ray absorption near edge structure spectroscopy. The growth of {Mo 132} cluster shows a typical sigmoid curve, suggesting a multi-step assembly mechanism for this reaction. The reaction starts with a lag phase period when partial MoVI centers of molybdate precursors are reduced to form {MoV2(acetate)} structures under the coordinationmore » effect of the acetate groups. Once the concentration of {Mo V 2(acetate)} reaches a critical value, it triggers the assembly of Mo V and Mo VI species into {Mo 132} clusters. Parameters such as the type and amount of reducing agent, the pH, the type of cation, and the type of organic ligand in the reaction buffer, have been studied for the roles they play in the formation of the target clusters.Understanding the formation mechanism of giant molecular clusters is essential for rational design and synthesis of cluster-based nanomaterials with required morphologies and functionalities. Here, typical synthetic reactions of a 2.9 nm spherical molybdenum oxide cluster, {Mo 132} (formula: [Mo VI 72Mo V 60O 372(CH 3COO) 30(H 2O) 72] 42), with systematically varied reaction parameters have been fully explored to determine the morphologies and concentration of products, reduction of metal centers, and chemical environments of the organic ligands. The growth of these clusters shows a typical sigmoid curve, suggesting a general multistep self-assembly mechanism for the formation of giant molecular clusters. The reaction starts with a lag phase period when partial MoVI centers of molybdate precursors are reduced to form {Mo V 2(acetate)} structures under the coordination effect of the acetate groups. Once the concentration of {MoV2(acetate)} reaches a critical value, it triggers the co-assembly of Mo V and Mo VI species into the giant clusters.« less

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7.

PubMed

Hosein, Althea M; Breidt, Frederick; Smith, Charles E

2011-02-01

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.

Salivary buffer capacity, pH, and stimulated flow rate of crack cocaine users.

PubMed

Woyceichoski, Iverson Ernani Cogo; Costa, Carlos Henrique; de Araújo, Cristiano Miranda; Brancher, João Armando; Resende, Luciane Grochocki; Vieira, Iran; de Lima, Antonio Adilson Soares

2013-08-01

Crack cocaine is the freebase form of cocaine that can be smoked. The use of this drug has been considered a public health problem in many countries. The aim of this study was to assess the stimulated salivary flow rate (SSFR), pH, and the buffer capacity of saliva in crack cocaine users. Stimulated whole saliva was collected from 54 selected crack cocaine users and 40 non-users. All samples were analyzed for SSFR, pH, and buffer capacity. SSFR was analyzed by gravimetric method. The buffer capacity and pH were determined using a digital pH meter. The crack cocaine users demonstrated higher buffer capacity than the control group (P > 0.05). Salivary pH was lower in crack cocaine users (P < 0.05). Mean values of the SSFR for the experimental and control groups were 1.1 and 1.3 mL/min, respectively (P > 0.05). Crack cocaine users might exhibit a significant decrease in salivary pH, but not in salivary flow rate or buffer capacity. © 2012 Blackwell Publishing Asia Pty Ltd.

Comparative in vitro antimicrobial efficacy of commercial ear cleaners.

PubMed

Swinney, Alison; Fazakerley, Jennifer; McEwan, Neil; Nuttall, Tim

2008-12-01

The aim of this study was to compare the antimicrobial efficacy of ear cleaners against Staphylococcus intermedius, Pseudomonas aeruginosa and Malassezia pachydermatis. Single isolates of each organism were incubated in duplicate at 38 degrees C for 30 min with each ear cleaner diluted 1/2 to 1/256 in phosphate-buffered saline. Positive and negative controls were included. Aliquots were then incubated for 16-18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud's dextrose agar (Malassezia) at 38 degrees C. The lowest dilutions exhibiting 100% antimicrobial efficacy for S. intermedius were: Cleanaural Dog 1/32; Sancerum 1/16; Otoclean 1/4; EpiOtic 1/2; MalAcetic 1/2; and Triz Plus 1/2. The results for P. aeruginosa were Sancerum and Triz Plus 1/16; Cleanaural Dog and EpiOtic 1/8; Otoclean 1/4; and MalAcetic 1/2. Results for M. pachydermatis were: Cleanaural Dog 1/32; Sancerum, Otoclean, EpiOtic and Triz Plus 1/8; and MalAcetic 1/4. Cleanaural Cat, MalAcetic HC and Triz EDTA did not display any antimicrobial activity at any dilution. Antimicrobial activity appeared to be associated with the presence of isopropyl alcohol, parachlorometaxylenol and a low pH. The results of this study may help clinicians make evidence-based decisions when selecting ear cleaners for use in individual cases.

Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase.

PubMed

Barron, Christiaan C; Sponagle, Brandon J D; Arivalagan, Pugazhendhi; D'Cunha, Godwin B

2017-01-01

Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO 4 ]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF 4 ]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF 6 ]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF 6 ]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0min) L -phenylalanine substrate concentration (30.0mM), nature of buffer (50.0mM Tris-HCl), pH (9.0), temperature (37°C), and speed of agitation (100 rev min -1 ). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290nm. PAL reverse reaction in ([BMIM][PF 6 ]) was determined spectrophotometrically at 520nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids. Copyright © 2016 Elsevier Inc. All rights reserved.

Boron-capped tris(glyoximato) cobalt clathrochelate as a precursor for the electrodeposition of nanoparticles catalyzing H2 evolution in water.

PubMed

Anxolabéhère-Mallart, Elodie; Costentin, Cyrille; Fournier, Maxime; Nowak, Sophie; Robert, Marc; Savéant, Jean-Michel

2012-04-11

Electrochemical investigation of a boron-capped tris(glyoximato)cobalt clathrochelate complex in the presence of acid reveals that the catalytic activity toward hydrogen evolution results from an electrodeposition of cobalt-containing nanoparticles on the electrode surface at a modest cathodic potential. The deposited particles act as remarkably active catalysts for H(2) production in water at pH 7. © 2012 American Chemical Society

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

PubMed Central

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-01-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å. PMID:16511316

Liquid-chromatographic determination of cephalosporins and chloramphenicol in serum.

PubMed

Danzer, L A

1983-05-01

A "high-performance" liquid-chromatographic technique involving a radial compression module is used for measuring chloramphenicol and five cephalosporin antibiotics: cefotaxime, cefoxitin, cephapirin, and cefamandol. Serum proteins are precipitated with acetonitrile solution containing 4'-nitroacetanilide as the internal standard. The drugs are eluted with a mobile phase of methanol/acetate buffer (30/70 by vol), pH 5.5. Absorbance of the cephalosporins is monitored at 254 nm. Standard curves are linear to at least 100 mg/L. The absorbance of chloramphenicol is monitored at 254 nm and 280 nm, and its standard curve is linear to at least 50 mg/L. The elution times for various other drugs were also determined, to check for potential interferents.

Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols.

PubMed

Cekiç, Sema Demirci; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2009-07-15

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na(2)EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-1). Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.

Effect of a low-moisture buffer block on ruminal pH in lactating dairy cattle induced with subacute ruminal acidosis.

PubMed

Krause, K M; Dhuyvetter, D V; Oetzel, G R

2009-01-01

The objective of this study was to evaluate the effect of a low-moisture buffer block on ruminal pH and milk production in cows induced with subacute ruminal acidosis (SARA). Sixteen ruminally cannulated cows were randomly assigned to treatment (access to buffer blocks) or control (no buffer blocks). Ruminal pH was recorded each minute; dry matter intake (DMI), milk yield, and milk composition were measured daily. The experiment lasted 12 d and consisted of a 3-d pre-SARA period (without access to buffer blocks; d 1 to 3), after which 8 cows were given access to buffer blocks and 8 cows continued without access to buffer blocks. The next 4 d (d 4 to 7) were for evaluating the response to buffer blocks. On d 8, cows were restricted to 50% of previous DMI, and on d 9 SARA was induced (addition of 4 kg of wheat/barley pellet to pre-SARA total mixed ration (TMR). Cows were then monitored for a 3-d recovery period (d 10 to 12). The SARA challenge was successful in decreasing mean ruminal pH and time and area below pH 5.6. Intake of buffer blocks averaged 0.33 kg of DM/cow per day and was greatest on d 4 and d 8. Total DMI (TMR plus buffer block) and yields of milk and milk components were not affected by treatment. Although there was no overall effect of treatment on any of the ruminal pH variables measured, there were significant treatment by period interactions for several ruminal pH variables. Cows on the control treatment tended to experience a greater decrease in mean ruminal pH when induced with SARA than cows with access to buffer blocks (-0.55 vs. -0.20 pH units). Cows on the control treatment also experienced a greater increase in time (9.7 vs. 4.1 h/d) and area (249 vs. 83 min x pH units/d) below pH 5.6 compared with cows with access to buffer blocks. Ruminal volatile fatty acids, lactate, ethanol, and succinate concentrations during the SARA challenge did not differ between treatments. Eating behavior was not affected by treatment. Size of the first meal of the day was greater on the SARA challenge day than during the pre-SARA period (11.0 vs. 5.7 kg, as fed). Giving cows access to a buffer-containing molasses block may reduce the duration and the severity of a 1-d SARA challenge.

Dominant Negative Mutants of the Estrogen Receptor as Probes of Estrogen Action and Inhibitors of Breast Cancer Growth

DTIC Science & Technology

1996-07-01

tetrazolium, inner salt; MTS; Promega], 1.9 mg/ml, and an electron coupling reagent ( phenazine methosulfate; PMS; Sigma), 0.044 mg/ml, in Dulbecco’s...acids PBS, phosphate buffered saline PCR, polymerase chain reaction PMS, phenazine methosulfate poly A, polyadenylation s.e., standard error TAE, tris

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: pH buffering properties of cheese.

PubMed

Upreti, P; Bühlmann, P; Metzger, L E

2006-03-01

The pH buffering capacity of cheese is an important determinant of cheese pH. However, the effects of different constituents of cheese on its pH buffering capacity have not been fully clarified. The objective of this study was to characterize the chemical species and chemical equilibria that are responsible for the pH buffering properties of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), residual lactose (2.4 vs. 0.78%), and salt-to-moisture ratio (6.4 vs. 4.8%) were manufactured. The pH-titration curves for these cheeses were obtained by titrating cheese:water (1:39 wt/wt) dispersions with 1 N HCl, and backtitrating with 1 N NaOH. To understand the role of different chemical equilibria and the respective chemical species in controlling the pH of cheese, pH buffering was modeled mathematically. The 36 chemical species that were found to be relevant for modeling can be classified as cations (Na+, Ca2+, Mg2+), anions (phosphate, citrate, lactate), protein-bound amino acids with a side-chain pKa in the range of 3 to 9 (glutamate, histidine, serine phosphate, aspartate), metal ion complexes (phosphate, citrate, and lactate complexes of Na+, Ca2+, and Mg2+), and calcium phosphate precipitates. A set of 36 corresponding equations was solved to give the concentrations of all chemical species as a function of pH, allowing the prediction of buffering curves. Changes in the calculated species concentrations allowed the identification of the chemical species and chemical equilibria that dominate the pH buffering properties of cheese in different pH ranges. The model indicates that pH buffering in the pH range from 4.5 to 5.5 is predominantly due to a precipitate of Ca and phosphate, and the protonation equilibrium involving the side chains of protein-bound glutamate. In the literature, the precipitate is often referred to as amorphous colloidal calcium phosphate. A comparison of experimental data and model predictions shows that the buffering properties of the precipitate can be explained, assuming that it consists of hydroxyapatite [Ca5(OH)(PO4)3] or Ca3(PO4)2. The pH buffering in the region from pH 3.5 to 4.5 is due to protonation of side-chain carboxylates of protein-bound glutamate, aspartate, and lactate, in order of decreasing significance. In addition, pH buffering between pH 5 to 8 in the backtitration results from the reprecipitation of calcium and phosphate either as CaHPO4 or Ca4H(PO4)3.

Calorimetric and Diffractometric Evidence for the Sequential Crystallization of Buffer Components and the Consequential pH Swing in Frozen Solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaramurthi, Prakash; Shalaev, Evgenyi; Suryanarayanan, Raj

2010-06-22

Sequential crystallization of succinate buffer components in the frozen solution has been studied by differential scanning calorimetry and X-ray diffractometry (both laboratory and synchrotron sources). The consequential pH shifts were monitored using a low-temperature electrode. When a solution buffered to pH < pK{sub a2} was cooled from room temperature (RT), the freeze-concentrate pH first increased and then decreased. This was attributed to the sequential crystallization of succinic acid, monosodium succinate, and finally disodium succinate. When buffered to pH > pK{sub a2}, the freeze-concentrate pH first decreased and then increased due to the sequential crystallization of the basic (disodium succinate) followedmore » by the acidic (monosodium succinate and succinic acid) buffer components. XRD provided direct evidence of the crystallization events in the frozen buffer solutions, including the formation of disodium succinate hexahydrate [Na{sub 2}(CH{sub 2}COO){sub 2} {center_dot} 6H{sub 2}O]. When the frozen solution was warmed in a differential scanning calorimeter, multiple endotherms attributable to the melting of buffer components and ice were observed. When the frozen solutions were dried under reduced pressure, ice sublimation was followed by dehydration of the crystalline hexahydrate to a poorly crystalline anhydrate. However, crystalline succinic acid and monosodium succinate were retained in the final lyophiles. The pH and the buffer salt concentration of the prelyo solution influenced the crystalline salt content in the final lyophile. The direction and magnitude of the pH shift in the frozen solution depended on both the initial pH and the buffer concentration. In light of the pH-sensitive nature of a significant fraction of pharmaceuticals (especially proteins), extreme care is needed in both the buffer selection and its concentration.« less

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

PubMed

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Accounting for dissociation and photolysis: a review of the algal toxicity of triclosan.

PubMed

Roberts, Jayne; Price, Oliver R; Bettles, Nicola; Rendal, Cecilie; van Egmond, Roger

2014-11-01

Triclosan, an antimicrobial agent commonly used in down-the-drain consumer products, is toxic to freshwater microalgae. However, the rapid photolysis and pH-dependent dissociation of this compound may give rise to uncertainty in growth inhibition tests with freshwater microalgae, if these are not well characterized. Methods are presented to minimize these uncertainties by stabilizing pH with an organic buffering agent (Bis-Tris) and by the application of ultraviolet (UV) covers to remove UV wavelengths. Toxicity tests with these methods were in compliance with the validity criteria of the Organisation for Economic Co-operation and Development test 201, and no negative effects were seen in controls relative to the unmodified method. The methods were used for toxicity tests with triclosan at pH levels of 7.0, 8.0, and 8.5, yielding effective concentration, 10% values of 0.5 µg/L, 0.6 µg/L, and 12.1 µg/L, respectively. The observed change in toxicity with pH was proportional to the change in bioconcentration factor (BCF) as calculated using the cell model (a dynamic flux model based on the Fick-Nernst-Planck equations, in this case parameterized for an algal cell). Effect concentrations produced with the methods presented in the present study offer robust data on which to base risk assessment, and it is suggested that similar approaches be used to minimize uncertainty when other compounds that dissociate and photolyse are tested. © 2014 SETAC.

Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.

PubMed

Ko, Chun-Han; Tsai, Chung-Hung; Lin, Po-Heng; Chang, Ko-Cheng; Tu, Jenn; Wang, Ya-Nang; Yang, Chien-Ying

2010-10-01

The Cel-BL11 gene from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli as a His-tag fusion protein. Zymographic analysis of the recombinant protein revealed cellulase activity corresponding to a protein with a 38-kDa molecular weight. The optimum temperature and pH for purified cellulase were 60 °C and pH 7.0, respectively. The enzyme retained more than 80% activity after 8h at 60 °C at pH 6 and 7. The cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 μmol/min/mg with carboxylmethyl cellulose (CMC). Then enzyme was active on Avicel, swollen Avicel, CMC, barley β-glucan, laminarin in the presence of 100 mM acetate buffer. It was inhibited by Hg²⁺, Cu²⁺ and Zn²⁺. Significant kraft pulp refining energy saving, 10%, was exhibited by the pretreatment of this cellulase applied at 2 IU per gram of oven-dried pulp. Broad pH and temperature stability render this cellulase a convenient applicability toward current mainstream biomass conversion and other industrial processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Matching phosphate and maleate buffer systems for dissolution of weak acids: Equivalence in terms of buffer capacity of bulk solution or surface pH?

PubMed

Cristofoletti, Rodrigo; Dressman, Jennifer B

2016-06-01

The development of in vitro dissolution tests able to anticipate the in vivo fate of drug products has challenged pharmaceutical scientists over time, especially in the case of ionizable compounds. In the seminal model proposed by Mooney et al. thirty-five years ago, the pH at the solid-liquid interface (pH0) was identified as a key parameter in predicting dissolution rate. In the current work it is demonstrated that the in vitro dissolution of the weak acid ibuprofen in maleate and phosphate buffer systems is a function of the pH0, which in turn is affected by properties of the drug and the medium. The reported pH0 for ibuprofen dissolution in bicarbonate buffer, the predominant buffer species in the human small intestine under fasting conditions, can be achieved by reducing the phosphate buffer concentration to 5.0mM or the maleate buffer concentration to 2.2mM. Using this approach to identify the appropriate buffer/buffer capacity combination for in vitro experiments in FaSSIF-type media, it would be possible to increase the physiological relevance of this important biopharmaceutics tool. However, the necessity of monitoring and adjusting the bulk pH during the experiments carried out in 5.0mM phosphate or 2.2mM maleate buffers must also be taken into consideration. Copyright © 2016 Elsevier B.V. All rights reserved.

Predicting the crystallization propensity of carboxylic acid buffers in frozen systems--relevance to freeze-drying.

PubMed

Sundaramurthi, Prakash; Suryanarayanan, Raj

2011-04-01

Selective crystallization of buffer components in frozen solutions is known to cause pronounced pH shifts. Our objective was to study the crystallization behavior and the consequent pH shift in frozen aqueous carboxylic acid buffers. Aqueous carboxylic acid buffers were cooled to -25°C and the pH of the solution was measured as a function of temperature. The thermal behavior of solutions during freezing and thawing was investigated by differential scanning calorimetry. The crystallized phases in frozen solution were identified by X-ray diffractometry. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartarate systems, at initial pH

Optimization of the SPME parameters and its online coupling with HPLC for the analysis of tricyclic antidepressants in plasma samples.

PubMed

Alves, Claudete; Fernandes, Christian; Dos Santos Neto, Alvaro José; Rodrigues, José Carlos; Costa Queiroz, Maria Eugênia; Lanças, Fernando Mauro

2006-07-01

Solid-phase microextraction (SPME)-liquid chromatography (LC) is used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. Extraction conditions are optimized using a 2(3) factorial design plus a central point to evaluate the influence of the time, temperature, and matrix pH. A Polydimethylsiloxane-divinylbenzene (60-mum film thickness) fiber is selected after the assessment of different types of coating. The chromatographic separation is realized using a C(18) column (150 x 4.6 mm, 5-microm particles), ammonium acetate buffer (0.05 mol/L, pH 5.50)-acetonitrile (55:45 v/v) with 0.1% of triethylamine as mobile phase and UV-vis detection at 214 nm. Among the factorial design conditions evaluated, the best results are obtained at a pH 11.0, temperature of 30 degrees C, and extraction time of 45 min. The proposed method, using a lab-made SPME-LC interface, allowed the determination of tricyclic antidepressants in in plasma at therapeutic concentration levels.

In vitro dissolution of proton-pump inhibitor products intended for paediatric and geriatric use in physiological bicarbonate buffer.

PubMed

Liu, Fang; Shokrollahi, Honaz

2015-05-15

Proton-pump inhibitor (PPI) products based on enteric coated multiparticulates are design to meet the needs of patients who cannot swallow tablets such as children and older adults. Enteric coated PPI preparations exhibit delays in in vivo absorption and onset of antisecretory effects, which is not reflected by the rapid in vitro dissolution in compendial pH 6.8 phosphate buffer commonly used for assessment of these products. A more representative and physiological medium, pH 6.8 mHanks bicarbonate buffer, was used in this study to evaluate the in vitro dissolution of enteric coated multiparticulate-based PPI products. Commercially available omeprazole, lansoprazole and esomeprazole products were subject to dissolution tests using USP-II apparatus in pH 4.5 phosphate buffer saline for 45 min (acid stage) followed by pH 6.8 phosphate buffer or pH 6.8 mHanks bicarbonate buffer. In pH 6.8 phosphate buffer, all nine tested products displayed rapid and comparable dissolution profiles meeting the pharmacopeia requirements for delayed release preparations. In pH 6.8 mHanks buffer, drug release was delayed and failed the pharmacopeia requirements from most enteric coated preparations. Despite that the same enteric polymer, methacrylic acid-ethyl acrylate copolymer (1:1), was applied to all commercial multiparticulate-based products, marked differences were observed between dissolution profiles of these preparations. The use of pH 6.8 physiological bicarbonate (mHanks) buffer can serve as a useful tool to provide realistic and discriminative in vitro release assessment of enteric coated PPI preparations and to assist rational formulation development of these products. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of pH on Drug Absorption from the Gastrointestinal Tract: A Simple Chemical Model

NASA Astrophysics Data System (ADS)

Hickman, Raymond J. S.; Neill, Jane

1997-07-01

A simple model of the gastrointestinal tract is obtained by placing ethyl acetate in contact with water at pH 2 and pH 8 in separate test tubes. The ethyl acetate corresponds to the lipid material lining the tract while the water corresponds to the aqueous contents of the stomach (pH 2) and intestine (pH 8). The compounds aspirin, paracetamol and 3-aminophenol are used as exemplars of acidic, neutral and basic drugs respectively to illustrate the influence which pH has on the distribution of each class of drug between the aqueous and organic phases of the model. The relative concentration of drug in the ethyl acetate is judged by applying microlitre-sized samples of ethyl acetate to a layer of fluorescent silica which, after evaporation of the ethyl acetate, is viewed under an ultraviolet lamp. Each of the three drugs, if present in the ethyl acetate, becomes visible as a dark spot on the silica layer. The observations made in the model system correspond well to the patterns of drug absorption from the gastrointestinal tract described in pharmacology texts and these observations are convincingly explained in terms of simple acid-base chemistry.

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

PubMed

Ow, Yan X; Uthicke, Sven; Collier, Catherine J

2016-01-01

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.

Light Levels Affect Carbon Utilisation in Tropical Seagrass under Ocean Acidification

PubMed Central

2016-01-01

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. PMID:26938454

Comparison of phage pVIII and KLH as vector in inducing the production of cytokines in C57BL/6J mice.

PubMed

Su, Quan-Ping; Wen, De-Zhong; Yang, Qiong; Zhang, Yan-Hui; Liu, Chong; Wang, Li

2007-01-22

We have demonstrated that phage display Candida albicans (C. albicans) LKVIRK epitope was protective in systemically infected C57BL/6J mice. The different development from precursor Ths, Th1 or Th2, will result in a protective or nonprotective immune response. To compare the types of cytokines induced by biologically and chemically synthesized vectors, C57BL/6J mice were immunized with hybrid phage displaying the epitope of LKVIRK and by synthesized peptide epitope LKVIRKNIVKKMIE conjugated through cysteine to keyhole limpet haemocyanin (KLH). The production of cytokines in spleens of immunized mice and in splenocytes culture supernatants stimulated by homologous immunogen in vitro was studied by RT-PCR and quantitative sandwich ELISA. The results showed that, compared to Tris-EDTA buffer (TE, 1 mM Tris, 0.1 mM EDTA, pH 8.0) injected mice, the expressions of Th1 type cytokine IFN-gamma, IL-2 and IL-12 were increased in hybrid phage, KLH-C, and wild phage immunized mice, and there were no differences between mice immunized with hybrid phage and KLH-C. While the expression of Th2 type cytokine IL-10 was similar in all mice, IL-4 was not detected. We obtained the same results in mRNA and protein level. These findings indicated that as carriers, phage and KLH were similar in inducing the Th1 type cytokines expression. Comparing to peptide synthesis couple with a carrier protein for injection, phage may be an inexpensive and simple route to the production of effective vaccines.

Influence of Bicarbonate, Sulfate, and Electron Donors on Biological reduction of Uranium and Microbial Community Composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Wensui; Zhou, Jizhong; Wu, Weimin

2007-01-01

A microcosm study was performed to investigate the effect of ethanol and acetate on uranium(VI) biological reduction and microbial community changes under various geochemical conditions. Each microcosm contained an uranium-contaminated sediment (up to 2.8 g U/kg) suspended in buffer with bicarbonate at concentrations of either 1 mM or 40 mM and sulfate at either 1.1 or 3.2 mM. Ethanol or acetate was used as an electron donor. Results indicate that ethanol yielded in significantly higher U(VI) reduction rates than acetate. A low bicarbonate concentration (1 mM) was favored for U(VI) bioreduction to occur in sediments, but high concentrations of bicarbonatemore » (40 mM) and sulfate (3.2 mM) decreased the reduction rates of U(VI). Microbial communities were dominated by species from the Geothrix genus and Proteobacteria phylum in all microcosms. However, species in the Geobacteraceae family capable of reducing U(VI) were significantly enriched by ethanol and acetate in low bicarbonate buffer. Ethanol increased the population of unclassified Desulfuromonales, while acetate increased the population of Desulfovibrio. Additionally, species in the Geobacteraceae family were not enriched in high bicarbonate buffer, but the Geothrix and the unclassified Betaproteobacteria species were enriched. This study concludes that ethanol could be a better electron donor than acetate for reducing U(VI) under given experimental conditions, and electron donor and geoundwater geochemistry alter microbial communities responsible for U(VI) reduction.« less

Studies on vaginal malodor. I. Study in humans.

PubMed

Chvapil, M; Eskelson, C; Jacobs, S; Chvapil, T; Russell, D H

1978-07-01

Forty-two percent of collagen sponges tested as an intravaginal barrier contraceptive method developed malodor when retained for 5 days. Only 4% developed odor when the sponge was removed within 24 hours after intercourse, rinsed, and reinserted. While sexually active volunteers found odor in 37% of the sponges, odor formed only in 4% of the sponges worn by sexually inactive users. No difference in the rate of odor formation was found when neutral pH (7.0) and acid pH (3.4) collagen sponges were tested, although we believe that a pH 3.4 is too acid and promotes odor formation. The optimal pH of the sponge should be 4.5 to 5.5. Malodor was efficiently extracted from sponges by washing in acid milieu of tap water and vinegar or 0.1 M acetate buffer, pH 4.0. Alkali extraction procedures were ineffective, and lukewarm water was slightly less effective than acid extraction of odor. At the time of malodor development, the high content of polyamines (putrescine, spermine, spermidine) in the ejaculate decreased to undetectable values. We conclude that the ejaculate is the major source of malodor formation in intravaginally worn collagen sponges. Removal, rinsing optimally in vinegar solution, and reinsertion within 24 hours after intercourse reduces the chance of malodor formation.

On the nature and origin of the calcium asymmetry arising during gravitropic response in etiolated pea epicotyls

NASA Technical Reports Server (NTRS)

Migliaccio, F.; Galston, A. W.

1987-01-01

Seven day old etiolated pea epicotyls were loaded symmetrically with 3H-indole 3-acetic acid (IAA) or 45Ca2+, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca2+ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca2+ asymmetries, but substances known to interfere with normal Ca2+ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca2+ asymmetries. These substances, however, are active in modifying both Ca2+ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the 45Ca2+ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca2+ movement secondary in gravitropism. We hypothesize that apoplastic Ca2+ changes during graviresponse because it is displaced by H+ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H+ efflux, increase Ca2+ release from pea epicotyl segments, whereas cycloheximide, which inhibits H+ efflux, has the reverse effect. We suggest that Ca2+ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca2+ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.

LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

PubMed

Trettin, Arne; Jordan, Jens; Tsikas, Dimitrios

2014-09-01

Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS. Copyright © 2013 Elsevier B.V. All rights reserved.

Out-of-equilibrium pH transients in the guinea-pig ventricular myocyte

PubMed Central

Leem, Chae-Hun; Vaughan-Jones, Richard D

1998-01-01

Following an intracellular alkali load (imposed by acetate prepulsing in CO2/HCO3− buffer), intracellular pH (pHi) of the guinea-pig ventricular myocyte (recorded from intracellular SNARF fluorescence) recovers to control levels. Recovery has two phases. An initial rapid phase (lasting up to 2 min) is followed by a later slow phase (several minutes). Inhibition of sarcolemmal acid-loading carriers (by removal of extracellular Cl−) inhibits the later, slow phase but the initial rapid recovery phase persists. It also persists in the absence of extracellular Na+ and in the presence of the HCO3− transport inhibitor DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid). The rapid recovery phase is not evident if the alkali load has been induced by reducing PCO2 (from 10 to 5 %), and it is inhibited in the absence of CO2/HCO3− buffer (i.e. Hepes buffer). It is also slowed by the carbonic anhydrase (CA) inhibitor acetazolamide (ATZ). We conclude that it is caused by buffering of the alkali load through the hydration of intracellular CO2 (CO2-dependent buffering). The time course of rapid recovery is consistent with an intracellular CO2 hydration rate constant (k1) of 0.36 s−1 in the presence of CA activity, and 0.14 s−1 in the absence of CA activity. This latter k1 value matches the literature value for uncatalysed CO2 hydration in free solution. Natural CO2 hydration is accelerated 2.6-fold in the ventricular myocyte by endogenous CA. The rapid recovery phase represents a period when the intracellular CO2/HCO3− buffer is out of equilibrium (OOE). Modelling of the recovery phase using our k1 value, indicates that OOE conditions will normally extend for at least 2 min following a step rise in pHi (at constant PCO2). If CA is inactive, this period can be as long as 5 min. During normal pHi regulation, the recovery rate during these periods cannot be used as a measure of sarcolemmal acid loading since it is a mixture of slow CO2-dependent buffering and transmembrane acid loading. The implication of this finding for quantification of pHi regulation during alkalosis is discussed. PMID:9575296

Voltammetric behaviour of free DNA bases, methylcytosine and oligonucleotides at disposable screen printed graphite electrode platforms.

PubMed

Brotons, Ariadna; Mas, Luis Alcaraz; Metters, Jonathan P; Banks, Craig E; Iniesta, Jesús

2013-09-21

Improvements in analytical methods for the determination and quantification of methylcytosine in DNA are vital since it has the potential to be used as a biomarker to detect different diseases in the first stage such as in the case of carcinomas and sterility. In this work we utilized screen printed graphite electrodes (SPGE) for studying the electrochemical response of all free DNA bases, methylcytosine and short oligonucleotides by cyclic voltammetry (CV) and square wave voltammetry (SWV). CV and SWV responses of free DNA bases and methylcytosine have been investigated by using SPGE platforms and the feasibility of detecting and quantifying cytosine and methylcytosine as free DNA moieties has been evaluated as a function of pH, concentration and the presence of the other free DNA bases in solution simultaneously. Repeatability of using SWV has been performed for the electrochemical behavior of both 250 μM cytosine and 250 μM methylcytosine in the presence of 25 μM guanine, with coefficient of variations of 6.9% and 2.6% respectively based upon peak current (N = 5). Six-mer oligonucleotides with a sequence 5'-XXXCGC-3', where the XXX motif corresponds to TTT, TTA, TAA and AAA have been performed using SWV in 0.1 M acetate buffer pH 5.0 to explore how the DNA base position effects the electrooxidation of guanine and cytosine into the oligonucleotide. Furthermore SWV comparisons of the electrooxidation of the oligonucleotides 5'-CGCGCG-3' and its methylated 5'-mCGmCGmCG-3' have been performed with concentrations in acetate buffer solutions, and the interaction of both oligonucleotides with the graphitic surface of the SPGE has been demonstrated by fitting well-known adsorption models such as Freundlich and Langmuir kinetics according to the SWV current response of guanine, cytosine and methylcytosine into the oligonucleotide.

Tunneling Injection and Exciton Diffusion of White Organic Light-Emitting Diodes with Composed Buffer Layers

NASA Astrophysics Data System (ADS)

Yang, Su-Hua; Wu, Jian-Ping; Huang, Tao-Liang; Chung, Bin-Fong

2018-02-01

Four configurations of buffer layers were inserted into the structure of a white organic light emitting diode, and their impacts on the hole tunneling-injection and exciton diffusion processes were investigated. The insertion of a single buffer layer of 4,4'-bis(carbazol-9-yl)biphenyl (CBP) resulted in a balanced carrier concentration and excellent color stability with insignificant chromaticity coordinate variations of Δ x < 0.023 and Δ y < 0.023. A device with a 2,9-Dimethyl-4,7-diphenyl-1,10-phenanthroline (BCP) buffer layer was beneficial for hole tunneling to the emission layer, resulting in a 1.45-fold increase in current density. The tunneling of holes and the diffusion of excitons were confirmed by the preparation of a dual buffer layer of CBP:tris-(phenylpyridine)-iridine (Ir(ppy)3)/BCP. A maximum current efficiency of 12.61 cd/A with a luminance of 13,850 cd/m2 was obtained at 8 V when a device with a dual-buffer layer of CBP:6 wt.% Ir(ppy)3/BCP was prepared.

Electrospun matrices for localised controlled drug delivery: release of tetracycline hydrochloride from layers of polycaprolactone and poly(ethylene-co-vinyl acetate).

PubMed

Alhusein, Nour; Blagbrough, Ian S; De Bank, Paul A

2012-12-01

We report the controlled release of tetracycline (Tet) HCl from a three-layered electrospun matrix for the first time. Five formulations of electrospun poly-ε-caprolactone (PCL) and poly(ethylene-co-vinyl acetate) (PEVA) have been designed, prepared as micro/nanofibre layers, and assayed for the controlled release of the clinically useful antibiotic Tet HCl with potential applications in wound healing and especially in complicated skin and skin-structure infections. Tet HCl was also chosen as a model drug possessing a good ultraviolet (UV) chromophore and capable of fluorescence together with limited stability. Tet HCl was successfully incorporated (essentially quantitatively at 3 %, w/w) and provided controlled release from multilayered electrospun matrices. The Tet HCl release test was carried out by a total immersion method on 2 × 2 cm(2) electrospun fibrous mats in Tris or phosphate-buffered saline heated to 37 °C. The formulation PCL/PEVA/PCL with Tet HCl in each layer gave a large initial (burst) release followed by a sustained release. Adding a third layer to the two-layered formulations led to release being sustained from 6 days to more than 15 days. There was no detectable loss of Tet chemical stability (as shown by UV and NMR) or bioactivity (as shown by a modified Kirby-Bauer disc assay). Using Tet HCl-sensitive bacteria, Staphylococcus aureus (ATCC 25923), the Tet HCl-loaded three-layered matrix formulations were still showing significantly higher antibacterial effects on days 4 and 5 than commercially available Antimicrobial Susceptibility Test Discs of Tet HCl. Electrospinning provides good encapsulation efficiency of Tet HCl within PCL/PEVA/PCL polymers in micro/nanofibre layers which display sustained antibiotic release.

Fluorimetric determination of some sulfur containing compounds through complex formation with terbium (Tb+3) and uranium (U+3).

PubMed

Taha, Elham Anwer; Hassan, Nagiba Yehya; Aal, Fahima Abdel; Fattah, Laila El-Sayed Abdel

2007-05-01

Two simple, sensitive and specific fluorimetric methods have been developed for the determination of some sulphur containing compounds namely, Acetylcysteine (Ac), Carbocisteine (Cc) and Thioctic acid (Th) using terbium Tb+3 and uranium U+3 ions as fluorescent probes. The proposed methods involve the formation of a ternary complex with Tb+3 in presence of Tris-buffer method (I) and a binary complex with aqueous uranyl acetate solution method (II). The fluorescence quenching of Tb+3 at 510, 488 and 540 nm (lambda(ex) 250, 241 and 268 nm) and of uranyl acetate at 512 nm (lambda(ex) 240 nm) due to the complex formation was quantitatively measured for Ac, Cc and Th, respectively. The reaction conditions and the fluorescence spectral properties of the complexes have been investigated. Under the described conditions, the proposed methods were applicable over the concentration range (0.2-2.5 microg ml(-1)), (1-4 microg ml(-1)) and (0.5-3.5 microg ml(-1)) with mean percentage recoveries 99.74+/-0.36, 99.70+/-0.52 and 99.43+/-0.23 for method (I) and (0.5-6 microg ml(-1)), (0.5-5 microg ml(-1)), and (1-6 microg ml(-1)) with mean percentage recoveries 99.38+/-0.20, 99.82+/-0.28 and 99.93+/-0.32 for method (II), for the three cited drugs, respectively. The proposed methods were successfully applied for the determination of the studied compounds in bulk powders and in pharmaceutical formulations, as well as in presence of their related substances. The results obtained were found to be in agree statistically with those obtained by official and reported ones. The two methods were validated according to USP guidelines and also assessed by applying the standard addition technique.

Rapid diagnosis of soybean mosaic virus N soybean by tissue-print immunoassay and DIBA in comparison to other serological methods.

PubMed

Ahangaran, A; Mohammadi, Gh Mosahebi; Habibi, M Koohi; Shahraeen, N; Khezri, S

2006-01-01

Soybean mosaic virus (SMV) is an important disease in soybean and is widely distributed in northern of Iran. SMV transmitted by soybean seed and detection of it is very important for disease management. In this study, several detection methods including DAS-ELISA, indirect-ELISA, tissue-print immunoassay (TPIA) and Dot immunobinding assay (DIBA) were optimized and compared with each other to identify the virus, using polyclonal antibody. For TPIA, nitrocellulose membrane was used to imprint fresh sections of healthy and infected plant materials, and for DIBA 10 microl of extracts was doted onto nitrocellulose membranes. Both membranes were incubated 1 hour in blocking buffer, and then incubated 2 h in 1:1000 dilution of IgG-conjugate. After incubation the membranes were washed three times with PBS-T buffer for 15 min. Then the membranes were incubated in substrate solution containing NBT/BCIP. After some minutes prints or blots of infected tissues turned dark violet, whereas prints or blots of healthy ones did not show any color changes. In some cases, substrate solution was Fast red, containing 0.2M Tris-HCl buffer and 2mM MgCl2, pH = 7.8, producing red color in infected prints or blots. Both methods are simple and TPIA is rapidly and easily applicable in the field. However, TPIA had some advantages over the others. TPIA is time-saving as there is no need for conventional sap extraction and also nitrocellulose membranes used for printing can be used in the field and stored for a long time or transported to another laboratory for process. These two methods can be used routinely for detection of SMV in many samples.

Ribonucleic Acid and Ribosomes of Bacillus stearothermophilus1

PubMed Central

Saunders, Grady F.; Campbell, L. Leon

1966-01-01

Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332–339. 1966.—The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10−2m MgCl2–10−2m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg++ concentration to 10−3m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10−2m Mg++ to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a Tm at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a Tm of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins. Images PMID:5903099

Transition state analogue imprinted polymers as artificial amidases for amino acid p-nitroanilides: morphological effects of polymer network on catalytic efficiency.

PubMed

Mathew, Divya; Thomas, Benny; Devaky, K S

2017-11-13

The morphology of the polymer network - porous/less porous - plays predominant role in the amidase activities of the polymer catalysts in the hydrolytic reactions of amino acid p-nitroanilides. Polymers with the imprints of stable phosphonate analogue of the intermediate of hydrolytic reactions were synthesized as enzyme mimics. Molecular imprinting was carried out in thermodynamically stable porogen dimethyl sulphoxide and unstable porogen chloroform, to investigate the morphological effects of polymers on catalytic amidolysis. It was found that the medium of polymerization has vital influence in the amidase activities of the enzyme mimics. The morphological studies of the polymer catalysts were carried out by scanning electron microscopy and Bruner-Emmett-Teller analysis. The morphology of the polymer catalysts and their amidase activities are found to be dependent on the composition of reaction medium. The polymer catalyst prepared in dimethyl sulphoxide is observed to be efficient in 1:9 acetonitrile (ACN)-Tris HCl buffer and that prepared in chloroform is noticed to be stereo specifically and shape-selectively effective in 9:1 ACN-Tris HCl buffer. The solvent memory effect in catalytic amidolysis was investigated using the polymer prepared in acetonitrile.

The Potential Role of Systemic Buffers in Reducing Intratumoral Extracellular pH and Acid-Mediated Invasion

PubMed Central

Silva, Ariosto S.; Yunes, Jose A.; Gillies, Robert J.; Gatenby, Robert A.

2013-01-01

A number of studies have shown that the extracellular pH (pHe) in cancers is typically lower than that in normal tissue and that an acidic pHe promotes invasive tumor growth in primary and metastatic cancers. Here, we investigate the hypothesis that increased systemic concentrations of pH buffers reduce intratumoral and peritumoral acidosis and, as a result, inhibit malignant growth. Computer simulations are used to quantify the ability of systemic pH buffers to increase the acidic pHe of tumors in vivo and investigate the chemical specifications of an optimal buffer for such purpose. We show that increased serum concentrations of the sodium bicarbonate (NaHCO3) can be achieved by ingesting amounts that have been used in published clinical trials. Furthermore, we find that consequent reduction of tumor acid concentrations significantly reduces tumor growth and invasion without altering the pH of blood or normal tissues. The simulations also show that the critical parameter governing buffer effectiveness is its pKa. This indicates that NaHCO3, with a pKa of 6.1, is not an ideal intratumoral buffer and that greater intratumoral pHe changes could be obtained using a buffer with a pKa of ~7. The simulations support the hypothesis that systemic pH buffers can be used to increase the tumor pHe and inhibit tumor invasion. PMID:19276380

Escherichia coli growth changes by the mediated effects after low-intensity electromagnetic irradiation of extremely high frequencies.

PubMed

Torgomyan, Heghine; Hovnanyan, Karlen; Trchounian, Armen

2013-04-01

Water is the major constituent of environmental medium and biological systems. The effects occurring in water as a result of low-intensity electromagnetic irradiation (EMI) in extremely high frequencies are supposed to be the primary mechanism to create conditions for biological responses. The EMI effects on Escherichia coli, after irradiation of their suspension, are most probably water-mediated. Indirect effects of EMI at 51.8, 53, 70.6, and 73 GHz frequencies on bacteria, through water, assay buffer (Tris-phosphate buffer with inorganic salts at low or moderate concentrations), or peptone growth medium were studied. The mediated effects of 70.6 and 73 GHz irradiated water, assay buffer, and growth medium on E. coli growth characteristics were insignificant. But the results were different for 51.8 and 53 GHz. EMI mediated effects on bacterial growth were clearly demonstrated. The effects were more strongly expressed with 53 GHz. Moreover, it was shown that 70.6 and 73 GHz similarly suppressed the cell growth after direct irradiation of E. coli in water or on solid medium. Interestingly, for 51.8 and 53 GHz the bacterial growth decreases after suspension irradiation was less, compared to the direct irradiation of bacteria on solid medium. Especially, it was also more expressed in case of 53 GHz. Also with electron microscopy, EMI-induced bacterial cell sizes and structure different changes were detected. In addition, the distinguished changes in surface tension, oxidation-reduction potential and pH of water, assay buffer, growth medium, and bacterial suspension were determined. They depended on EMI frequency used. The differences could be associated with the partial absorbance of EMI energy by the surrounding medium, which depends on a specific frequency. The results are crucial to understand biophysical mechanisms of EMI effects on bacteria.

Enantioseparation on cellulose dimethylphenylcarbamate-modified zirconia monolithic columns by reversed-phase capillary electrochromatography.

PubMed

Kumar, Avvaru Praveen; Park, Jung Hag

2010-06-25

This work reports the preparation of monolithic zirconia chiral columns for separation of enantiomeric compounds by capillary electrochromatography (CEC). Using sol-gel technology, a porous monolith having interconnected globular-like structure with through-pores is synthesized in the capillary column as a first step in the synthesis of monolithic zirconia chiral capillary columns. In the second step, the surface of the monolith is modified by coating with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) as the chiral stationary phase to obtain a chiral column (CDMPCZM). The process of the preparation of the zirconia monolithic capillary column was investigated by varying the concentrations of the components of the sol solution including polyethylene glycol, water and acetic acid. CDMPCZM is mechanically stable and no bubble formation was detected with the applied current of up to 30 microA. The enantioseparation behavior of the CDMPCZM columns was investigated by separating a set of 10 representative chiral compounds by varying the applied voltage and pH and organic composition of the aqueous organic mobile phases. Copyright 2010 Elsevier B.V. All rights reserved.

Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase from Bacillus thuringiensis.

PubMed

Wang, Yung Lin; Lin, Yi Ting; Chen, Chia Lin; Shaw, Gwo Chyuan; Liaw, Shwu Huey

2014-10-01

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5 at 288 K. The crystals belonged to space group P1, with unit-cell parameters a = 42.97, b = 83.23, c = 85.50 Å, α = 73.45, β = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Å resolution with an Rmerge of 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure of Streptomyces lividans chloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

Quantitative aspects of microchip isotachophoresis for high precision determination of main components in pharmaceuticals.

PubMed

Hradski, Jasna; Chorváthová, Mária Drusková; Bodor, Róbert; Sabo, Martin; Matejčík, Štefan; Masár, Marián

2016-12-01

Although microchip electrophoresis (MCE) is intended to provide reliable quantitative data, so far there is only limited attention paid to these important aspects. This study gives a general overview of key aspects to be followed to reach high-precise determination using isotachophoresis (ITP) on the microchip with conductivity detection. From the application point of view, the procedure for the determination of acetate, a main component in the pharmaceutical preparation buserelin acetate, was developed. Our results document that run-to-run fluctuations in the sample injection volume limit the reproducibility of quantitation based on the external calibration. The use of a suitable internal standard (succinate in this study) improved the repeatability of the precision of acetate determination from six to eight times. The robustness of the procedure was studied in terms of impact of fluctuations in various experimental parameters (driving current, concentration of the leading ions, pH of the leading electrolyte and buffer impurities) on the precision of the ITP determination. The use of computer simulation programs provided means to assess the ITP experiments using well-defined theoretical models. A long-term validity of the calibration curves on two microchips and two MCE equipments was verified. This favors ITP over other microchip electrophoresis techniques, when chip-to-chip or equipment-to-equipment transfer of the analytical method is required. The recovery values in the range of 98-101 % indicate very accurate determination of acetate in buserelin acetate, which is used in the treatment of hormone-dependent tumors. This study showed that microchip ITP is suitable for reliable determination of main components in pharmaceutical preparations.

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

PubMed Central

Wang, Hengwei; Wang, Fengqing; Wang, Wei; Yao, Xueling; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

2014-01-01

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars. PMID:25402470

Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

PubMed

Wang, Hengwei; Wang, Fengqing; Wang, Wei; Yao, Xueling; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

2014-01-01

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

Homoleptic organolanthanide compounds supported by the bis(dimethylsilyl)benzyl ligand

DOE PAGES

Boteju, Kasuni C.; Ellern, Arkady; Sadow, Aaron D.

2016-12-19

Here, a β-SiH functionalized benzyl anion [C(SiHMe 2) 2Ph]$-$s obtained by deprotonation of HC(SiHMe 2) 2Ph with KCH 2Ph or by reaction of KOtBu and (Me 2HSi) 3CPh; LnI 3(THF)n and three equivalents of this carbanion combine to provide homoleptic tris(alkyl)lanthanide compounds Ln{C(SiHMe 2) 2Ph} 3 (Ln = La, Ce, Pr, Nd) containing secondary metal–ligand interactions.

A Reliable and Non-destructive Method for Monitoring the Stromal pH in Isolated Chloroplasts Using a Fluorescent pH Probe.

PubMed

Su, Pai-Hsiang; Lai, Yen-Hsun

2017-01-01

The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpH env ), whether the concentration of ionophores used can effectively abolish the ΔpH env is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpH env can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r -square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpH env can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin required to collapse the ΔpH env was then studied. The establishment of a non-destructive method of monitoring the stromal pH will be valuable for studying the roles of the ΔpH env in chloroplast physiology.

Influence of variation in mobile phase pH and solute pK(a) with the change of organic modifier fraction on QSRRs of hydrophobicity and RP-HPLC retention of weakly acidic compounds.

PubMed

Han, Shu-ying; Liang, Chao; Zou, Kuan; Qiao, Jun-qin; Lian, Hong-zhen; Ge, Xin

2012-11-15

The variation in mobile phase pH and ionizable solute dissociation constant (pK(a)) with the change of organic modifier fraction in hydroorganic mobile phase has seemingly been a troublesome problem in studies and applications of reversed phase high performance liquid chromatography (RP-HPLC). Most of the early studies regarding the RP-HPLC of acid-base compounds have to measure the actual pH of the mixed mobile phase rigorously, sometimes bringing difficulties in the practices of liquid chromatographic separation. In this paper, the effect of this variation on the apparent n-octanol/water partition coefficient (K(ow)″) and the related quantitative structure-retention relationship (QSRR) of logK(ow)″ vs. logk(w), the logarithm of retention factor of analytes in neat aqueous mobile phases, was investigated for weakly acidic compounds. This QSRR is commonly used as a classical method for K(ow) measurement by RP-HPLC. The theoretical and experimental derivation revealed that the variation in mobile phase pH and solute pK(a) will not affect the QSRRs of acidic compounds. This conclusion is proved to be suitable for various types of ion-suppressors, i.e., strong acid (perchloric acid), weak acid (acetic acid) and buffer salt (potassium dihydrogen phosphate/phosphoric acid, PBS). The QSRRs of logK(ow)″ vs. logk(w) were modeled by 11 substituted benzoic acids using different types of ion-suppressors in a binary methanol-water mobile phase to confirm our deduction. Although different types of ion-suppressor all can be used as mobile phase pH modifiers, the QSRR model obtained by using perchloric acid as the ion-suppressor was found to have the best result, and the slightly inferior QSRRs were obtained by using acetic acid or PBS as the ion-suppressor. Copyright © 2012 Elsevier B.V. All rights reserved.

Comb-like amphiphilic copolymers bearing acetal-functionalized backbones with the ability of acid-triggered hydrophobic-to-hydrophilic transition as effective nanocarriers for intracellular release of curcumin.

PubMed

Zhao, Junqiang; Wang, Haiyang; Liu, Jinjian; Deng, Liandong; Liu, Jianfeng; Dong, Anjie; Zhang, Jianhua

2013-11-11

The pH-responsive micelles have enormous potential as nanosized drug carriers for cancer therapy due to their physicochemical changes in response to the tumor intracellular acidic microenvironment. Herein, a series of comb-like amphiphilic copolymers bearing acetal-functionalized backbone were developed based on poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate-co-poly(ethylene glycol) methyl ether methacrylate] [P(TTMA-co-mPEGMA)] as effective nanocarriers for intracellular curcumin (CUR) release. P(TTMA-co-mPEGMA) copolymers with different hydrophobic-hydrophilic ratios were prepared by one-step reversible addition fragmentation chain transfer (RAFT) copolymerization of TTMA and mPEGMA. Their molecular structures and chemical compositions were confirmed by (1)H NMR, Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). P(TTMA-co-mPEGMA) copolymers could self-assemble into nanosized micelles in aqueous solution and displayed low critical micelle concentration (CMC). All P(TTMA-co-mPEGMA) micelles displayed excellent drug loading capacity, due to the strong π-π conjugate action and hydrophobic interaction between the PTTMA and CUR. Moreover, the hydrophobic PTTMA chain could be selectively hydrolyzed into a hydrophilic backbone in the mildly acidic environment, leading to significant swelling and final disassembly of the micelles. These morphological changes of P(TTMA-co-mPEGMA) micelles with time at pH 5.0 were determined by DLS and TEM. The in vitro CUR release from the micelles exhibited a pH-dependent behavior. The release rate of CUR was significantly accelerated at mildly acidic pH of 4.0 and 5.0 compared to that at pH 7.4. Toxicity test revealed that the P(TTMA-co-mPEGMA) copolymers exhibited low cytotoxicity, whereas the CUR-loaded micelles maintained high cytotoxicity for HepG-2 and EC-109 cells. The results indicated that the novel P(TTMA-co-mPEGMA) micelles with low CMC, small and tunable sizes, high drug loading, pH-responsive drug release behavior, and good biocompatibility may have potential as hydrophobic drug delivery nanocarriers for cancer therapy with intelligent delivery.

Variation of power generation at different buffer types and conductivities in single chamber microbial fuel cells.

PubMed

Nam, Joo-Youn; Kim, Hyun-Woo; Lim, Kyeong-Ho; Shin, Hang-Sik; Logan, Bruce E

2010-01-15

Microbial fuel cells (MFCs) are operated with solutions containing various chemical species required for the growth of electrochemically active microorganisms including nutrients and vitamins, substrates, and chemical buffers. Many different buffers are used in laboratory media, but the effects of these buffers and their inherent electrolyte conductivities have not been examined relative to current generation in MFCs. We investigated the effect of several common buffers (phosphate, MES, HEPES, and PIPES) on power production in single chambered MFCs compared to a non-buffered control. At the same concentrations the buffers produced different solution conductivities which resulted in different ohmic resistances and power densities. Increasing the solution conductivities to the same values using NaCl produced comparable power densities for all buffers. Very large increases in conductivity resulted in a rapid voltage drop at high current densities. Our results suggest that solution conductivity at a specific pH for each buffer is more important in MFC studies than the buffer itself given relatively constant pH conditions. Based on our analysis of internal resistance and a set neutral pH, phosphate and PIPES are the most useful buffers of those examined here because pH was maintained close to the pK(a) of the buffer, maximizing the ability of the buffer to contribute to increase current generation at high power densities. Copyright 2009 Elsevier B.V. All rights reserved.

Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae.

PubMed

Casey, Elizabeth; Sedlak, Miroslav; Ho, Nancy W Y; Mosier, Nathan S

2010-06-01

A current challenge of the cellulosic ethanol industry is the effect of inhibitors present in biomass hydrolysates. Acetic acid is an example of one such inhibitor that is released during the pretreatment of hemicellulose. This study examined the effect of acetic acid on the cofermentation of glucose and xylose under controlled pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engineered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 g L(-1), representing the range of concentrations expected in actual biomass hydrolysates, were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic acid in the fermentation media led to a significant decrease in the observed maximum cell biomass concentration. Glucose- and xylose-specific consumption rates decreased as the acetic acid concentration increased, with the inhibitory effect being more severe for xylose consumption. The ethanol production rates also decreased when acetic acid was present, but ethanol metabolic yields increased under the same conditions. The results also revealed that the inhibitory effect of acetic acid could be reduced by increasing media pH, thus confirming that the undissociated form of acetic acid is the inhibitory form of the molecule.

Preliminary findings on the correlation of saliva pH, buffering capacity, flow, Consistency and Streptococcus mutans in relation to cigarette smoking.

PubMed

Voelker, Marsha A; Simmer-Beck, Melanie; Cole, Molly; Keeven, Erin; Tira, Daniel

2013-02-01

The purpose of this preliminary study was to examine the relationship of caries risk, salivary buffering capacity, salivary pH, salivary quality (flow, consistency) and levels of Streptococcus mutans in relation to cigarette smoking. This clinical trial consisted of 53 volunteer patients receiving care in a university based dental hygiene clinic. Participants completed a questionnaire specific to their social history in regards to tobacco use, oral health and dietary history. Measurements of unstimulated saliva were collected followed by collection of stimulated saliva samples. These samples were used to measure salivary pH, buffering capacity and Streptococcus mutans levels. The subject's smoking status was significantly associated with caries risk (p= 0.001), with 25% of the variability of caries risk attributed to smoking. The smoking status was significantly associated with buffering capacity (p=0.025), with 9% of the variability of buffering status attributed to the smoking. Associations between smoking status and salivary pH were not statistically significant. The subject's caries risk was significantly associated with buffering capacity (p= 0.001), with 25% of the variability of caries risk attributed to the buffering capacity. The subject's caries risk was significantly associated with salivary pH (p= 0.031), with 9% of the variability of caries risk attributed to the salivary pH. The Streptococcus mutans test showed no statistical significance (p>0.05) possibly due to the number and low variance in the subjects. A relationship between caries risk and smoking, buffering capacity and smoking, and stimulated salivary pH and smoking were concluded. No significance difference (p>0.05) between caries risk and salivary pH, salivary quality and smoking, S. mutans and smoking were noted from the preliminary results.

Development of soft extraction method for structural characterization of boreal forest soil proteins with MALDI-TOF/MS

NASA Astrophysics Data System (ADS)

Kanerva, Sanna; Ketola, Raimo A.; Kitunen, Veikko; Smolander, Aino; Kotiaho, Tapio

2010-05-01

Nitrogen (N) is usually the nutrient restricting productivity in boreal forests. Forest soils contain a great amount of nitrogen, but only a small part of it is in mineral form. Most part of soil N is bound in the structures of different organic compounds such as proteins, peptides, amino acids and more stabilized, refractory compounds. Due to the fact that soil organic N has a very important role in soil nutrient cycling and in plant nutrition, there is a need for more detailed knowledge of its chemistry in soil. Conventional methods to extract and analyze soil organic N are usually very destructive for structures of higher molecular weight organic compounds, such as proteins. The aim of this study was to characterize proteins extracted from boreal forest soil by "soft" extraction methods in order to maintain their molecular structure. The organic layer (F) from birch forest floor containing 78% of organic matter was sieved, freeze dried, pulverized, and extracted with a citrate or phosphate buffer (pH 6 or 8). Sequential extraction with the citrate or phosphate buffer and an SDS buffer (pH 6.8), slightly modified from the method of Chen et al. (2009, Proteomics 9: 4970-4973), was also done. Proteins were purified from the soil extract by extraction with buffered phenol and precipitated with methanol + 0.1M ammonium acetate at -20°C. Characterization of proteins was performed with matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/MS) and the concentration of total proteins was measured using Bradford's method. Bovine serum albumin (BSA) was used as a positive control in the extractions and as a standard protein in Bradford's method. Our results showed that sequential extraction increased the amount of extracted proteins compared to the extractions without the SDS-buffer; however, it must be noted that the use of SDS-buffer very probably increased denaturization of proteins. Purification of proteins from crude soil extracts by phenol extraction was essential prior to measurement of total proteins; there seemed to be a lot of compounds in crude soil extracts that interfere with the analysis of total proteins, causing overestimation in protein concentration. pH of the buffer solution did not seem to be very crucial for the extractability of soil natural proteins, but at the higher pH, the amount of interfering compounds increased. However, the recovery of BSA added was clearly higher at the higher pH. When the protein precipitates were analyzed with MALDI-TOF/MS, a large curve, most likely formed from wide peaks of several compounds, indicate that most of the compounds in the precipitate were <15 kDa or ~20-50 kDa in molecular weight. It seems that in order to identify individual proteins from mass spectra, a separation of compounds with varying molecular weight is needed before the MALDI-TOF/MS analysis. Due to the fact that a relatively high amount of BSA added was not recovered by the extractions and that the intensity of the signals observed in mass spectra was low, it is questionable whether it is possible to extract soil natural proteins effectively from soils containing a high amount of organic matter without destructing the structures of proteins.

An evaluation of MES (2(N-Morpholino)ethanesulfonic acid) and Amberlite IRC-50 as pH buffers for nutrient solution studies

NASA Technical Reports Server (NTRS)

Bugbee, B. G.; Salisbury, F. B.

1985-01-01

All buffering agents used to stabilize pH in hydroponic research have disadvantages. Inorganic buffers are absorbed and may become phytotoxic. Solid carbonate salts temporarily mitigate decreasing pH but provide almost no protection against increasing pH, and they alter nutrient absorption. Exchange resins are more effective, but we find that they remove magnesium and manganese from solution. We have tested 2(N-Morpholino)ethanesulfonic acid (MES) as a buffering agent at concentrations of 1 and 10 mol m-3 (1 and 10 mM) with beans, corn, lettuce, tomatoes, and wheat. MES appears to be biologically inert and does not interact significantly with other solution ions. Relative growth rates among controls and MES treatments were nearly identical for each species during the trial period. The pH was stabilized by 1 mol m-3 MES. This buffer warrants further consideration in nutrient research.

Effect of moisture content on the invertase activity of freeze-dried S. cerevisiae.

PubMed

Pitombo, R N; Spring, C; Passos, R F; Tonato, M; Vitolo, M

1994-08-01

The invertase activity of intact Saccharomyces cerevisiae submitted to freezing-thawing was affected by pH, the chemical nature of the buffer, and the freezing cooling rate (CR), leading in some cases to a complete invertase inactivation (acetate buffer, pH 4.0, CR = 0.5 degree C/min). Once established under adequate freezing conditions the invertase activity remained unchanged after freeze-drying. Nevertheless, in some cases the cell-growing capability after freeze-drying diminished around 70%, mainly if the frozen cell suspension was attained through freezing carried out at CR = 0.5 degree C/min. Water sorption isotherms of freeze-dried samples (freeze-dryer Edwards L-4KR; 30 degrees C and 0.1 mB) were determined at 10 and 25 degrees C. The monolayer moisture content (MMC) at each temperature (12.7 and 3.71 for 10 and 25 degrees C, respectively) was calculated from isotherms by applying BET and GAB models. Freeze-dried yeast with water activity (Aw) between 0 and 0.33 (about the MMC value) maintained at 25 degrees C for 235 days and at 89 degrees C for 15 min retained at least 85% of its original invertase activity (IA), whereas samples with Aw > MMC lost at least 60% of its IA. X ray diffraction showed that the freeze-dried cake before and after storage presented an amorphous structure.

Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection.

PubMed

Sajid, Muhammad; Na, Na; Safdar, Muhammad; Lu, Xin; Ma, Lin; He, Lan; Ouyang, Jin

2013-11-01

A sensitive and inexpensive quantification method with online extraction using a short C-18 column for sulfonamide residues in honey by high performance liquid chromatography with fluorescence detector was developed and validated. In sample preparation, acid hydrolysis was used to break the N-glycoside bond between the honey sugar and sulfonamide drugs and derivatization of sulfonamide residues with fluorescamine was conducted at pH 3.5 using a citrate buffer (0.5M) in the honey matrix. The chromatography was carried out on Zorbax Extended C-18 (250mm×4.6mm; 5μm) column, using a mixture of acetonitrile and an acetate buffer (pH 4.50, 20mM) as a mobile phase. A Zorbax Extended C-18 (12mm×4.6mm; 5μm) column was used for online extraction of fifteen sulfonamide residues from honey sample with the help of a two position valve. The limit of quantification of sulfonamide residues in honey was less than 3ngg(-1), and the percentage recovery of study compounds in spiked honey sample was from 80% for sulfacetamide to 100% of sulfachloropyridazine. The developed method has excellent linearity for all studied sulfonamides with a correlation coefficient 0.993. Copyright © 2013 Elsevier B.V. All rights reserved.

Intestinal absorption of calcium from calcium ascorbate in rats.

PubMed

Tsugawa, N; Yamabe, T; Takeuchi, A; Kamao, M; Nakagawa, K; Nishijima, K; Okano, T

1999-01-01

The intestinal absorption of calcium (Ca) from Ca ascorbate (Ca-AsA) was investigated in normal rats. Each animal was perorally administered either 5mg (low dose) or 10mg (high dose) of Ca in 1ml of distilled water as Ca-AsA, Ca carbonate (CaCO3), or Ca chloride (CaCl2), which were intrinsically labeled with 45Ca using 45CaCl2. The amount of radioactivity in plasma was measured periodically up to 34h after dosing, and pharmacokinetic parameters were calculated from the radioactivity in plasma. The time taken to reach the maximum 45Ca level (Tmax) did not differ among the three groups. The area under the plasma 45Ca level/time curve (AUCinfinity) value for the Ca-AsA group was significantly higher than those for the CaCO3 and the CaCl2 groups. The radioactivity at Tmax (Cmax) for the Ca-AsA group was significantly higher than those for the CaCO3 and the CaCl2 groups for the low dose, and comparable with or significantly higher than those for the CaCl2 and CaCO3 groups for the high dose. Similar results were observed for whole-body 45Ca retention. Radioactivity in the femur 34h after dosing was the highest in the Ca-AsA group and the lowest in the CaCO3 group. The rank order of solubility in water, the first fluid (pH 1.2, JP-1) of JPXIII disintegration medium, acetate buffer solution (pH 4.0), triethanolamine-malate buffer solution (pH 7.0) and ammonium chloride buffer solution (pH 10.0) at 37 degrees C was CaCl2 > Ca-AsA > CaCO3. In contrast, the rank order of the solubility in the second fluid (pH 6.8, JP-2) of JPXIII disintegration medium at 37 degrees C was Ca-AsA > CaCl2 > CaCO3. These results indicate that the absorbability of Ca from Ca-AsA is almost comparable with, or higher than, that from CaCl2 and significantly higher than that from CaCO3 because of its high degree of solubility in the intestine. Therefore, Ca-AsA would be useful as a Ca supplement with relatively high absorption from intestine.

Inactivation of viruses using novel protein A wash buffers.

PubMed

Bolton, Glen R; Selvitelli, Keith R; Iliescu, Ionela; Cecchini, Douglas J

2015-01-01

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers.

Three-Stream, Bicarbonate-Based Hemodialysis Solution Delivery System Revisited: With an Emphasis on Some Aspects of Acid-Base Principles.

PubMed

Lew, Susie Q; Kohn, Orly F; Cheng, Yuk-Lun; Kjellstrand, Carl M; Ing, Todd S

2017-06-01

Hemodialysis patients can acquire buffer base (i.e., bicarbonate and buffer base equivalents of certain organic anions) from the acid and base concentrates of a three-stream, dual-concentrate, bicarbonate-based, dialysis solution delivery machine. The differences between dialysis fluid concentrate systems containing acetic acid versus sodium diacetate in the amount of potential buffering power were reviewed. Any organic anion such as acetate, citrate, or lactate (unless when combined with hydrogen) delivered to the body has the potential of being converted to bicarbonate. The prescribing physician aware of the role that organic anions in the concentrates can play in providing buffering power to the final dialysis fluid, will have a better knowledge of the amount of bicarbonate and bicarbonate precursors delivered to the patient. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Identification of inorganic improvised explosive devices by analysis of postblast residues using portable capillary electrophoresis instrumentation and indirect photometric detection with a light-emitting diode.

PubMed

Hutchinson, Joseph P; Evenhuis, Christopher J; Johns, Cameron; Kazarian, Artaches A; Breadmore, Michael C; Macka, Miroslav; Hilder, Emily F; Guijt, Rosanne M; Dicinoski, Greg W; Haddad, Paul R

2007-09-15

A commercial portable capillary electrophoresis (CE) instrument has been used to separate inorganic anions and cations found in postblast residues from improvised explosive devices (IEDs) of the type used frequently in terrorism attacks. The purpose of this analysis was to identify the type of explosive used. The CE instrument was modified for use with an in-house miniaturized light-emitting diode (LED) detector to enable sensitive indirect photometric detection to be employed for the detection of 15 anions (acetate, benzoate, carbonate, chlorate, chloride, chlorite, cyanate, fluoride, nitrate, nitrite, perchlorate, phosphate, sulfate, thiocyanate, thiosulfate) and 12 cations (ammonium, monomethylammonium, ethylammonium, potassium, sodium, barium, strontium, magnesium, manganese, calcium, zinc, lead) as the target analytes. These ions are known to be present in postblast residues from inorganic IEDs constructed from ammonium nitrate/fuel oil mixtures, black powder, and chlorate/perchlorate/sugar mixtures. For the analysis of cations, a blue LED (470 nm) was used in conjunction with the highly absorbing cationic dye, chrysoidine (absorption maximum at 453 nm). A nonaqueous background electrolyte comprising 10 mM chrysoidine in methanol was found to give greatly improved baseline stability in comparison to aqueous electrolytes due to the increased solubility of chrysoidine and its decreased adsorption onto the capillary wall. Glacial acetic acid (0.7% v/v) was added to ensure chrysoidine was protonated and to enhance separation selectivity by means of complexation with transition metal ions. The 12 target cations were separated in less than 9.5 min with detection limits of 0.11-2.30 mg/L (calculated at a signal-to-noise ratio of 3). The anions separation system utilized a UV LED (370 nm) in conjunction with an aqueous chromate electrolyte (absorption maximum at 371 nm) consisting of 10 mM chromium(VI) oxide and 10 mM sodium chromate, buffered with 40 mM tris(hydroxymethyl)aminomethane at pH 8.05. All 15 target anions were baseline separated in less than 9 min with limits of detection ranging from 0.24 to 1.15 mg/L (calculated at a signal-to-noise ratio of 3). Use of the portable instrumentation in the field was demonstrated by analyzing postblast residues in a mobile laboratory immediately after detonation of the explosive devices. Profiling the ionic composition of the inorganic IEDs allowed identification of the chemicals used in their construction.

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

PubMed

Chétrite, G; Cassoly, R

1985-10-05

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Salivary pH and Buffering Capacity as Risk Markers for Early Childhood Caries: A Clinical Study.

PubMed

Jayaraj, D; Ganesan, S

2015-01-01

The diagnostic utility of saliva is currently being explored in various branches of dentistry, remarkably in the field of caries research. This study was aimed to determine if assessment of salivary pH and buffering capacity would serve as reliable tools in risk prediction of early childhood caries (ECC). Paraffin-stimulated salivary samples were collected from 50 children with ECC (group I) and 50 caries free children (group II). Salivary pH and buffering capacity (by titration with 0.1 N hydrochloric acid) were assessed using a handheld digital pH meter in both groups. The data obtained were subjected to statistical analysis. Statistically, no significant difference was observed between both the groups for all salivary parameters assessed, except for the buffering capacity level at 150 μl titration of 0.1 N hydrochloric acid (p = 0.73; significant at 1% level). Salivary pH and buffering capacity may not serve as reliable markers for risk prediction of ECC. How to cite this article: Jayaraj D, Ganesan S. Salivary pH and Buffering Capacity as Risk Markers for Early Childhood Caries: A Clinical Study. Int J Clin Pediatr Dent 2015;8(3):167-171.

Aqueous photolysis of niclosamide

USGS Publications Warehouse

Graebing, P.W.; Chib, J.S.; Hubert, T.D.; Gingerich, W.H.

2004-01-01

The photodegradation of [14C]niclosamide was studied in sterile, pH 5, 7, and 9 buffered aqueous solutions under artificial sunlight at 25.0 A? 1.0 A?C. Photolysis in pH 5 buffer is 4.3 times faster than in pH 9 buffer and 1.5 times faster than in pH 7 buffer. In the dark controls, niclosamide degraded only in the pH 5 buffer. After 360 h of continuous irradiation in pH 9 buffer, the chromatographic pattern of the degradates was the same regardless of which ring contained the radiolabel. An HPLC method was developed that confirmed these degradates to be carbon dioxide and two- and four-carbon aliphatic acids formed by cleavage of both aromatic rings. Carbon dioxide was the major degradate, comprising 40% of the initial radioactivity in the 360 h samples from both labels. The other degradates formed were oxalic acid, maleic acid, glyoxylic acid, and glyoxal. In addition, in the chloronitroaniline-labeled irradiated test solution, 2-chloro-4-nitroaniline was observed and identified after 48 h of irradiation but was not detected thereafter. No other aromatic compounds were isolated or observed in either labeled test system.

High-performance liquid chromatography analysis methods developed for quantifying enzymatic esterification of flavonoids in ionic liquids.

PubMed

Lue, Bena-Marie; Guo, Zheng; Xu, Xuebing

2008-07-11

Methods using reversed-phase high-performance liquid chromatography (RP-HPLC) with ELSD were investigated to quantify enzymatic reactions of flavonoids with fatty acids in the presence of diverse room temperature ionic liquids (RTILs). A buffered salt (preferably triethylamine-acetate) was found essential for separation of flavonoids from strongly polar RTILs, whereby RTILs were generally visible as two major peaks identified based on an ion-pairing/exchanging hypothesis. C8 and C12 stationary phases were optimal while mobile phase pH (3-7) had only a minor influence on separation. The method developed was successfully applied for primary screening of RTILs (>20), with in depth evaluation of substrates in 10 RTILs, for their evaluation as reaction media.

Quantification of urinary uric acid in the presence of thymol and thimerosal by high-performance liquid chromatography

NASA Technical Reports Server (NTRS)

Chen, Y.; Pietrzyk, R. A.; Whitson, P. A.

1997-01-01

A high-performance liquid chromatographic method was developed as an alternative to automated enzymatic analysis of uric acid in human urine preserved with thymol and/or thimerosal. Uric acid (tR = 10 min) and creatinine (tR = 5 min) were separated and quantified during isocratic elution (0.025 M acetate buffer, pH 4.5) from a mu Bondapak C18 column. The uric-acid peak was identified chemically by incubating urine samples with uricase. The thymol/thimerosal peak appeared at 31 min during the washing step and did not interfere with the analysis. We validated the high-performance liquid chromatographic method for linearity, precision and accuracy, and the results were found to be excellent.

Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution

PubMed Central

Creamer, Kaitlin E.; Ditmars, Frederick S.; Basting, Preston J.; Kunka, Karina S.; Hamdallah, Issam N.; Bush, Sean P.; Scott, Zachary; He, Amanda; Penix, Stephanie R.; Gonzales, Alexandra S.; Eder, Elizabeth K.; Camperchioli, Dominic W.; Berndt, Adama; Clark, Michelle W.; Rouhier, Kerry A.

2016-01-01

ABSTRACT Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA. Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques. PMID:27793830

Evaluation of sonication treatment and buffer composition on rumen bacteria protein extraction and carboxymethylcellulase activity.

PubMed

Prauchner, Carlos A; Kozloski, Gilberto V; Farenzena, Roberta

2013-05-01

The methodological procedures for studying the fibrolytic activity of rumen bacteria are not clearly established. In this study the efficiency of sonication treatment and buffer composition (i.e. buffer varying in tonicity or pH) on the level of protein extraction from the residue of forage samples incubated in the rumen of a grazing steer and the effect of buffer composition or CaCl₂ concentration on the carboxymethylcellulase (CMCase) activity of the released protein were evaluated. The amount of protein released from the residue of incubation was higher (P < 0.05) for the sonicated material and increased linearly with increasing buffer pH (P < 0.05). The CMCase activity of the released protein was not improved by sonication treatment, whereas it was higher (P < 0.05) for hypotonic than for hypertonic buffer. Both linear and quadratic effects (P < 0.05) of buffer pH on CMCase activity were significant, with CMCase activity being maximal at pH 5.4-6.1. CMCase activity was higher (P < 0.05) at a CaCl₂ concentration of 1 mmol L(-1) compared with lower values. Although sonication treatment increases the amount of protein extracted from rumen bacteria adhered to the residue of incubation, the CMCase activity of the released protein might be measured without sonication treatment and should be carried out with a hypotonic buffer solution that includes a calcium source. When pH is not a treatment factor, the buffer pH should be between 5.5 and 6. © 2012 Society of Chemical Industry.

Feasibility of reusing the black liquor for enzymatic hydrolysis and ethanol fermentation.

PubMed

Wang, Wen; Chen, Xiaoyan; Tan, Xuesong; Wang, Qiong; Liu, Yunyun; He, Minchao; Yu, Qiang; Qi, Wei; Luo, Yu; Zhuang, Xinshu; Yuan, Zhenhong

2017-03-01

The black liquor (BL) generated in the alkaline pretreatment process is usually thought as the environmental pollutant. This study found that the pure alkaline lignin hardly inhibited the enzymatic hydrolysis of cellulose (EHC), which led to the investigation on the feasibility of reusing BL as the buffer via pH adjustment for the subsequent enzymatic hydrolysis and fermentation. The pH value of BL was adjusted from 13.23 to 4.80 with acetic acid, and the alkaline lignin was partially precipitated. It deposited on the surface of cellulose and negatively influenced the EHC via blocking the access of cellulase to cellulose and adsorbing cellulase. The supernatant separated from the acidified BL scarcely affected the EHC, but inhibited the ethanol fermentation. The 4-times diluted supernatant and the last-time waste wash water of the alkali-treated sugarcane bagasse didn't inhibit the EHC and ethanol production. This work gives a clue of saving water for alkaline pretreatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analytical Enantioseparation of β-Substituted-2-Phenylpropionic Acids by High-Performance Liquid Chromatography with Hydroxypropyl-β-Cyclodextrin as Chiral Mobile Phase Additive.

PubMed

Tong, Shengqiang; Zhang, Hu; Yan, Jizhong

2016-04-01

Analytical enantioseparation of five β-substituted-2-phenylpropionic acids by high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral mobile phase additive was established in this paper, and chromatographic retention mechanism was studied. The effects of various factors such as the organic modifier, different ODS C18 columns and concentration of HP-β-CD were investigated. The chiral mobile phase was composed of methanol or acetonitrile and 0.5% triethylamine acetate buffer at pH 3.0 added with 25 mmol L(-1) of HP-β-CD, and baseline separations could be reached for all racemates. As for chromatographic retention mechanism, it was found that there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor under constant pH value and column temperature. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Controlled release of acidic drugs in compendial and physiological hydrogen carbonate buffer from polymer blend-coated oral solid dosage forms.

PubMed

Wulff, R; Rappen, G-M; Koziolek, M; Garbacz, G; Leopold, C S

2015-09-18

The objective of this study was to investigate the suitability of "Eudragit® RL/Eudragit® L55" (RL/L55) blend coatings for a pH-independent release of acidic drugs. A coating for ketoprofen and naproxen mini tablets was developed showing constant drug release rate under pharmacopeial two-stage test conditions for at least 300 min. To simulate drug release from the mini tablets coated with RL/L55 blends in the gastrointestinal (GI) tract, drug release profiles in Hanks buffer pH 6.8 were recorded and compared with drug release profiles in compendial media. RL/L55 blend coatings showed increased drug permeability in Hanks buffer pH 6.8 compared to phosphate buffer pH 6.8 due to its higher ion concentration. However, drug release rates of acidic drugs were lower in Hanks buffer pH 6.8 because of the lower buffer capacity resulting in reduced drug solubility. Further dissolution tests were performed in Hanks buffer using pH sequences simulating the physiological pH conditions in the GI tract. Drug release from mini tablets coated with an RL/L55 blend (8:1) was insensitive to pH changes of the medium within the pH range of 5.8-7.5. It was concluded that coatings of RL/L55 blends show a high potential for application in coated oral drug delivery systems with a special focus on pH-independent release of acidic drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

Stopped-flow studies of the reaction of D-tartronate semialdehyde-2-phosphate with human neuronal enolase and yeast enolase 1.

PubMed

Brewer, John M; McKinnon, Jared S; Phillips, Robert S

2010-03-05

We determined the kinetics of the reaction of human neuronal enolase and yeast enolase 1 with the slowly-reacting chromophoric substrate D-tartronate semialdehyde phosphate (TSP), each in tris (tris (hydroxymethyl) aminomethane) and another buffer at several Mg2+ concentrations, 50 or 100 microM, 1 mM and 30 mM. All data were biphasic, and could be satisfactorily fit, assuming either two successive first-order reactions or two independent first-order reactions. Higher Mg2+ concentrations reduce the relative magnitude of the slower reaction. The results are interpreted in terms of a catalytically significant interaction between the two subunits of these enzymes. Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Separation of attogram terpenes by the capillary zone electrophoresis with fluorometric detection.

PubMed

Kubesová, Anna; Horká, Marie; Růžička, Filip; Slais, Karel; Glatz, Zdeněk

2010-11-12

An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium. Copyright © 2010 Elsevier B.V. All rights reserved.

Photocatalytic effect of anodic titanium oxide nanotubes on various cell culture media

NASA Astrophysics Data System (ADS)

Yu, Chun-Kang; Hu, Kan-Hung; Wang, Shing-Hoa; Hsu, Todd; Tsai, Huei-Ting; Chen, Chien-Chon; Liu, Shiu-Mei; Lin, Tai-Yuan; Chen, Chin-Hsing

2011-02-01

The use of titanium dioxide (TiO2) in photodynamic therapy for the treatment of cancer cells has been proposed following studies of cultured cancer cells. In this work, an ordered channel array of anodic titanium oxide (ATO) was fabricated by anodizing titanium foil. The ATO layer of nanotubes with diameters of 100 nm was made in NH4F electrolyte by anodization. The photocatalytic effect of ATO was examined on various culture media by ultraviolet A (UV-A) (366 nm) irradiation. After UV-A irradiation of the ATO layer, redox potential of Tris-HCl buffer (pH 7.5) and dilute acrylamide solution increased instantaneously. The redox potential of the serum-containing RPMI1640 medium also increased dramatically, while that of serum-containing MEM and DMEM media increased slightly. The UVA-induced high redox potential was correlated with the greater ability to break down plasmid DNA strands. These phenomena suggest that a culture medium, such as RPMI1640, with a greater ability to produce free radical may be associated with a stronger photocatalytic effect of ATO on cultured cancer cells reported previously.

DNA interaction studies of sesamol (3,4-methylenedioxyphenol) food additive.

PubMed

Kashanian, Soheila; Tahmasian Ghobadi, Ameneh; Roshanfekr, Hamideh; Shariati, Zohreh

2013-02-01

The interaction of native calf thymus DNA (CT-DNA) with sesamol (3,4-methylenedioxyphenol) in Tris-HCl buffer at neutral pH 7.4 was monitored by absorption spectrophotometry, viscometry and spectrofluorometry. It is found that sesamol molecules could interact with DNA outside and/or groove binding modes, as are evidenced by: hyperchromism in UV absorption band, very slow decrease in specific viscosity of DNA, and small increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of sesamol, which indicates that it is able to partially release the bound MB. Furthermore, the enthalpy and entropy of the reaction between sesamol and CT-DNA showed that the reaction is enthalpy-favored and entropy-disfavored (ΔH = -174.08 kJ mol(-1); ΔS = -532.92 J mol(-1) K(-1)). The binding constant was determined using absorption measurement and found to be 2.7 × 10(4) M(-1); its magnitude suggests that sesamol interacts to DNA with a high affinity.

The voltammetric behaviour of lead at a microband screen-printed carbon electrode and its determination in acetate leachates from glazed ceramic plates.

PubMed

Honeychurch, Kevin C; Al-Berezanchi, Saman; Hart, John P

2011-05-15

Microband screen-printed carbon electrodes (μBSPCEs) without further modification have been investigated as disposable sensors for the measurement of lead in acetate leachates from ceramic glazed plates. Cyclic voltammetry was employed to elucidate the electrochemical behaviour of Pb(2+) at these electrodes in a variety of supporting electrolytes. The anodic peaks obtained on the reverse scans, showed that Pb had been deposited as a thin layer on the surface of the μBSPCE. The anodic peak of greatest magnitude was obtained in 0.1M pH 4.1 acetate buffer containing 13 mM Cl(-). The effect of chromium, copper, phosphate, sulphate and tin was examined and under the conditions employed, no significant change in current was found. The μBSPCEs were evaluated by carrying out lead determinations for acetate leachates from glazed ceramic plates. A highly decorated ornamental plate was found to leach 400 μg Pb(2+) (%CV=1.91%). A second plate, designed for dinnerware was found not to leach any detectable levels of Pb(2+). However, once fortified with 2.10 μg of Pb (equivalent to 100 ng/ml in the leachate), a mean recovery of 82.08% (%CV=4.07%) was obtained. The performance characteristics indicate that reliable data has been obtained for this application which could identify potentially toxic sources of lead. Copyright © 2011 Elsevier B.V. All rights reserved.

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications.

PubMed

Tomcik, Kristyen; Ibarra, Rafael A; Sadhukhan, Sushabhan; Han, Yong; Tochtrop, Gregory P; Zhang, Guo-Fang

2011-03-01

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs-Ringer bicarbonate buffer only, 0-1mM [3,4-(13)C(2)]-4-hydroxynonanoate, and 0-2mM [5,6,7-(13)C(3)]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-(13)C(3)]heptanoate perfusates. 2010 Elsevier Inc. All rights reserved.

A dynamic system for the simulation of fasting luminal pH-gradients using hydrogen carbonate buffers for dissolution testing of ionisable compounds.

PubMed

Garbacz, Grzegorz; Kołodziej, Bartosz; Koziolek, Mirko; Weitschies, Werner; Klein, Sandra

2014-01-23

The hydrogen carbonate buffer is considered as the most biorelevant buffer system for the simulation of intestinal conditions and covers the physiological pH range of the luminal fluids from pH 5.5 to about pH 8.4. The pH value of a hydrogen carbonate buffer is the result of a complex and dynamic interplay of the concentration of hydrogen carbonate ions, carbonic acid, the concentration of dissolved and solvated carbon dioxide and its partial pressure above the solution. The complex equilibrium between the different ions results in a thermodynamic instability of hydrogen carbonate solutions. In order to use hydrogen carbonate buffers with pH gradients in the physiological range and with the dynamics observed in vivo without changing the ionic strength of the solution, we developed a device (pHysio-grad®) that provides both acidification of the dissolution medium by microcomputer controlled carbon dioxide influx and alkalisation by degassing. This enables a continuous pH control and adjustment during dissolution of ionisable compounds. The results of the pH adjustment indicate that the system can compensate even rapid pH changes after addition of a basic or acidic moiety in amounts corresponding up to 90% of the overall buffer capacity. The results of the dissolution tests performed for a model formulation containing ionizable compounds (Nexium 20mg mups) indicate that both the simulated fasting intraluminal pH-profiles and the buffer species can significantly affect the dissolution process by changing the lag time prior to initial drug release and the release rate of the model compound. A prediction of the in vivo release behaviour of this formulation is thus most likely strongly related to the test conditions such as pH and buffer species. Copyright © 2013 Elsevier B.V. All rights reserved.

Carbon nanotube/poly(ethylene-co-vinyl acetate) composite electrode for capillary electrophoretic determination of esculin and esculetin in Cortex Fraxini.

PubMed

Chen, Zhi; Zhang, Luyan; Chen, Gang

2009-10-01

In this report, a novel carbon nanotube/poly(ethylene-co-vinyl acetate) (CNT/EVA) composite electrode was developed for the amperometric detection in CE. The composite electrode was fabricated by packing a mixture of CNTs and melted EVA in a piece of fused-silica capillary under heat. It was coupled with CE for the separation and detection of esculin and esculetin in Cortex Fraxini, a traditional Chinese medicine, to demonstrate its feasibility and performance. Esculin and esculetin have been well separated within 9 min in a 40 cm long capillary at a separation voltage of 12 kV using a 50 mM borate buffer (pH 9.2). The new CNT-based CE detector offered significantly lower detection potentials, yielded enhanced S/N characteristics, and exhibited high resistance to surface fouling and enhanced stability. It showed long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n=15) and should also find a wide range of applications in other microfluidic analysis systems.

Electrochemical Behavior and Determination of Chlorogenic Acid Based on Multi-Walled Carbon Nanotubes Modified Screen-Printed Electrode

PubMed Central

Ma, Xiaoyan; Yang, Hongqiao; Xiong, Huabin; Li, Xiaofen; Gao, Jinting; Gao, Yuntao

2016-01-01

In this paper, the multi-walled carbon nanotubes modified screen-printed electrode (MWCNTs/SPE) was prepared and the MWCNTs/SPE was employed for the electrochemical determination of the antioxidant substance chlorogenic acids (CGAs). A pair of well-defined redox peaks of CGA was observed at the MWCNTs/SPE in 0.10 mol/L acetic acid-sodium acetate buffer (pH 6.2) and the electrode process was adsorption-controlled. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods for the determination of CGA were proposed based on the MWCNTs/SPE. Under the optimal conditions, the proposed method exhibited linear ranges from 0.17 to 15.8 µg/mL, and the linear regression equation was Ipa (µA) = 4.1993 C (×10−5 mol/L) + 1.1039 (r = 0.9976) and the detection limit for CGA could reach 0.12 µg/mL. The recovery of matrine was 94.74%–106.65% (RSD = 2.92%) in coffee beans. The proposed method is quick, sensitive, reliable, and can be used for the determination of CGA. PMID:27801797

Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification.

PubMed

Emaus, Miranda N; Clark, Kevin D; Hinners, Paige; Anderson, Jared L

2018-04-28

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P 6,6,6,14 + ][Ni(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac) 3 - ]), or [P 6,6,6,14 + ] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac) 4 - ]) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1%). High enrichment factors were achieved using the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples. Graphical abstract Magnetic ionic liquid solvents are shown to preconcentrate sufficient KRAS DNA template from an aqueous solution in as short as 2 min without using chaotropic salts or toxic organic solvents. By using custom-designed qPCR buffers, DNA can be directly amplified and quantified from four MILs examined in this study.

Changes in composition and enamel demineralization inhibition activities of gallic acid at different pH values.

PubMed

Zhang, Jingyang; Huang, Xuelian; Huang, Shengbin; Deng, Meng; Xie, Xincheng; Liu, Mingdong; Liu, Hongling; Zhou, Xuedong; Li, Jiyao; Ten Cate, Jacob Martien

2015-01-01

Gallic acid (GA) has been shown to inhibit demineralization and enhance remineralization of enamel; however, GA solution is highly acidic. This study was to investigate the stability of GA solutions at various pH and to examine the resultant effects on enamel demineralization. The stability of GA in H2O or in phosphate buffer at pH 5.5, pH 7.0 and pH 10.0 was evaluated qualitatively by ultraviolet absorption spectra and quantified by high performance liquid chromatography with diode array detection (HPLC-DAD). Then, bovine enamel blocks were subjected to a pH-cycling regime of 12 cycles. Each cycle included 5 min applications with one of the following treatments: 1 g/L NaF (positive control), 4 g/L GA in H2O or buffered at pH 5.5, pH 7.0 and pH 10.0 and buffers without GA at the same pH (negative control), followed by a 60 min application with pH 5.0 acidic buffers and a 5 min application with neutral buffers. The acidic buffers were analysed for dissolved calcium. GA was stable in pure water and acidic condition, but was unstable in neutral and alkaline conditions, in which ultraviolet spectra changed and HPLC-DAD analysis revealed that most of the GA was degraded. All the GA groups significantly inhibited demineralization (p < 0.05) and there was no significant difference of the inhibition efficacy among different GA groups (p > 0.05). GA could inhibit enamel demineralization and the inhibition effect is not influenced by pH. GA could be a useful source as an anti-cariogenic agent for broad practical application.

D-alanine carboxypeptidase activity of Micrococcus lysodeikticus released into the protoplasting medium.

PubMed

Linder, R; Salton, M R

1975-06-16

Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.

Evaluation of buffers toxicity in tobacco cells: Homopiperazine-1,4-bis (2-ethanesulfonic acid) is a suitable buffer for plant cells studies at low pH.

PubMed

Borgo, Lucélia

2017-06-01

Low pH is an important environmental stressor of plant root cells. Understanding the mechanisms of stress and tolerance to acidity is critical; however, there is no widely accepted pH buffer for studies of plant cells at low pH. Such a buffer might also benefit studies of Al toxicity, in which buffering at low pH is also important. The challenge is to find a buffer with minimal cellular effects. We examined the cytotoxicity and possible metabolic disturbances of four buffers that have adequate pK a values and potential use for studies in the pH range of 4.0-5.0. These were homopipes (homopiperazine-1,4-bis (2-ethanesulfonic acid); pK a1 4.4), 3,3-dimethylglutaric acid (pK a1 3.73), β-alanine (pK a1 3.70) and potassium biphthalate (pK a1 2.95; pK a2 5.41). First, tobacco BY-2 cells were grown in a rich medium containing 10 mM of each buffer or MES (2-(N-morpholino) ethanesulfonic acid) as a control, with the pH initially adjusted to 5.7. β-alanine was clearly toxic and dimethylgluturate and biphthalate were found to be cytostatic, in which no culture growth occurred but cell viability was either unaffected or decreased only after 5 days. Only homopipes allowed normal culture growth and cell viability. Homopipes (10 mM) was then tested in cell cultures with an initial pH of 4.3 ± 0.17 in minimal medium to examine whether its undissociated species (H 2 A) displayed any cellular effects and no cytotoxic effects were observed. It is possible to conclude that among tested buffers, homopipes is the most suitable for studies at low pH, and may be especially useful for aluminum toxicity experiments. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

PubMed

Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

2010-03-01

The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

Taka-amylase A in the conidia of Aspergillus oryzae RIB40.

PubMed

Nguyen, Cong Ha; Tsurumizu, Ryoji; Sato, Tsutomu; Takeuchi, Michio

2005-11-01

A study of Taka-amylase A of conidia from Aspergillus oryzae RIB40 was done. During the research, proteins from conidia and germinated conidia were analyzed using SDS-PAGE, 2-D gel electrophoresis, Western blot analysis, MALDI-TOF Mass spectrometry, and native-PAGE combined with activity staining of TAA. The results showed that TAA exists not only in germinated conidia but also in conidia. Some bands representing degraded products of TAA were detected. Conidia, which formed on starch (SCYA), glucose (DCYA), and glycerol (GCYA) plates, contained mature TAA. Only one active band of TAA was detected after native-PAGE activity staining. In addition, TAA activity was detected in cell extracts of conidia using 0.5 M acetate buffer, pH 5.2, as extraction buffer, but was not detected in whole conidia or cell debris. The results indicate that TAA exists in conidia in active form even when starch, glucose, or glycerol is used as carbon source. TAA might belong to a set of basal proteins inside conidia, which helps in imbibition and germination of conidia.

21 CFR 114.90 - Methodology.

Code of Federal Regulations, 2011 CFR

2011-04-01

... buffer. First standardize the electrodes using a pH 4.0 buffer at or near 25 °C. Standardization control..., the optimum being 25 °C. Any temperature determinations made without meter compensation may affect pH... approximately ±0.01 pH unit and a reproducibility of ±0.005 pH units. (4) General procedure for determining pH...

21 CFR 114.90 - Methodology.

Code of Federal Regulations, 2010 CFR

2010-04-01

... buffer. First standardize the electrodes using a pH 4.0 buffer at or near 25 °C. Standardization control..., the optimum being 25 °C. Any temperature determinations made without meter compensation may affect pH... approximately ±0.01 pH unit and a reproducibility of ±0.005 pH units. (4) General procedure for determining pH...

Procedure for rapid concentration and detection of enteric viruses from berries and vegetables.

PubMed

Butot, S; Putallaz, T; Sánchez, G

2007-01-01

Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID(50)), 54 RT-PCR units, and 0.02 TCID(50) per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.

Stability of cefozopran hydrochloride in aqueous solutions.

PubMed

Zalewski, Przemysław; Skibiński, Robert; Paczkowska, Magdalena; Garbacki, Piotr; Talaczyńska, Alicja; Cielecka-Piontek, Judyta; Jelińska, Anna

2016-01-01

The influence of pH on the stability of cefozopran hydrochloride (CZH) was investigated in the pH range of 0.44-13.00. Six degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The degradation of CZH as a result of hydrolysis was a pseudo-first-order reaction. As general acid-base hydrolysis of CZH was not occurred in the solutions of hydrochloric acid, sodium hydroxide, acetate, borate and phosphate buffers, kobs = kpH because specific acid-base catalysis was observed. Specific acid-base catalysis of CZH consisted of the following reactions: hydrolysis of CZH catalyzed by hydrogen ions (kH+), hydrolysis of dications (k1H2O), monocations (k2H2O) and zwitter ions (k3H2O) and hydrolysis of zwitter ions (k1OH-) and monoanions (k2OH-) of CZH catalyzed by hydroxide ions. The total rate of the reaction was equal to the sum of partial reactions: [Formula: see text]. CZH similarly like other fourth generation cephalosporin was most stable at slightly acidic and neutral pH and less stable in alkaline pH. The cleavage of the β-lactam ring resulting from a nucleophilic attack on the carbonyl carbon in the β-lactam moiety is the preferred degradation pathway of β-lactam antibiotics in aqueous solutions.

The potential effects of pH and buffering capacity on dental erosion.

PubMed

Owens, Barry M

2007-01-01

Soft drink pH (initial pH) has been shown to be a causative factor--but not necessarily the primary initiating factor--of dental erosion. The titratable acidity or buffering capacity has been acknowledged as playing a significant role in the etiology of these lesions. This in vitro study sought to evaluate five different soft drinks (Coca-Cola Classic, Diet Coke, Gatorade sports drink, Red Bull high-energy drink, Starbucks Frappucino coffee drink) and tap water (control) in terms of initial pH and buffering capacity. Initial pH was measured in triplicate for the six beverages. The buffering capacity of each beverage was assessed by measuring the weight (in grams) of 0.10 M sodium hydroxide necessary for titration to pH levels of 5.0, 6.0, 7.0, and 8.3. Coca-Cola Classic produced the lowest mean pH, while Starbucks Frappucino produced the highest pH of any of the drinks except for tap water. Based on statistical analysis using ANOVA and Fisher's post hoc tests at a P < 0.05 level of significance, Red Bull had the highest mean buffering capacity (indicating the strongest potential for erosion of enamel), followed by Gatorade, Coca-Cola Classic, Diet Coke, and Starbucks Frappucino.

Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: from protein buffer capacity prediction to bioprocess applications.

PubMed

Bahrenburg, Sven; Karow, Anne R; Garidel, Patrick

2015-04-01

Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Programmable pH buffers

DOEpatents

Gough, Dara Van; Huber, Dale L.; Bunker, Bruce C.; Roberts, Mark E.

2017-01-24

A programmable pH buffer comprises a copolymer that changes pK.sub.a at a lower critical solution temperature (LCST) in water. The copolymer comprises a thermally programmable polymer that undergoes a hydrophobic-to-hydrophilic phase change at the LCST and an electrolytic polymer that exhibits acid-base properties that are responsive to the phase change. The programmable pH buffer can be used to sequester CO.sub.2 into water.

Effect of pH buffer molecules on the light-induced currents from oriented purple membrane.

PubMed Central

Liu, S Y; Kono, M; Ebrey, T G

1991-01-01

The effect of pH buffers on the microsecond photocurrent component, B2, of oriented purple membranes has been studied. We found that under low salt conditions (less than 10 mM monovalent cationic salt) pH buffers can dramatically alter the waveform of the B2 component. The effect is induced by the protonation process of the buffer molecules by protons expelled from the membrane. These effects can be classified according to the charge transition upon protonation of the buffer. Buffers that carry two positive charges in their protonated form add a negative current component (N component) to B2. Almost all of the other buffers add a positive current component (P component) to B2, which is essentially a mirror image of the N component. Buffers with a pK less than 5.5 have only a small positive buffer component. The pH dependence of the buffer effect is closely related to the pK of the buffer; it requires that the buffer be in its unprotonated form. The rise time of the buffer component increases with the concentration of the buffer molecules. All the buffer effects can be inhibited by the addition of 5 mM of a divalent cation such as Ca2+. Reducing the surface potential slows down the N component but accelerates the P component without affecting the amplitude of the buffer effect significantly. Many of the buffer effects can be explained if we assume that upon protonation of the buffer by a proton expelled from the membrane by light, the buffer molecules move toward the membrane. This backward movement of buffer molecules forms a counter current very similar to that due to cations discussed in Liu, S. Y., R. Govindjee, and T. G. Ebrey. (1990. Biophys. J. 57:951-963). PMID:1883939

Fluorophotometric measurement of the buffering action of human tears in vivo.

PubMed

Yamada, M; Kawai, M; Mochizuki, H; Hata, Y; Mashima, Y

1998-10-01

The buffering action of human tears is thought to be important to keep its pH constant. We measured the change in pH in the precorneal tear film in vivo when the acidic solution is challenged, using a fluorophotometric technique. Twelve eyes from 6 healthy subjects were entered in this study. Each subject was pretreated with either one drop of 0.4% oxybuprocaine for once (light anesthesia), three times (deep anesthesia), or none (controls). The measurement was initiated by instilling 20 microl of 0.067 M phosphate buffer at pH 5.5 containing 2 mM bis-carboxyethyl-carboxyfluorescein free acid, a pH sensitive dye, into the subject's eye. The pH was determined by the ratio of fluorescent intensities at two excitation wavelengths (490 and 430 nm). pH recovery time (PHRT) as defined by the time required for pH to reach 95% of pH at equilibrium was used for the marker of tear buffering action. Tear turnover rate was also determined using the fluorescent decay curve at 430 nm, which was independent of pH, but dependent on dye concentration. Immediately after the instillation, the pH value in the tear film was around 6.0-6.5 in all cases. The tear film rapidly became more alkaline, reaching its normal value in 2.3 +/- 0.5 min in untreated eyes. The pretreatment with 0.4% oxybuprocaine retarded the neutralization process. A single regression analysis revealed that the PHRT had a significant negative correlation with the tear turnover rate (r = -0.78). Our results suggest that the neutralization process of tears largely depends on the tear turnover rate. The buffering action of tears in vivo consists of the tear turnover as well as its chemical buffering capacity.

Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases.

PubMed

Van Der Heyden, N; Docampo, R

2000-02-05

Regulation of intracellular pH (pHi) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pHi was determined to be 7.33 +/- 0.08 and 7.35 +/- 0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH, between the two stages. Steady-state pHi, recovery of pHi from acidification, and H+-efflux were all decreased markedly by the H+-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H+-ATPase in the regulation of pHi. pHi was maintained at neutrality over a range of external pH (pHe) from 5-8 in parasites suspended in a buffer containing Na+ and K+ (standard buffer) but was acidified at low pHe in the absence of these cations (choline buffer). The pHi of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pHi by acidified parasites was similar in Na+-free buffer and standard buffer but was slower in the absence of K+ (K+-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba2+ and Cs+ decreased the pHi of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K+ channel. The pHi of amastigotes and trypomastigotes suspended in Cl(-)-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na+/H+ or Cl-/HCO3- exchanger was found. These results are consistent with the presence of a plasma membrane H+-ATPase that regulates pHi and is supported by K+ and Cl- channels.

Final report of the key comparison APMP.QM-K91: APMP comparison on pH measurement of phthalate buffer

NASA Astrophysics Data System (ADS)

Hioki, Akiharu; Asakai, Toshiaki; Maksimov, Igor; Suzuki, Toshihiro; Miura, Tsutomu; Ketrin, Rosi; Nuryatini; Thanh, Ngo Huy; Truong Chinh, Nguyen; Vospelova, Alena; Bastkowski, Frank; Sander, Beatrice; Matzke, Jessica; Prokunin, Sergey; Frolov, Dmitry; Aprelev, Alexey; Dobrovolskiy, Vladimir; Uysal, Emrah; Liv, Lokman; Velina Lara-Manzano, Judith; Montero-Ruiz, Jazmin; Ortiz-Aparicio, JosÉ Luis; Ticona Canaza, Galia; Anuar Mohd Amin, Khirul; Abd Kadir, Haslina; Bakovets, Nickolay; Wong, Siu-Kay; Lam, Wai-Hing

2017-01-01

The APMP.QM-K91 was organised by TCQM of APMP to test the abilities of the national metrology institutes in the APMP region to measure a pH value of a phthalate buffer. This APMP comparison on pH measurement was proposed by the National Metrology Institute of Japan at the APMP-TCQM meeting held September 22-23, 2014. After approval by TCQM, the comparison has been conducted by NMIJ. The comparison is a key comparison following CCQM-K91. The comparison material was a phthalate buffer of pH around 4.0 and the measurement temperatures were 15 °C, 25 °C and 37 °C. This is the third APMP key comparison on pH measurement and the fifth APMP comparison on pH measurement following APMP.QM-P06 (two phosphate buffers) in 2004, APMP.QM-P09 (a phthalate buffer) in 2006, APMP.QM-K9/APMP.QM-P16 (a phosphate buffer) in 2010-2011 and APMP.QM-K19/APMP.QM-P25 (a borate buffer) in 2013-2014. The results can be used further by any participant to support its CMC claim at least for a phthalate buffer. That claim will concern the pH method employed by the participant during this comparison and will cover the used temperature(s) or the full temperature range between 15°C and 37 °C for the participant which measured pH values at the three temperatures. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Improved pH buffering agent for sodium hypochlorite

NASA Technical Reports Server (NTRS)

Nash, J. R.; Veeder, L. N.

1969-01-01

Sodium citrate/citric acid was found to be an effective buffer for pH control when used with sodium hypochlorite. The mixture does not corrode aluminum. The buffer appears to form a type of conversion coating that may provide corrosion-resistant properties to aluminum in other applications.

Effect of Sample Preparation on the Discrimination of Bacterial Isolates Cultured in Liquid Nutrient Media Using Laser-Induced Breakdown Spectroscopy (LIBS).

PubMed

Gamble, Gary R; Park, Bosoon; Yoon, Seung-Chul; Lawrence, Kurt C

2016-03-01

Laser-induced breakdown spectroscopy (LIBS) is used as the basis for discrimination between two genera of gram-negative bacteria and two genera of gram-positive bacteria representing pathogenic threats commonly found in poultry processing rinse waters. Because LIBS-based discrimination relies primarily upon the relative proportions of inorganic cell components including Na, K, Mg, and Ca, this study aims to determine the effects of trace mineral content and pH found in the water source used to isolate the bacteria upon the reliability of the resulting discriminant analysis. All four genera were cultured using tryptic soy agar (TSA) as the nutrient medium, and were grown under identical environmental conditions. The only variable introduced is the source water used to isolate the cultured bacteria. Cultures of each bacterium were produced using deionized (DI) water under two atmosphere conditions, reverse osmosis (RO) water, tap water, phosphate buffered saline (PBS) water, and TRIS buffered water. After 3 days of culture growth, the bacteria were centrifuged and washed three times in the same water source. Bacteria were then freeze dried, mixed with microcrystalline cellulose, and a pellet was made for LIBS analysis. Principal component analysis (PCA) was used to extract related variations in LIBS spectral data among the four bacteria genera and six water types used to isolate the bacteria, and Mahalanobis discriminant analysis (MDA) was used for classification. Results indicate not only that the four genera can be discriminated from each other in each water type, but that each genus can be discriminated by water type used for isolation. It is concluded that in order for LIBS to be a reliable and repeatable method for discrimination of bacteria grown in liquid nutrient media, care must be taken to insure that the water source used in purification of the culture be precisely controlled regarding pH, ionic strength, and proportionate amounts of mineral cations present. © The Author(s) 2016.

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes

PubMed Central

1983-01-01

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium- stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient. PMID:6411737

Visualization of Buffer Capacity with 3-D "Topo" Surfaces: Buffer Ridges, Equivalence Point Canyons and Dilution Ramps

ERIC Educational Resources Information Center

Smith, Garon C.; Hossain, Md Mainul

2016-01-01

BufCap TOPOS is free software that generates 3-D topographical surfaces ("topos") for acid-base equilibrium studies. It portrays pH and buffer capacity behavior during titration and dilution procedures. Topo surfaces are created by plotting computed pH and buffer capacity values above a composition grid with volume of NaOH as the x axis…

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.

PubMed

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. © 2013 Elsevier B.V. All rights reserved.

Sample storage conditions significantly influence faecal microbiome profiles

PubMed Central

Choo, Jocelyn M; Leong, Lex EX; Rogers, Geraint B

2015-01-01

Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to −80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants’ homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to −80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to −80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available. PMID:26572876

Sample storage conditions significantly influence faecal microbiome profiles.

PubMed

Choo, Jocelyn M; Leong, Lex E X; Rogers, Geraint B

2015-11-17

Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to -80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants' homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to -80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to -80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.

Multivariate Approaches for Simultaneous Determination of Avanafil and Dapoxetine by UV Chemometrics and HPLC-QbD in Binary Mixtures and Pharmaceutical Product.

PubMed

2016-04-07

Multivariate UV-spectrophotometric methods and Quality by Design (QbD) HPLC are described for concurrent estimation of avanafil (AV) and dapoxetine (DP) in the binary mixture and in the dosage form. Chemometric methods have been developed, including classical least-squares, principal component regression, partial least-squares, and multiway partial least-squares. Analytical figures of merit, such as sensitivity, selectivity, analytical sensitivity, LOD, and LOQ were determined. QbD consists of three steps, starting with the screening approach to determine the critical process parameter and response variables. This is followed by understanding of factors and levels, and lastly the application of a Box-Behnken design containing four critical factors that affect the method. From an Ishikawa diagram and a risk assessment tool, four main factors were selected for optimization. Design optimization, statistical calculation, and final-condition optimization of all the reactions were Carried out. Twenty-five experiments were done, and a quadratic model was used for all response variables. Desirability plot, surface plot, design space, and three-dimensional plots were calculated. In the optimized condition, HPLC separation was achieved on Phenomenex Gemini C18 column (250 × 4.6 mm, 5 μm) using acetonitrile-buffer (ammonium acetate buffer at pH 3.7 with acetic acid) as a mobile phase at flow rate of 0.7 mL/min. Quantification was done at 239 nm, and temperature was set at 20°C. The developed methods were validated and successfully applied for simultaneous determination of AV and DP in the dosage form.

Arrested α-hydride migration activates a phosphido ligand for C–H insertion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hickey, Anne K.; Muñoz, Salvador B.; Lutz, Sean A.

Bulky tris(carbene)borate ligands provide access to high spin iron(II) phosphido complexes. The complex PhB(MesIm) 3FeP(H)Ph is thermally unstable, and we observed [PPh] group insertion into a C–H bond of the supporting ligand. An arrested α-hydride migration mechanism suggests increased nucleophilicity of the phosphorus atom facilitates [PPh] group transfer reactivity.

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.

PubMed

Nagai, Satoshi; Yamamoto, Keigo; Hata, Naotugu; Itakura, Shigeru

2012-09-01

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP. Copyright © 2012 Elsevier B.V. All rights reserved.

The determination of levofloxacin by flow injection analysis using UV detection, potentiometry, and conductometry in pharmaceutical preparations.

PubMed

Altiokka, G; Atkosar, Z; Can, N O

2002-10-15

A flow injection analysis (FIA) using UV detection, potentiometry and conductometry for levofloxacin (LVF) are described in this study. The best solvent system was found to consist of 0.2 M acetate buffer at pH 3 having 10% MeOH. A flow rate of 1 ml min(-1) was pumped and active material was detected at 288 nm. The detection limit (LOD) and limit of quantification (LOQ) for FIA were calculated to be 3 x 10(-7) M (S/N = 3) and 1 x 10(-7) M (S/N = 10), respectively. In the analysis of tablets, the RSD values were found to be 0.83, 0.98 and 0.99 for FIA, potentiometric and conductometric methods, respectively. Copyright 2002 Elsevier Science B.V.

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase.

PubMed

Matsuo, Takemasa; Kobayashi, Takashi; Kimura, Yukitaka; Tsuchiyama, Moriyasu; Oh, Tadanobu; Sakamoto, Tatsuji; Adachi, Shuji

2008-12-01

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL "Amano" from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50 degrees C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25 degrees C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.

[Relationship between blood glucose levels and salivary pH and buffering capacity in type II diabetes patients].

PubMed

Elkafri, I H; Mashlah, A; Shaqifa, A

2014-03-13

This study was evaluated the relationship between blood glucose levels and salivary pH and buffering capacity in type II diabetic patients. The sample comprised 210 participants (age ranged 40-60 years). Based on fasting blood glucose levels the participants were divided into 3 groups: controls with normal blood glucose levels; diabetic patients with levels ≤ 200 mg/dL; and diabetic patients with levels > 200 mg/dL. Salivary pH and buffering capacity were determined in a sample of resting (non-stimulated) saliva taken from each participant. Salivary pH levels in diabetic patients with blood glucose levels > 200 mg/dL were lower than in the controls and diabetic patients with levels ≤ 200 mg/dL. Salivary pH levels were comparable in controls and diabetic patients with blood glucose levels ≤ 200 mg/dL. Salivary buffering capacity in the 3 groups was comparable.

[Indirect and repeated electromagnetic irradiation of extremely high freguency of bacteria Escherichia coli].

PubMed

Isakhanian, V; Trchunian, A

2005-01-01

It has been shown that separate irradiation of distilled water and tris-phosphate buffer containing some inorganic ions, with Escherichia coli K12 grown in anaerobic conditions upon fermentation of sugar (glucose) with "noise" electromagnetic radiation of extremely high frequencies (53.5-68 gHz) or millimeter waves (wavelength of 3 to 8 mm) with low flux capacity (0.01 mW) for 10, 30 and 60 min caused opposite effects, changing the growth of these bacteria. The irradiation of water has a bactericide effect, whereas the irradiation of the buffer stimulates bacterial growth although the buffer itself inhibits the growth. These results point out the role of water in the bactericide action of "noise" electromagnetic radiation of extremely high frequencies, and confirm the significance of membranotropic effects. The bactericide action disappeared after repeated irradiation for 10 and 30 min with 2-h intervals. This indicates the operation of some compensatory mechanisms in bacteria.

Internal Active Thermal Control System (IATCS) Sodium Bicarbonate/Carbonate Buffer in an Open Aqueous Carbon Dioxide System and Corollary Electrochemical/Chemical Reactions Relative to System pH Changes

NASA Technical Reports Server (NTRS)

Stegman, Thomas W.; Wilson, Mark E.; Glasscock, Brad; Holt, Mike

2014-01-01

The International Space Station (ISS) Internal Active Thermal Control System (IATCS) experienced a number of chemical changes driven by system absorption of CO2 which altered the coolant’s pH. The natural effects of the decrease in pH from approximately 9.2 to less than 8.4 had immediate consequences on system corrosion rates and corrosion product interactions with specified coolant constituents. The alkalinity of the system was increased through the development and implementation of a carbonate/bicarbonate buffer that would increase coolant pH to 9.0 – 10.0 and maintain pH above 9.0 in the presence of ISS cabin concentrations of CO2 up to twenty times higher than ground concentrations. This paper defines how a carbonate/bicarbonate buffer works in an open carbon dioxide system and summarizes the analyses performed on the buffer for safe and effective application in the on-orbit system. The importance of the relationship between the cabin environment and the IATCS is demonstrated as the dominant factor in understanding the system chemistry and pH trends before and after addition of the carbonate/bicarbonate buffer. The paper also documents the corollary electrochemical and chemical reactions the system has experienced and the rationale for remediation of these effects with the addition of the carbonate/bicarbonate buffer.

Electrochemical study of the interaction between dsDNA and copper(I) using carbon paste and hanging mercury drop electrode.

PubMed

Stanić, Z; Girousi, S

2008-06-30

The interaction of copper(I) with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer solution (pH 5.0). As a result of the interaction of Cu(I) between the base pairs of the dsDNA, the characteristic peaks of dsDNA, due to the oxidation of guanine and adenine, increased and after a certain concentration of Cu(I) a new peak at +1.37 V appeared, probably due to the formation of a purine-Cu(I) complex (dsDNA-Cu(I) complex). Accordingly, the interaction of copper(I) with calf thymus dsDNA was studied in solution as well as at the electrode surface using hanging mercury drop electrode (HMDE) by means of alternating current voltammetry (AC voltammetry) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. Its interaction with DNA is shown to be time dependent. Significant changes in the characteristic peaks of dsDNA were observed after addition of higher concentration of Cu(I) to a solution containing dsDNA, as a result of the interaction between Cu(I) and dsDNA. All the experimental results indicate that Cu(I) can bind to DNA by electrostatic binding and form an association complex.

Reactions of hypoiodous acid with model compounds and the formation of iodoform in absence/presence of permanganate.

PubMed

Zhao, Xiaodan; Ma, Jun; von Gunten, Urs

2017-08-01

The kinetics for the reactions of hypoiodous acid (HOI) with various phenols (phenol, 4-nitrophenol, 4-hydroxybenzoic acid), 3-oxopentanedioic acid (3-OPA) and flavone were investigated in the pH range of 6.0-11.0. The apparent second order rate constants for the reactions of HOI with phenolic compounds, 3-OPA, flavone and citric acid at pH 8.0 are 10-10 7  M -1 s -1 , (4.0 ± 0.3) × 10 3  M -1 s -1 , (2.5 ± 0.2) × 10 3  M -1 s -1 and <1 M -1 s -1 , respectively. The effect of buffer type and concentration was investigated with acetate, phosphate and borate. All tested buffers promote the HOI reactions with phenols. The percentage of iodine incorporation for various (hydroxyl)phenolic compounds and two NOM extracts ranges from 5% to 98%, indicating that electrophilic aromatic substitution and/or electron transfer can occur. The extent of these reactions depends on the number and relative position of the hydroxyl moieties on the phenolic compounds. Iodoform formation rates increase with increasing pH and iodoform yields increase from 9% to 67% for pH 6.0-10.0 for the HOI/3-OPA reactions. In the permanganate/HOI/3-OPA and permanganate/iodide/3-OPA system at pH < 8.0, iodoform formation is elevated compared to the HOI/3-OPA system in absence of permanganate. For pH > 8.0, in presence of permanganate, iodoform formation is significantly inhibited and iodate formation enhanced, which is due to a faster permanganate-mediated HOI disproportionation to iodate compared to the iodination process. The production of reactive iodine in real waters containing iodide in contact with permanganate may lead to the formation of iodinated organic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hydrogen-bonded structures from adamantane-based catechols

NASA Astrophysics Data System (ADS)

Kawahata, Masatoshi; Matsuura, Miku; Tominaga, Masahide; Katagiri, Kosuke; Yamaguchi, Kentaro

2018-07-01

Adamantane-based bis- and tris-catechols were synthesized to examine the effect of hydrogen bonds on the arrangement and packing of the components in the crystalline state. Single-crystal X-ray crystallographic analysis revealed that hydrogen bonds formed by the hydroxyl groups of catechol groups play essential roles in the production of various types of unique structures. 1,3-Bis(3,4-dihydroxyphenyl)adamantane (1) provided hydrogen-bonded network structures composed of helical chains in crystal from chloroform/methanol, and layer structures in crystal from ethyl acetate/hexane. The complexation of 1 with 1,3,5-trinitrobenzene or 1,2,4,5-tetracyanobenzene resulted in the formation of co-crystals, respectively. One-dimensional hydrogen-bonded structures were constructed from the adamantane-based molecules, which participated in charge-transfer interactions with guests. 1,3,5-Tris(3,4-dihydroxyphenyl)adamantane also afforded crystal, and the components were assembled into infinite polymers.

Expanding pH screening space using multiple droplets with secondary buffers for protein crystallization

NASA Astrophysics Data System (ADS)

Zhang, Chen-Yan; Dong, Chen; Lu, Xiao-Li; Wang, Bei; He, Tian-Yuan; Yang, Rui-Zeng; Lin, Hua-Long; Yang, Xue-Zhou; Yin, Da-Chuan

2017-04-01

We have proposed a rational strategy for selecting a suitable pH of protein solution based on protein biochemical properties. However, it is difficult to use this strategy for biochemical properties unknown proteins. In this paper, a simpler and faster pH buffer strategy was proposed. An additional pH-controlling buffer was added to crystallization droplet mixed with protein solution and commercial crystallization reagents to adjust its pH. The results revealed that protein crystallization success rates were enhanced by this strategy due to expansion of the pH screening space, which was closely related with protein solubility. Thus, the possibility of reaching supersaturation was increased by using this strategy.

Free flow cell electrophoresis using zwitterionic buffer

NASA Technical Reports Server (NTRS)

Rodkey, R. Scott

1990-01-01

Studies of a zwitterionic buffer formulated for cell electrophoresis were done using the McDonnell-Douglas Continuous Flow Electrophoresis System. Standard buffers were analyzed for their stability in the electrical field and the results showed that both buffers tested were inherently unstable. Further, titration studies showed that the standards buffers buffered poorly at the pH employed for electrophoresis. The zwitterionic buffer buffered well at its nominal pH and was shown to be stable in the electrical field. Comparative studies of the buffer with standard cell separation buffers using formalin fixed rabbit and goose red blood cells showed that the zwitterionic buffer gave better resolution of the fixed cells. Studies with viable hybridoma cells showed that buffer Q supported cell viability equal to Hank's Balanced Salt Solution and that hybridoma cells in different stages of the growth cycle demonstrated reproducible differences in electrophoretic mobility.

Deduction of a calcium ion circuit affecting rooster sperm in vitro.

PubMed

Froman, D P

2016-08-01

Four premises for rooster sperm preservation were outlined previously. Understanding mitochondrial Ca cycling in terms of whole-cell Ca flux was one premise. The present work tested the hypothesis that sperm mitochondria can be damaged by intracellular as well as extracellular Ca. Sperm were washed by centrifugation through 12% (wt/vol) Sperm were washed by centrifugation through 12%(at/vol) Accudenz to procure sperm at a physiological concentration within a chemically-defined suspension. Five solutions were tested. Each solution contained 30 m glucose, and had an osmolality of 320 mmol/kg and a pH of 7.4. Washed sperm were diluted to 2.0 × 10 sperm/mL. Each replicate sperm suspension was cooled to 10°C. Sperm mobility was measured after 1, 2, 4, 8, 12, and 24 h. Data were plotted as a function of time in each experiment. Function type was confirmed by lack of fit analysis. A parabola with a maximum at 3.7 h was observed when sperm were suspended in 205 m taurine buffered with 50 m-tris[hydroxyl-methyl]methyl-2-amino-ethanesulfonic acid (TES). This effect was attributed to a Ca flux from the nuclear envelope into mitochondria. An exponential decay was observed when TES-buffered taurine contained 2 m Ca. This effect was attributed to mitochondrial Ca overload induced by uptake of extracellular Ca. Exponential decay also was observed when TES-buffered taurine contained a Ca chelator. This effect was attributed to a Ca flux from the nuclear envelope through mitochondria and then into an extracellular Ca sink. This possibility was supported by the response of sperm to thapsigargin. Specifically, inhibition of sarcoendoplasmic reticulum Ca-ATPase compromised sperm mobility relative to a buffer control. Finally, a 60 m phosphate buffer containing 2 m citrate yielded a linear relationship in contrast to the TES-buffered solutions tested. Sperm mobility after 24 h of storage in the phosphate buffer was 92% of that observed for prewashed sperm. The linear response was attributed to weak chelators providing resistance within a Ca circuit and thereby preventing mitochondrial Ca overload. Fertility, however, was compromised when hens were inseminated with mobile sperm recovered after either 8 or 24 h of storage at 10°C. In conclusion, sperm cell Ca homeostasis was proven to be critical for maintaining sperm mobility in vitro, but mitochondrial Ca uptake is not the sole phenomenon that compromises sperm function during in vitro storage.

Assessment of polyelectrolyte coating stability under dynamic buffer conditions in CE.

PubMed

Swords, Kyleen E; Bartline, Peter B; Roguski, Katherine M; Bashaw, Sarah A; Frederick, Kimberley A

2011-09-01

Dynamic buffer conditions are present in many electrophoretically driven separations. Polyelectrolyte multilayer coatings have been employed in CE because of their chemical and physical stability as well as their ease of application. The goal of this study is to measure the effect of dynamic changes in buffer pH on flow using a real-time method for measuring EOF. Polyelectrolyte multilayers (PEMs) were composed of pairs of strong or completely ionized polyelectrolytes including poly(diallyldimethylammonium) chloride and poly(styrene sulfonate) and weak or ionizable polyelectrolytes including poly(allylamine) and poly(methacrylic acid). Polyelectrolyte multilayers of varying thicknesses (3, 4, 7, 8, 15, or 16 layers) were also studied. While the magnitude of the EOF was monitored every 2 s, the buffer pH was exchanged from a relatively basic pH (7.1) to increasingly acidic pHs (6.6, 6.1, 5.5, and 5.1). Strong polyelectrolytes responded minimally to changes in buffer pH (<1%), whereas substantial (>10%) and sometimes irreversible changes were measured with weak polyelectrolytes. Thicker coatings resulted in a similar magnitude of response but were more likely to degrade in response to buffer pH changes. The most stable coatings were formed from thinner layers of strong polyelectrolytes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of pravastatin acid, lactone and isomers in human plasma by UHPLC-MS/MS and its application to a pediatric pharmacokinetic study.

PubMed

van Haandel, Leon; Gibson, Kim T; Leeder, J Steven; Wagner, Jonathan B

2016-02-15

An ultra high pressure liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantitation of pravastatin and major metabolites, 3'α-hydroxy-pravastatin, pravalactone and 3'α-hydroxy-pravalactone, in human plasma has been developed and validated. Aliquots of (100μL) plasma in EDTA were diluted in pH 4.5 (0.1M buffer) to stabilize the analytes and subjected to hydrophilic lipophilic balance (HLB) solid phase extraction on 96 well μelution plates. Extracted samples were evaporated to dryness and reconstituted in pH 4.5 buffer. Chromatographic separation was performed on a Cortecs™ C18 column (2.1×100mm, 1.8μm), using gradient elution with a blend of acetonitrile and 10mM methylammonium acetate buffer (pH 4.5) at a flow rate of 0.4mL/min. Mass spectrometric detection was performed using multiple reaction monitoring (MRM) switching between positive/negative electrospay ionization (ESI). Pravastatin, 3'α-hydroxy-pravastatin, and internal standards [(2)H3]-pravastatin, and [(2)H3]-3'α-hydroxy-pravastatin were monitored in negative ESI mode at ion transitions m/z 423.2→321.1 and 426.2→321.1, respectively. Positive ESI mode was used for the detection of pravalactone, 3'α-hydroxy-pravalactone, and internal standards [(2)H3]-pravalactone, and [(2)H3]-3'α-hydroxy-pravalactone at ion transitions m/z 438.2→183.1 and 441.2→269.1 respectively. The method was linear for all analytes in the concentration range 0.5-200nM with intra- and inter-day precisions (as relative standard deviation) of ≤5.2% and accuracy (as relative error) of ≤8.0% at all quality control levels. The method was successfully applied to the investigation of pharmacokinetic properties of pravastatin and its metabolites in children after an oral dose of 20-40mg. Copyright © 2016 Elsevier B.V. All rights reserved.

Report of the key comparison APMP.QM-K19. APMP comparison on pH measurement of borate buffer

NASA Astrophysics Data System (ADS)

Hioki, Akiharu; Asakai, Toshiaki; Maksimov, Igor; Suzuki, Toshihiro; Miura, Tsutomu; Obromsook, Krairerk; Tangpaisarnkul, Nongluck; Rodruangthum, Patumporn; Wong, Siu-Kay; Lam, Wai-Hing; Zakaria, Osman; Anuar Mohd. Amin, Khirul; Thanh, Ngo Huy; Máriássy, Michal; Vyskocil, Leos; Hankova, Zuzana; Fisicaro, Paola; Stoica, Daniela; Singh, Nahar; Soni, Daya; Ticona Canaza, Galia; Kutovoy, Viatcheslav; Barbieri Gonzaga, Fabiano; Dias, Júlio Cesar; Vospelova, Alena; Bakovets, Nickolay; Zhanasbayeva, Bibinur

2015-01-01

The APMP.QM-K19 was organised by TCQM of APMP to test the abilities of the national metrology institutes in the APMP region to measure a pH value of a borate buffer. This APMP comparison on pH measurement was proposed by the National Metrology Institute of Japan (NMIJ) and the National Institute of Metrology (Thailand) (NIMT) at the APMP-TCQM meeting held 26-27 November 2012. After approval by TCQM, the comparison has been conducted by NMIJ and NIMT. The comparison is a key comparison following CCQM-K19 and CCQM-K19.1. The comparison material was a borate buffer of pH around 9.2 and the measurement temperatures were 15 °C, 25 °C and 37 °C. This is the second APMP key comparison on pH measurement and the fourth APMP comparison on pH measurement following APMP.QM-P06 (two phosphate buffers) in 2004, APMP.QM-P09 (a phthalate buffer) in 2006 and APMP.QM-K9/APMP.QM-P16 (a phosphate buffer) in 2010-2011. The results can be used further by any participant to support its CMC claim at least for a borate buffer. That claim will concern the pH method employed by the participant during this comparison and will cover the used temperature(s) or the full temperature range between 15°C and 37 °C for the participant which measured pH values at the three temperatures. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).