Tris Solution Research Articles

HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA

The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a approximately 450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.

Combination of the optical waveguide lightmode spectroscopy method with electrochemical measurements

Optical waveguides are normally sensitive to the surrounding media and also to the surface contaminations. The effective refractive index changes at the surface. Various sensor systems were developed based on this effect. One of the most sensitive and effective methods is the optical waveguide lightmode spectroscopy (OWLS). At the same time, electrochemical methods are widely used both in inorganic and organic chemistry, and also advantages in microbiology were demonstrated. In this work, efforts are made and results are presented for the combination of these two methods for simultaneous measurement of refractive index and electrical current changes caused by the presence of cells/molecules/ions to be investigated. An electrically conductive indium tin oxide (ITO) nanolayer is deposited and activated on the top of the OWLS planar waveguide oxide layer. ITO layers serve as working electrodes in the electrochemical measurements. The basic setup and an integrated system are demonstrated here. Measurements using H 2O 2, toluidine blue solutions, and KCl and TRIS solutions as buffer and transport media are represented. Measurements show both the changes detected by the sensor layer and the effect of the applied potential in cyclic and chrono voltammetric measurements. Results demonstrate an effective combination of optical and electrochemical methods.

Synthesis and characterization of hydroxyapatite nanoparticles

Hydroxyapatite (HA) nanoparticles were synthesized by precipitation method in the presence of simulated body fluid (SBF) solution or calcium phosphor tris (CaPTris) solution which was used for the first time. The synthesized powders were characterized in terms of structure (Fourier transform infrared spectrograph, FTIR and X-ray diffraction, XRD), particle size and morphology (zeta sizer and scanning electron microscopy, SEM). HA crystallites were in hexagonal structure (space group: P63/m). Sizes of crystallites determined at the (002) and (300) crystal planes varied from 15.88 to 16.12nm for CaPTris solution. Although particle size distributions were significantly different for both samples, SEM images of the samples showed that the particles were in nano-sized. Characterization results depicted that HA nanoparticles were also synthesized using CaPTris solution by precipitation method as well as in the presence of SBF solution.

Rapid quantification of total genomic DNA methylation degree by short-end injection capillary zone electrophoresis

We propose a new capillary zone electrophoresis method applying short-end injection technique for the fast evaluation of methylcystosine/total cytosine ratio after acidic DNA hydrolysis. By short-end injection and by using a 100 mmol/l Tris solution titrated with 1 mol/l phosphoric acid to pH 3.75 as background electrolyte, cytosine and methylcytosine were separated with a good resolution in less than 1.5 min. Stepwise multiple linear regression with DNA methylation degree as the dependent variable and age, cysteine, homocysteine and methionine as independent variables, showed a negative association with age and that total cysteine is the most important determinant of DNA methylation.

Synthesis of chitin calcium phosphate composite in different growth media

AbstractThis paper presents some results concerning chitin calcium phosphate composites obtained in different growth media. The synthesis of chitin calcium phosphate composite was carried out by phosphorylation, calcification, and soaking in different calcium phosphate growth media. This research is focused on studying and understanding the effect of using different growth media on composite samples. Hydroxyapatite was determined in the composite samples synthesized by using both simulated body fluid (SBF) and calcium phosphorus tris (Ca‐PTris) solution. Calcium/phosphorus (Ca:P) ratio of the composite synthesized by using SBF was found higher than that of composite synthesized by using Ca‐PTris solution as calcium phosphate growth media. The Ca:P ratio of the composite (1.72) immersed in 1.5× SBF for 35 day is similar to the theoretical value of hydroxyapatite (1.67) and closer to the theoretical value of human bone (1.75). POLYM. COMPOS., 29:84–91, 2008. © 2007 Society of Plastics Engineers

Selective Incorporation of Polyanionic Molecules into Hamster Prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

Development of Alkali Containing Calcium Phosphate Cements

Commercially available calcium phosphate cements set by precipitation of nanoapatite or brushite. The goal of this study was to elucidate the most suitable conditions for forming cements from calcium potassium sodium phosphate. Furthermore, the behaviour of these cements after immersion in SBF and/or TRIS solution was investigated. Using varying additives resulted in differences in solubility kinetics. The XRD spectra of all investigated cement compositions displayed Ca2KNa(PO4)2 after setting. However, the various cement compositions differed with respect to apatite formation when immersed in TRIS buffer in and/or SBF solution. Therefore, when investigating calcium phosphate cements we consider it necessary to clearly differentiate between the phases which form after completion of the final setting time when these materials set in air, and the phases which form in a time dependant manner after immersion in different biological fluids.

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose‐scented geranium (Pelargonium sp.), a hyper‐acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.

Change in the volume of orthophosphoric acid solutions in the neutralization with solutions of various bases

Volume changes in the reactions of orthophosphoric acid neutralization with solutions of NaOH, NH4OH, and tris(hydroxymethyl)methanamine in the mixed solvent H2O-dimethylformamide were studied by dilatometric titration. Volume effects of the reaction depending on the mixed solvent composition and base strength were discussed.

Characterization of polyelectrolyte multilayers on mica and oxidized titanium by streaming potential and wetting angle measurements

Multilayer adsorption of polyelectrolytes on mica and electrochemically oxidized titanium was studied by the streaming potential method using a parallel-plate channel arrangement. Streaming potential data were complemented by wetting angle measurements. Two types of polyelectrolytes (PE) were used: polyallylamine hydrochloride (PAH), of a cationic type, and polysodium 4-styrenesulfonate (PSS) of an anionic type (both having molecular weight of ∼70,000). Layer by layer adsorption of polyelectrolytes from NaCl and TRIS solutions of various concentrations was studied in situ. It was demonstrated that after completing a bilayer, periodic variations in the apparent zeta potential between positive and negative values appeared for multilayers terminated by PAH and PSS, respectively. This was in accordance with zeta potential of the polymers in the bulk measured by electrophoresis. It also was observed that streaming potential data correlated well with the wetting angle characteristics of dried polyelectrolyte films. Variations in the contact angle between 70° for PAH and 40° for PSS were exactly in phase with the variation in the zeta potential. The stability of polyelectrolyte films against prolong washing (reaching 24 h) was determined using the streaming potential method. It was revealed that both for mica and titanium, the PSS layer was considerably more resistant to washing, compared to the PAH layer. The experimental data obtained were consistent with a model postulating particle-like adsorption of polyelectrolytes.

Kinetic and Mutational Analyses of the Major Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.

Amplified fluorescence quenching in high ionic strength media.

We report a new cationic poly(phenylene ethynylene) (PPE) derivative that exhibits strong amplified fluorescence quenching in the presence of electron-deficient species, yielding high Stern-Volmer coefficients of 4.7 × 10 M in aqueous solutions. Importantly, with the addition of appropriate non-ionic surfactants, the polymer is found to retain its excellent sensitivity even when transferred to high ionic strength buffered media, which have previously been shown to suppress the amplified quenching effect in other polyelectrolyte systems. The cationic PPE derivative yields Stern-Volmer coefficients as high as 10 M in 25 mM buffer solutions of both tris(hydroxymethyl) aminomethane (Tris) and sodium acetate containing 150 mM sodium chloride, the optimal conditions for many enzymes such as phosphatases. The ability to maintain high Stern-Volmer coefficients in high ionic strength buffered media extends the applicability of ionic conjugated polymers to high sensitivity detection in biological media, and thus greatly enhances their versatility as biological sensors.

Characterization of dual-wavelength seminaphthofluorescein and seminapthorhodafluor dyes for pH sensing under high hydrostatic pressures

Hydrostatic pressure is an important physical parameter in biology, with pressures in the few-hundred-atm range having significant effects on cellular morphology, metabolism, and viability. To ensure valid results when studying pressure effects using fluorescence spectroscopy and imaging methods, metabolic probes need to be characterized for high-pressure use. Of interest is the sensing of pH at high pressures due to the key role that pH plays in cellular function. Despite the availability of pH-sensitive dyes, only a few have been characterized for high-pressure use. Here we present the effects of pressure on the acid–base equilibria of four dual-wavelength seminaphthorhodafluor and seminaphthofluorescein dyes (p K a = 6.6–7.8). Using phosphate buffers as high-pressure pH references, we investigate the pressure dependence of p K a for these dyes and determine the volume change associated with the acid-dissociation reaction. We find that if pressure-induced p K a changes are not accounted for during interpretation of emission spectra, systematic errors of up to 0.02 pH units per 100 atm would result, comparable to previously measured pressure-induced pH changes in vivo. Results are validated by correctly sensing pH changes in Tris and acetate solutions. Methods presented here are applicable to other metabolic probes utilizing dual-wavelength ratiometric sensing modes.

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Separation and determination of perfluorinated carboxylic acids using capillary zone electrophoresis with indirect photometric detection

Perfluorinated carboxylic acids (PFCAs) belong to anthropogenic fluoroorganic compounds that have been detected in the natural environment and living organisms including humans. A capillary zone electrophoretic method with indirect UV detection using 2,4-dinitrobenzoic acid (2,4-DNBA) as a chromophore probe has been developed for analysis of PFCAs (C6–C12) in water. Optimal analyte resolution and detection sensitivity was obtained with 50 mM Tris solution of pH 9.0 and 50% methanol as a background electrolyte (BGE). The baseline separation of C6–C12 PFCAs was obtained within 20 min with detection limits in the range from 0.6 to 2.4 ppm.

Synthesis of Twisted Intercalating Nucleic Acids Possessing Acridine Derivatives. Thermal Stability Studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.

Fabrication of PMMA micro- and nanofluidic channels by proton beam writing: electrokinetic and morphological characterization

The fabrication of microfluidic channels in poly(methylmethacrylate) (PMMA) by proton beam writing and the characterization of their electrokinetic behavior are reported. Microchannels down to 200 nm width have been fabricated in high-molecular-weight, thick PMMA sheets and a surface smoothness of 2.45 nm for the sidewalls of the channels was measured using atomic force microscopy. The polymeric channels were sealed using thermal bonding. The methods of digital video microscopy and current monitoring were used for characterization of the electrokinetic phenomena. The electrokinetic properties of the PMMA rectangular microchannels were measured in 20 mM buffered solutions of phosphate (pH 7.20), tris (pH 8.00) and borate (pH 9.05) at different voltages. In addition, particle image velocimetry was used to determine the electroosmotic flow profile and the electrophoretic mobility of 600 nm polystyrene microspheres. A theoretical model was developed to predict the bulk electroosmotic flow of phosphate buffer solution in the PMMA microchannels and this model showed good agreement with the measured electroosmotic mobilities.

Caffeine modulates potassium currents in Drosophila neurons

We investigated the effects of caffeine on the delayed-rectifier potassium current (IKDR) which is important in repolarizing the membrane potential, and the transient A-type potassium current (IKA) which regulates neuronal firing threshold and the rate of repetitive action potentials. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The currents were measured from neurons before and after the application of 1mM caffeine to the external saline of the same neuron. IKDR measured in the caffeine-containing solution (470±36pA, n=18), was smaller than that measured in the control 6K/0Ca Tris solution (745±51pA, n=18). IKA measured in the caffeine-containing solution (17±2pA, n=16) was smaller than that measured in the control 6K/0Ca Tris solution (35±4pA, n=16).These results indicate that caffeine reduces IKDR and IKA amplitudes and possibly leads to increased action potential frequency and enhanced neuronal excitability.

Newly synthesized tetraoxa-diaza crown ether derivatives versus commercialized crown ethers in the separation of positional isomers with capillary electrophoresis

Three new tetraoxa-diaza derivatives of 1,4,10,13-tetraoxa-7,16-diazacyclo-octadecane ( R-1, R-2 and R-3) and three commercially available crown ethers, 18-crown-6 (18C6), (+)-18-crown-6-tetracarboxilic acid (18C6H 4) and 1,4,10,13-tetraoxa-7,16-diazacyclo-octadecane, were investigated to separate the positional isomers of aminophenol, aminobenzoic acid and aminocresol. The running electrolyte, in which the crown ethers were dissolved, was a 50 mM Tris solution adjusted to pH 2.0 with hydrochloric acid. Using 50 mM H 3PO 4 buffer, whose pH was adjusted to 2.0 with Tris, or only hydrochloric acid solution with the same pH, did not allow good separations for the tested components. The effect of the crown ether concentration on the separation of the 11 positional isomers was studied in the concentration range of 10–50 mM. The best separations were achieved using the 18C6 and the 18C6H 4 crown ethers: 9 isomers out of 11 could be separated within one run. The m- and p-aminophenol isomers could not be separated under the investigated experimental conditions. The newly synthesized tetraoxa-diaza crown ether derivatives were only found suitable for the separation of aminobenzoic acid positional isomers. The macrocyclic ring of the tetraoxa-diaza crown ethers was not able to form a stable inclusion complex with the tested positional isomers. Consequently, the aminophenol and aminocresol isomers were not separated, the isomers migrated with the same or very similar velocities.