Edit My Alerts
Alerts for
Analytical biochemistry
Title: Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins.
2008, Vol. 373 Issue 2 p. 207 Authors: Na, Dong Hee Authors: Park, Eun Ji Authors: Jo, Yeong Woo Authors: Lee, Kang Choon Abstract: This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS-CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG(40K)-, PEG(43.5K)-, and PEG(47K)-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS-CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.
Find Text at NCSU
Title: An optimized GC-MS method detects nanomolar amounts of anandamide in mouse brain.
2008, Vol. 373 Issue 2 p. 220 Authors: Muccioli, Giulio G Authors: Stella, Nephi Abstract: The endocannabinoids anandamide and 2-arachidonoylglycerol, as well as several anandamide-related N-acylethanolamines, belong to a family of lipid transmitter that regulate fundamental physiological processes, including neurotransmission and neuroinflammation. Their precise quantification in biological matrices can be achieved by gas chromatography-mass spectrometry (GC-MS), but this method typically requires multiple time-consuming purification steps such as solid-phase extraction followed by HPLC. Here we report a novel solid-phase extraction procedure allowing for single-step, and thus higher throughput, purification of endocannabinoids and N-acylethanolamines before GC-MS quantification. We determined the minimal amount of mouse brain tissue required to reliably detect endocannabinoids and N-acylethanolamines when using this approach and provide direct evidence for quantification accuracy by using radioactive and deuterated standards spiked into mouse brain samples. Using this method, we found that mouse brain contains much higher levels of anandamide (>1nmol/g tissue) than previously reported, whereas levels of 2-arachidonoylglycerol and other N-acylethanolamines are well within the range of previous reports. In addition, we show that mouse brain amounts of endocannabinoids and N-acylethanolamines differ depending on animal gender as well as on whether the tissue was fixed or not. Our study shows that endocannabinoid and N-acylethanolamine levels quantified in mouse brain by GC-MS depend closely on tissue amount and preparation as well as on animal gender and that, depending on such parameters, anandamide levels could be underestimated.
Find Text at NCSU
Title: Block copolymer-oligonucleotide conjugates for genotyping on microarrays.
2008, Vol. 373 Issue 2 p. 229 Authors: de Lambert, Bertrand Authors: Chaix, Carole Authors: Charreyre, Marie-Th©♭r©·se Authors: Martin, Thibault Authors: Aigoui, Arnaud Authors: Perrin-Rubens, Agn©·s Authors: Pichot, Christian Authors: Mandrand, Bernard Abstract: Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.
Find Text at NCSU
Title: Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L.
2008, Vol. 373 Issue 2 p. 239 Authors: Rhoden, Kerry J Authors: Cianchetta, Stefano Authors: Duchi, Serena Authors: Romeo, Giovanni Abstract: The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35muM and a V(max) of 55muM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43muM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.
Find Text at NCSU
Title: Microcalorimetric study of the inhibition of butyrylcholinesterase by carbamates.
2008, Vol. 373 Issue 2 p. 247 Authors: Debord, Jean Authors: Laubarie, C©♭cile Authors: Dantoine, Thierry Abstract: The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by three carbamates (eserine, neostigmine, and rivastigmine) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5). The kinetics of carbamylation was studied in the absence or presence of the substrate, butyrylcholine, using an extension of the model described by Stojan and coworkers (FEBS Lett. 440 (1998) 85-88). The model was fitted to the data by a nonlinear regression procedure using simulated annealing followed by Marquardt's method. The affinity of the carbamates for the free enzyme increased in the order neostigmine
Find Text at NCSU
Title: Adsorption and elution characteristics of nucleic acids on silica surfaces and their use in designing a miniaturized purification unit.
2008, Vol. 373 Issue 2 p. 253 Authors: Poeckh, Tyson Authors: Lopez, Santiago Authors: Fuller, A Oveta Authors: Solomon, Michael J Authors: Larson, Ronald G Abstract: We report nucleic acid (NA) adsorption isotherms and elution profiles for silica surfaces and use these to design a miniaturized NA purification unit based on solid-phase extraction with silica beads. The procedure is based on a pressure drop equation for flow through a packed bed and allows estimation of key design parameters such as channel dimensions, liquid flow rates, sample volume, and amount of silica needed. The usefulness of this design procedure is demonstrated by applying it to a column-based NA purification device for influenza detection for a case study of Madin-Darby canine kidney cells infected with influenza A virus.
Find Text at NCSU
Title: Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins.
2008, Vol. 373 Issue 2 p. 281 Authors: Obata, T Authors: Nomura, M Authors: Kase, Y Authors: Sasaki, H Authors: Shirasawa, Y Abstract: The gelation of Limulus amebocyte lysate (LAL) evoked by bacterial endotoxins can be detected earlier than with usual methods by using laser scattering photometry to recognize the formation of small particles of clotted enzyme produced when the reaction mixture is agitated. The appearance of these small particles means that the influence of endotoxins has stimulated activation of the clotting enzyme across the LAL cascade, and the timing of their appearance is related to endotoxin concentration. This new method can be used for quick and sensitive endotoxin assay. The average endotoxin level of healthy volunteers was assayed to be 0.0738 pg/ml [0.0312-0.3445 pg/ml] (n = 11) within 70 min from the start of the assay.
Find Text at NCSU
Title: A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation.
2008, Vol. 373 Issue 2 p. 287 Authors: Lane, Darius J R Authors: Lawen, Alfons Abstract: Ferricyanide reduction frequently is analyzed to determine the activity of membraneous reductases. An improved, highly sensitive, and rapid method for quantitative endpoint determination of ferrocyanide is presented. Ferrocyanide is oxidized by Fe(3+) in the presence of Ferene-S under acid conditions to form a chromogenic Ferene-S/Fe(2+) complex. The latter is quantitated at 593 nm with a sensitivity of 33.2 mM(-1) . cm(-1). The assay is 60% more sensitive to ferrocyanide (and with a 50% lower detection limit) than the prevailing method of Avron and Shavit, which employs sulfonated bathophenanthroline as the ferrous chromogen. Both pH dependence and potential sources of interference are discussed. Using the method, a sulfhydryl-sensitive, ascorbate-stimulated transplasma membrane ferricyanide reductase was assayed in human chronic myeloid (K562) leukemia cells. Furthermore, malonate-sensitive succinate dehydrogenase activity of heart mitochondria was easily assayed with ferricyanide as terminal electron acceptor. The current method will suit routine applications demanding high throughput, robustness, and sensitivity in a 96-well plate format.
Find Text at NCSU
Title: Detection and quantification of tau aggregation using a membrane filter assay.
2008, Vol. 373 Issue 2 p. 330 Authors: Chang, Edward Authors: Kuret, Jeff Abstract: Aggregation of the microtubule-associated protein tau contributes to the formation of neurofibrillary lesions in Alzheimer's disease and is a useful marker of disease progression. Although filter trap assays have been employed to assess the extent of tau aggregation in cells and tissues as well as in vitro, their performance relative to other assay modalities has not been reported. To clarify this issue, the ability of the filter trap approach to quantify aggregation of purified recombinant full-length tau protein in vitro was examined as a function of membrane chemistry in a 96-well format. Results showed that nitrocellulose yielded the greatest assay sensitivity relative to polyvinylidene fluoride or cellulose acetate at equal membrane porosity. However, all combinations of filter chemistries, porosities, and monoclonal detection antibodies yielded nonlinear correlations between signal intensity and analyte concentration. When corrected for nonlinearity, the filter trap assay determined a value for the critical monomer concentration for tau aggregation that was statistically identical to determinations made by electron microscopy assay. The data suggest conditions under which filter trap assays can be used to estimate tau aggregation kinetics.
Find Text at NCSU
Title: Electrocatalytic oxidation and determination of deferasirox and deferiprone on a nickel oxyhydroxide-modified electrode.
2008, Vol. 373 Issue 2 p. 337 Authors: Hajjizadeh, M Authors: Jabbari, A Authors: Heli, H Authors: Moosavi-Movahedi, A A Authors: Shafiee, A Authors: Karimian, K Abstract: The electrocatalytic oxidation of two orally administered iron chelator drugs (deferiprone, CP20, and deferasirox, ICL670) was investigated on a nickel oxyhydroxide-modified nickel electrode in alkaline solution. The oxidation process involved and its kinetics were investigated using cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy techniques, as well as steady-state polarization measurements. Voltammetric studies indicated that in the presence of the drugs under study, the anodic peak current of low-valence nickel species increased, followed by a decrease in the corresponding cathodic current. This result indicates that the drugs were oxidized via oxyhydroxide species immobilized on the electrode surface via an EC' mechanism. A mechanism based on the electrochemical generation of Ni(III) active sites and their subsequent consumption by the drugs in question was also investigated. The corresponding rate law under the control of charge transfer was developed, and kinetic parameters were derived. In this context, the charge-transfer resistance accessible both theoretically and through impedancemetry was used as a criterion. The rate constants of the catalytic oxidation of the drugs and the electron-transfer coefficients are reported. A sensitive, simple, and time-saving amperometric procedure was developed for the analysis of deferasirox and deferiprone, with detection limits of 28 and 19muM, respectively. The electrode was used for the direct assay of deferasirox and deferiprone tablets.
Find Text at NCSU
Title: Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data.
2008, Vol. 373 Issue 2 p. 370 Authors: Swillens, St©♭phane Authors: Dessars, Barbara Authors: Housni, Hakim El Abstract: Amplification of a cDNA product by quantitative polymerase chain reaction (qPCR) gives rise to fluorescence sigmoidal curves from which absolute or relative target gene content of the sample is inferred. Besides comparative C(t) methods that require the construction of a reference standard curve, other methods that focus on the analysis of the sole amplification curve have been proposed more recently. Among them, the so-called sigmoidal curve fitting (SCF) method rests on the fitting of an empirical sigmoidal model to the experimental amplification data points, leading to the prediction of the amplification efficiency and to the calculation of the initial copy number in the sample. The implicit assumption of this method is that the sigmoidal model may describe an amplification curve quantitatively even in the portion of the curve where the fluorescence signal is hidden in the noise band. The theoretical basis of the SCF method was revisited here for defining the class of experimental amplification curves for which the method might be relevant. Applying the SCF method to six well-characterized different PCR assays illustrated possible pitfalls leading to biased estimates of the amplification efficiency and, thus, of the target gene content of a sample.
Find Text at NCSU
Title: DNA digestion to deoxyribonucleoside: A simplified one-step procedure.
2008, Vol. 373 Issue 2 p. 383 Authors: Quinlivan, Eoin P Authors: Gregory, Jesse F Abstract: We present a simple and inexpensive 'one-step' protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, this new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol.
Find Text at NCSU
Title: PseAAC: A flexible web server for generating various kinds of protein pseudo amino acid composition.
2008, Vol. 373 Issue 2 p. 386 Authors: Shen, Hong-Bin Authors: Chou, Kuo-Chen Abstract: The pseudo amino acid (PseAA) composition can represent a protein sequence in a discrete model without completely losing its sequence-order information, and hence has been widely applied for improving the prediction quality for various protein attributes. However, dealing with different problems may need different kinds of PseAA composition. Here, we present a web-server called PseAAC at http://chou.med.harvard.edu/bioinf/PseAA/, by which users can generate various kinds of PseAA composition to best fit their need.
Find Text at NCSU
Title: Strategies to evade false positives in the in situ analysis of peptide antibiotics in SDS-PAGE gels.
2008, Vol. 373 Issue 2 p. 401 Authors: Anandaraj, Balaiah Authors: Selvamanikandan, Athinarayanan Authors: Mittal, Prabakar Authors: Elavarasi, Natarajan Authors: Thangadurai, Chinnathambi Authors: Murugan, Vadivel Abstract: In situ activity assay is one of the promising techniques for the characterization of peptide antibiotics. This assay was carried out for the peptide purified from a new bacterial isolate Paenibacillus alvei and commercial peptide antibiotic polymixin E. Towards this, the routine and new protocols were tried. Interestingly, the unexpected result of these experiments - the "switch over activity" has led us to have further investigations. Here, we have addressed the potential problem in the methodology of in situ assay and demonstrated a fool proof protocol to evade the false positive results.
Find Text at NCSU
Title: Determination of protein oligomerization state: Two approaches based on glutaraldehyde crosslinking.
2008, Vol. 373 Issue 2 p. 404 Authors: Fadouloglou, Vasiliki E Authors: Kokkinidis, Michael Authors: Glykos, Nicholas M Abstract: Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking procedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two approaches based on glutaraldehyde that readily give reliable results.
Find Text at NCSU
Title: Do zwitterions contribute to the ionic strength of a solution?
2008, Vol. 373 Issue 2 p. 407 Authors: Stellwagen, Earle Authors: Prantner, Jason D Authors: Stellwagen, Nancy C Abstract: Capillary electrophoresis has been used to determine whether zwitterions contribute to the ionic strength of a solution, by measuring the mobility of a double-stranded DNA oligomer in cacodylate-buffered solutions containing various concentrations of the ionic salt tetraethylammonium chloride (TEA(+)Cl(-)) or the zwitterion tricine(+/-). The mobility of the DNA decreased as the square root of ionic strength, as expected from the Debye-H©ơckel-Onsager theory of electrophoresis, when TEA(+)Cl(-) was added to the buffer. However, the mobility was independent of the concentration of added tricine(+/-). Hence, zwitterions do not contribute to the ionic strength of a solution.
Find Text at NCSU
Edit My Alerts
For technical assistance or for help finding articles listed above, contact us at:
LibraryAlerts@ncsu.edu.